Description
BSC 300-001 Exam 1 Studyguide (Ch. 1,2,3,4,7)
I. Chapter 1 – Molecules, Cells and Model Organisms
a. The Molecules of Life (Cells: The fundamental units of life)
i. Common ancestral cell that lived 3.9-2.5 billion years ago
ii. Three Domain Theory (Carl Woese 1977) replaced the Five Kingdom Theory
1. Life branched into three major groups: Archaea, Bacteria, and
Eukaryotes
iii. Cell Theory – every cell came from a pre-existing cell
1. Never been discounted
2. Articulated in the mid-1800’s by Schleiden, Schwann, Virchow
(and Remak)
3. NEED TO KNOW THE THREE PARTS:
a. All organisms are composed of one or more cells
We also discuss several other topics like What type of biomolecules are sex hormones?
b. The cell is the structural unit of life Don't forget about the age old question of How does kepler's laws apply?
c. Cells arise from pre-existing cells by division
iv. All life carries genetic info in the form of DNA
1. Genes, composed of DNA, define biological structure and maintain the integration of cellular function
a. Many genes encode proteins, the primary molecules that
make up cell structure and carry out cellular activities
2. Within DNA, genes provide into to carry our cellular activities
3. Evolution driven by alterations to genetic info (mutations, gene
duplication, and genome duplication followed by mutation)
a. This is what makes life so diverse
v. Major Cellular Macromolecules – the polymers and their monomers 1. Proteins are made of amino acids
a. Structure and perform most cellular tasks
i. Workhorses of the cell
b. Most abundant and functionally versatile of the cellular If you want to learn more check out What is sociology?
macromolecules
c. Many proteins are enzymes
d. Cytoskeletal proteins serve as structural components We also discuss several other topics like What is marketing mix?
e. Many proteins that are embedded in the plasma membrane
import and export small molecules
f. Some proteins are hormones or hormone receptors
2. Nucleic Acids such as RNA and DNA are made of nucleotides
(bases)
a. Cary coded info for making proteins at the right time and
place
b. DNA made of A with T and C with G
i. Complementary matching of strands
ii. Genes – specific sequences of DNA
1. Genes contain two parts
a. Coding region specifies amino acid
sequence of a protein
BSC 300-001 Exam 1 Studyguide (Ch. 1,2,3,4,7)
b. Regulatory region binds specific
proteins and controls when and in
which cells the gene’s protein is
made
iii. Convert coded info in DNA into proteins:
1. Transcription – protein coding region of a
gene is copied into RNA strand whose
sequence is the same as one of the two in the
DNA
a. RNA polymerase catalyzes linkage
of nucleotides into an RNA chain
using DNA as a template
b. In eukaryotic cells the initial ENA If you want to learn more check out Where can you get proteins?
product is processed into a smaller
messenger RNA molecule, which
moves out of the nucleus into the
cytoplasm
2. Ribosome carries out translation
a. Ribosome assembles and links
together amino acids in the precise
order dictated by the mRNA
sequence
iv. Transcription factors bind to specific sequences of DNA and act as switches, either activating or
repressing transcription of particular genes
c. The Sequence Hypothesis
i. The sequence of bases in DNA or RNA determines
the sequence of amino acids for which that segment
of nucleic acid codes, and this amino acid sequence
determines the 3D structure into which protein folds
d. Central Dogma (WRONG IN THE BOOK)
i. Deals with detailed residue-by residue transfer of
sequential info
ii. States that such info cannot be transferred back
from protein to either protein or nucleic acid
1. AKA genetic info flows unidirectionally,
from nucleic acid to protein Don't forget about the age old question of What is homologies?
2. Info flows from one generation to the next
3. Carbohydrates are made of sugars
a. Provide structural support, energy storage, and are the source of many small molecules
b. Simply 5-6 Carbon sugars
c. Can be added to molecules to modify function
4. Non-polymer macromolecule: lipids
a. Structural in cell membranes and energy source (fatty acids)
BSC 300-001 Exam 1 Studyguide (Ch. 1,2,3,4,7)
b. Amorphous – insoluble in water but soluble in oils
c. Phospholipids are the conserved building blocks of all
cellular membranes
i. Serve as building materials for other molecules
ii. Important in cell signaling
iii. Non-phospholipid cholesterol alters membrane
dynamics and is the source of all steroid hormones
iv. Phospholipids have a charge and build the double
membrane of the cell
v. Has a water loving hydrophilic head and a water
hating hydrophobic tail
vi. Impermeable to water, all ions, and virtually all
hydrophilic small molecules
b. Prokaryotic Cell Structure and Function
i. Origin of Eukaryotic Cells
1. Prokaryotic cells evolved first and gave rise to Eukaryotic cells a. Endosymbiont – sort of parasitism – ingested something
making ATP, which stayed there and acted as a parasite
2. Endosymbiont Theory – organelles in eukaryotic cells
(mitochondria and chloroplasts) evolved from smaller prokaryotic cells
ii. Types of Prokaryotic Cells
1. Domain Archaea
a. Extremophiles, methanogens (oxygen is poisonous to
them), halophiles (high salt environments), acidophiles,
thermophiles (cannot live at low temps), endoliths (live
inside rocks, can utilize minerals or metals as an energy
source)
i. Ocean vents, high temps, no O2
1. Shows us that life has the capacity to live
anywhere
2. Domain bacteria
a. Includes smallest known cells – mycoplasma – as well as
cyanobacteria – photosynthetic bacteria
b. Possess a cell wall composed of peptidoglycan
3. Defining features of Prokaryotes
a. Unicellular
b. Small circular genome – many copies per cell
c. No nucleus, but the genome is folded and condensed into
the nucleoid
d. Additional genetic info in small, circular plasmids that can
be exchanged through cells
e. No significant processing of messenger RNA (mRNA)
f. Complex extracellular cell wall composed of peptidoglycan
(protein and sugar)
BSC 300-001 Exam 1 Studyguide (Ch. 1,2,3,4,7)
g. Gram negative: thin cell wall with second exterior lipid
membrane
i. Doesn’t stain
h. Gram positive: think cell wall with no second membrane
i. 0.5-5 million bp
ii. 500-5000 genes
4. Escherichia coli: the original lab rat
a. We learn about ourselves from E. coli
b. Commonly found in lower intestine of warm-blooded
animals
c. Dozens of types (strains) – most harmless
i. A few with mild to severe pathogenicity
d. Critical model for early molecular biology work (The
Sequence Hypothesis, the Central Dogma, enzyme
function, etc)
c. Eukaryotic Cell Structure and Function we need to know cell parts and be able to point them out and tell their functions
i. Key Features of Eukaryotes
1. Many unicellular
a. Some where multicellularity evolved, including animals,
the metazoan
2. Membrane enclosed subcellular compartments, organelles that separate cell processes
3. Cytosol – organelle-free solution of water, dissolved ions, small molecules and proteins
4. Cytoplasm – cytosol and organelles
a. NEED TO KNOW the difference between cytosol and
cytoplasm
5. True nucleus containing several linear DNA chromosomes
associated with proteins called histones forming chromatin
6. Nuclear envelope consists of 2 lipid membranes (outer and inner) that are continuous with one another
a. Fuse at nuclear pore complexes
7. Membranes fused at nuclear pore complexes, sites where transport into and out of nucleus is regulated
8. Rigid shape of nucleus maintained by internal network of
intermediate filaments called lamins, generating the nuclear lamina 9. Chromosomes only visible by light microscope during cell
division, when they are fully compacted
10. During interphase chromosomes vary in degree of compactions 11. Densely packed heterochromatin is inactive – not being transcribed 12. More loosely packed euchromatin can be transcribed
13. Nucleolus – site of ribosomal gene transcription and assembly of ribosomes (RNA and protein)
14. Diverse specialized organelles perform discrete cellular activities
BSC 300-001 Exam 1 Studyguide (Ch. 1,2,3,4,7)
a. Endoplasmic Reticulum (ER) – system of interconnected
flattened sacs (cisternae) – continuous with outer nuclear
membrane
i. Synthesis of lipids, fatty acids, membrane bound
proteins and protein exported from the cell
ii. Smooth ER – produces fatty acids lipids that
comprise of the cell membrane system
1. Smooth because it lacks ribosomes
iii. Rough ER – produces proteins found in the
endomembrane system
1. Proteins to be secreted gather in the lumen,
the aqueous interior of the Rough ER
d. Cell cycle
i. Unicellular eukaryotes, animals, and plants all use essentially the same cell cycle, the series of events that prepares a cell to divide, and the same actual division process, called mitosis
ii. S (synthesis) phase – chromosomes and the DNA they carry are duplicated iii. M (mitotic) phase – replicated chromosomes separate
iv. G1 and G2 phase – separate M and S phases
1. mRNAs, proteins, lipids, and other cell constituents are made and the cell increases in size
e. Model Organisms – a non-human species that is extensively studied to understand particular biological phenomena, with the expectation that discoveries made in the model will provide general insight into the workings of other organisms
i. Used to be only 5 model organisms, but now that we can change a genome at will, any organism can be a model organism
ii. Yeast – one of the most helpful model organisms
1. Saccharomyces cerevisiae (brewer’s yeast) – the budding years a. Endomembrane studies
2. Schizosaccharomyces pombe – the fission yeast
3. Yeast genetics have been critical for discovering key components of endomembrane trafficking and cell cycle
4. What are the advantages of using yeast?
a. Can be grown easily and cheaply in culture from a single
cell
b. May be haploid or diploid, and both can divide by mitosis
c. Sexual cycle allows exchange of genes between cells
f. Metazoan Structure, Differentiation, and Model Organisms
i. Metazoa – animals
1. Multicellularity and embryonic development
a. Multicellularity – subdivision of labor requires cells to be
organized into different tissue types
i. Epithelia – sheets of tightly associated cells
ii. Mesenchyme – autonomous free moving cells
iii. Neuronal – autonomous and conductive
iv. Muscle – contractile
BSC 300-001 Exam 1 Studyguide (Ch. 1,2,3,4,7)
2. Metazoans
a. Evolved 575 mya
i. Started with Cambrian explosion
b. Thought to have evolved in an ocean-like, saline
environment
c. 30 extant phyla, each defined by distinct body plan
d. Shared characteristics include multicellularity and
embryonic development
e. Vary greatly in genome size and gene number
f. Deeply conserved set of key developmental genes that
drive the processes of cell division, growth, organization
and differentiation
3. Gene Homology
a. Genes with shared ancestry and function are called
homologs
i. Eyeless – gene for eye development in flies
ii. Human version of this gene is Pax6 – mutations in
this gene result in the defect aniridia in which the
iris fails to form
iii. Cloned Pax6 and put it in the legs of flies – created
6 functioning eyes in the legs of the flies
II. Chapter 2 – Chemical Foundations, Part 1
a. Key Concepts
i. Polymerization – small molecules forming macromolecules
ii. Molecular Complementarity – molecules with complementary shapes can form biomolecular interactions
1. Allows tight association between molecules through additive effect of many noon-covalent interactions
2. Lock-and-key kind of fit between their shapes, charges, or other
physical properties
3. Can form multiple noncovalent interactions at close range
4. Depending on the number and strength of the noncovalent
interactions between two molecules and on their environment, their
binding may be tight or loose and lasting or transient
5. Induced fit – if the shape of the binding partner changes and the
molecular complementarity increase after the interaction
b. Covalent Bonds and Noncovalent Interactions
i. Covalent bonds
1. Shared electron pairs
2. Strongest atomic interactions
3. Important in structure of organic molecules
4. Molecules – combinations of atoms held together by covalent
bonds
ii. Non-covalent interactions – ionic bonds, hydrogen bonds, van der Waal’s interactions, and the hydrophobic effect
1. Weaker than covalent bonds
BSC 300-001 Exam 1 Studyguide (Ch. 1,2,3,4,7)
2. Important in shape and chemistry of biological molecules 3. Hydrophobic effect – because water can’t form hydrogen bonds with nonpolar substances, they form “cages” of rigid hydrogen bonded pentagons and hexagons around nonpolar molecules a. Energetically unfavorable – decreases entropy of the
system
b. Like dissolves like
i. Polar molecules dissolve in polar solvents such as
water
ii. Nonpolar molecules dissolve in nonpolar solvents
such as hexane
iii. Electronic structure determines number and geometry of the covalent bonds an atom can make
1. Non-polar bonds
a. Electrons shared equally
i. Electrons spend an equal amount of time around
both nuclei in the bonds
b. Identical or similar electronegativities
2. Polar bonds
a. Electrons pulled toward one of the nuclei and spend more time there – polar bonds (partial positive and partial
negative charges)
b. Different electronegativities
3. Electronegativity – the extent on an atom’s ability to attract an electron
a. Covalent bond between different electronegativities results in polar covalent bonds
4. Polar Covalent Bonds
a. Electric dipole – positive charge separated by negative
charge
i. Dipole moment (m) – quantitative measurement of
the separation of the charges
1. Combination of charge strength and distance
of separation (bond length)
b. Functional Groups – covalently attached molecular units that change the chemical characteristics and properties of
the molecule
i. Establish the chemical properties of all organic
molecules
5. Noncovalent bonds – much weaker than covalent bonds
a. Easy broken and reformed
b. Important in dynamic cellular processes
i. Ex: transient interactions between cellular
macromolecules
c. Four major types:
i. Ionic bonds – between fully charged atoms
BSC 300-001 Exam 1 Studyguide (Ch. 1,2,3,4,7)
1. Cations (+) and anions (-)
2. Weakened in the presence of water
(hydration shell)
3. Much weaker than covalent bonds
4. Contribute greatly to the shape of most
biological molecules
5. Pack together in opposite directions to form
ordered crystalline arrays when in solid dry
state
ii. Hydrogen bonds – covalently bound hydrogen has a
partial positive charge and attracts electrons of a
second atom (usually O) that has a partial negative
charge
1. H- bonds determine the structure and
properties of water
2. H-bonds in biological molecules promote
conformation:
a. Protein structure
b. Annealing strands of DNA
c. Interaction between two molecules
3. Longer and weaker than covalent bonds
iii. Van der Waals Interactions – hydrophobic
attractions between nonpolar molecules due to
transient dipole formation
1. When any two atoms approach each other
2. Temporary atom charges
3. Electron distribution not equal
4. Responsible for interactions such as heptane
where hydrogen or ionic bonds cannot form
5. Strength decreases with distance
c. Chemical Building Blocks of Cells
i. Peptide bond links amino acids into proteins
ii. Phosphodiester bond links nucleotides into nucleic acids
iii. Glycosidic bond links monosaccharides (sugars) into polysaccharides iv. Lipids – poorly soluble, or completely insoluble, in water
1. Have large hydrophobic (hydrocarbon – virtually insoluble in water) regions
a. Lipids must be packaged into special hydrophilic carriers
called lipoproteins that can dissolve into the blood and be
transported throughout the body
2. Include:
a. Fats – alcohol glycerol linked by ester bonds to three fatty
acids
b. Sterols – cholesterol and its derivatives
c. Phospholipids – like fats, but one fatty acid chain is
replaced by a charged phosphate group
BSC 300-001 Exam 1 Studyguide (Ch. 1,2,3,4,7)
i. Amphipathic – large hydrophobic region and a
charged hydrophilic (water liking- readily dissolve
in water) region
ii. Fatty acids – hydrocarbon chain attached to a
carboxyl group
1. Important energy source for cells
2. Fatty acids are covalently attached to
another molecule by esterification
d. Steroids – animal lipids derived from cholesterol
i. Hydrocarbon skeleton with 4 rings
ii. Different groups attached to each steroid
v. Amino Acids
R
-
-
Amino Group ----- Carbon ----- Carboxyl Group
-
-
Hydrogen
1. Basic Structure
a. Central alpha carbon
i. Only L forms of amino acids are found in proteins
ii. D amino acids are prevalent in bacterial cell walls
and other microbial products
b. -H atom
c. Amino group (-NH2)
d. Carboxyl group (-COOH)
e. Variable R group
i. Provides unique chemical characteristics to each
amino acid
1. Residue – when incorporated into a protein
polymer
2. May be polar charged, polar uncharged, or
nonpolar
f. Linked together by peptide bonds – amide type covalent
bonds between amino group of one monomer and the
carboxylic acid group of another
g. Polymers – peptides/polypeptides
h. Proteins – when they fold into a functional 3D shape
2. We need to be able to:
a. Find the side chain if given an image of an amino acid
b. Determine whether an amino acid is hydrophobic, polar
(hydrophilic uncharged), or charged (hydrophilic
charged – acid or base)
BSC 300-001 Exam 1 Studyguide (Ch. 1,2,3,4,7)
i. Polar charged – R groups act as strong organic acids or bases
1. Almost always fully charged at pH 7
2. Can form ionic bonds
3. Histidine – partially charged at pH 7
ii. Polar uncharged – R groups weakly acidic or basic
1. Not ionized at pH 7
2. Can form hydrogen bonds with atoms of
other molecules that have partial charge
(part of polar covalent bonds)
iii. Nonpolar – R groups hydrophobic; lack O and N
1. Cannot interact with water or for
electrostatic bonds
2. Vary in size and shape
3. Can pack tightly into protein core
4. Associate with one another via hydrophobic
and van der Waals interactions in protein
interior
5. Known as hydrophobic amino acids – poorly
soluble in water
iv. Unique amino acids: glycine, cysteine, and histidine 1. 20 biologically relevant amino acids
a. We can synthesize 11
b. Other nine are essential amino acids
that must be included in our diet:
i. Phenylalanine
ii. Valine
iii. Threonine
iv. Tryptophan
v. Isoleucine
vi. Methionine
vii. Leucine
viii. Lysine
ix. Histidine
2. Histidine – imidazole ring can shift from
positively charged in acidic environment to
unprotonated in slightly alkaline conditions
a. Activities of many proteins
modulated by shifts in pH through
protonation or deprotonation of
histidine residues
3. Basic Structure of a nucleotide:
a. Each consists of three parts:
i. Five-carbon sugar
ii. Nitrogenous base on the 1’ Carbon
iii. Phosphate group on the 5’ Carbon
BSC 300-001 Exam 1 Studyguide (Ch. 1,2,3,4,7)
iv. All of this is linked to a nitrogen and carbon
containing ring structure commonly referred to as a
base
b. May be mono-, di-, or tri-phosphate
c. Acidic and at physiological pH, the phosphate group is
deprotonated
4. Nitrogenous bases
a. Nucleosides – covalently attached to a sugar (ribose or
deoxyribose)
i. Combo of a base and sugar without a phosphate
ii. Role as building blocks of other organic molecules
iii. Ribose in RNA
iv. Deoxyribose in DNA
1. Has a proton rather than a hydroxyl group as
position 2’
b. Nucleotides – covalently attached to a sugar (ribose or
deoxyribose) and one to three phosphate groups
5. Bases
a. Purines
i. Adenine and guanine
ii. Double ring
iii. Found in DNA and RNA
b. Pyrimidines
i. Cytosine and thymine
ii. Single ring
iii. Thymine is replaced by Uracil in RNA
6. Nucleotide Base Pairing
a. Nucleotides polymerize through phosphodiester bonds
producing single-stranded nucleic acid polymers
i. Ester bonds – dehydration reaction between
carboxylic acid and an alcohol
b. In DNA two polymers anneal to one another cia
complementary base pairing and hydrogen bonds between
purines and pyrimidines
i. A to R (or U) – 2 hydrogen bonds
ii. G to C – 3 hydrogen bonds
1. Greater stability
vi. Carbohydrates
1. Include simple sugars and sugar polymers
2. Energy storage and structural molecules
3. General formula – (CH2O)n
4. -ose suffix denotes sugars
a. Hexoses and pentoses are the most common ones
5. Linear in no-aqueous state
6. All but one carbon linked to an -OH and H’s
7. Remaining Carbon is a carbonyl (C=O)
BSC 300-001 Exam 1 Studyguide (Ch. 1,2,3,4,7)
8. Examples
a. Ketones – carbonyl on an internal carbon (ketose)
b. Aldehydes – carbonyl on a terminal carbon (aldose)
c. Glycosidic bonds – are -C-O-C- links between sugar -OH
groups
d. Disaccharides – source of readily available energy (like
lactose and sucrose)
e. Oligosaccharides (3-10 sugars) – bound to cells surface
proteins and lipids and used for cell recognition
i. Alter protein solubility and ability to interact with
other proteins
f. Polysaccharides (more than 10 sugars) – energy storage and
structural molecules
*Isomers – molecules with the same chemical formula but different structure
--Stereoisomers different in arrangements of atoms/molecules attached to a central atom (stereogenic center)
----Stereoisomers with a single different stereogenic center – epimers
9. In aqueous solution sugars spontaneously form rings (hemiacetals
or hemiketals), linkage between carbonyl and hydroxyl group
10. Structural isomers generates by different C=O + -OH interactions
a. Additional stereoisomers generated from orientation of -
OH and -H around the linkage
b. Enzymes catalyze chemical reactions in cells and recognize
only specific isomers
11. Polysaccharides – identical sugar monomers but dramatically
different properties
a. Storage polysaccharides – polymers of sugars joined by
glycosidic alpha (1-4) linkage with branches formed by
alpha (1-6 linkages)
i. Glycogen – animal product made of branched
glucose polymers
ii. Starch – plant product made of both branched and
unbranched glucose polymers
b. Structural polysaccharides – polymers joined by beta (1-4)
glycosidic linkage
i. Cellulose – principal plant structural polysaccharide
ii. Chitin – exoskeleton of invertebrates
iii. *Most animals lack digestive enzymes that can
hydrolyze beta glycosidic bonds*
BE FAMILIAR WITH GYLSINE, PROLINE, CYSTINE, AND HISTIDINE
BSC 300-001 Exam 1 Studyguide (Ch. 1,2,3,4,7)
III. Chapter 2 – Chemical Foundations, Part 2
a. Chemical Reactions and Chemical Equilibrium
i. Chemical reactions are reversible, and direction is concentration
dependent
1. The extent and rate at which a chemical reaction proceeds
determines the chemical composition of the cell
2. Initial forward/reverse reaction rates depend on the initial
concentrations of reactants and products
a. Net forward reaction rate slows as the concentration of
reactants decreases
b. Net reverse reaction rate increases as the concentration of
products increases
3. Chemical equilibrium – rates of forward and reverse reactions are
equal
a. No net change in concentrations of reactants and products
b. Equilibrium constant (Keq)
i. Fixed value, dependent on the chemical nature of
the species involved, temperature, and pressure
ii. Useful in determining the amount of energy
released or absorbed by chemical reactions
iii. A catalyst will NOT change Keq
�� + �� + �� ↔ �� + �� + ��
c. Upper case – molarity – and lowercase – reaction
coefficient
��� = [�]6[�]7[�]8
[�]9[�]:[�];
d. Brackets denote concentration of molecules
e. Rates of forward and reverse reactions can be written:
����>?@A9@B = �>[�]6[�]7[�]8
����@DED@FD = �@[�]9[�]:[�];
i. Where kf and kr are empirically determined rate
constants that do not change with altered conditions
or concentrations
ii. Chemical reactions in cells are at steady state – the rate of formation of a substance is equal to the rate of its consumption
1. In cells, reactions are linked in pathways
2. Products of one reaction are reactants of other reactions
3. Equilibrium is never reached for individual reactions
a. The concentrations of cellular molecules remain relatively
constant over time (steady state AKA dynamic equilibrium)
i. Prevents build up of toxic intermediates
BSC 300-001 Exam 1 Studyguide (Ch. 1,2,3,4,7)
ii. Promotes only a certain direction of reaction
b. A system at equilibrium cannot perform work – in the case of cells, this means death
4. Homeostasis is a consequence of steady state
iii. Dissociation constants of binding reactions reflect affinity of interacting molecules
1. If the equilibrium constant of a binding reaction is known, the stability of the resulting complex can be predicted
2. Dissociation constant (Kd) – the reciprocal of the equilibrium constant (measured in molarity M (moles/L)
a. Describes binding reactions
3. Example of specific protein binding to DNA:
� + � ↔ ��
a. PD = protein/DNA complex
i. Do NOT do [P] + [D] – test question
�B = [�][�]
[��]
b. Kd is determined at equilibrium, when the rates of
association and disassociation are equal
4. Usefulness of Kd
a. Tells us relative affinity of molecules for one another
b. Based on the protein example above, when half of the DNA is bound to protein, the concentration of protein = Kd
i. The lower the Kd, the stronger is the affinity of the
protein for the DNA
iv. Biological fluids have characteristic pH values
1. Amphoteric molecules – act as acids or bases
a. Ex: water
i. In pure water [H+] = [OH-] = 10-7 M
b. Buffers are amphoteric
2. Acids, Bases, and Buffers
a. As [H+] increases, [OH-] decreases so that the product
equals 10-14
i. pH =7 is neutral
ii. Below 7 is acidic (excesses H+)
iii. Above 7 is basic (excess OH-)
iv. Cells compartmentalize pH to regulate enzyme
activity
b. Many biological molecules have acid and base groups
i. Amino acids have carboxylic acid and amino base
groups
1. At pH=7 both groups are charged
a. Carboxylic acid is deprotonated, and
the amino acid group is protonated
BSC 300-001 Exam 1 Studyguide (Ch. 1,2,3,4,7)
b. Called a zwitterion
i. Neutral – no charge
c. At extreme pH only one of the
groups is charged
c. Acid Dissociation and Acid Dissociation Constant (Ka) �� ↔ �J + �K
�9 = ��9 + ��� [�K]
[��]
*where pKa = -logKa
i. Can be rearranged to the Henderson-Hasselbalch
equation – shows the pKa of any acid is the pH
where half of the molecules are dissociated and half
are protonated
1. Do NOT need to memorize H-H equation –
just understand what it means and how to
use it
2. pKa provides important description of a
molecule’s chemical traits and an
understanding of how biological systems
and physiological pH can alter molecules
(critical in pharmaceutical industry)
a. pKa of histidine = 6.4
3. Lower pKa = stronger acid
a. NEED TO KNOW
b. Higher pKa is a stronger base
d. Biological processes are very sensitive to pH
i. A living, actively metabolizing cell must maintain
a constant pH in the cytoplasm of about 7.2-7.4
1. Must do so even as its metabolism is
producing many acids
ii. pH changes affect ion state and function of proteins iii. Buffers resist pH changes
1. Soak up excess H+ and OH- when these
ions are added to the cell or are produced by
metabolism
2. Bicarbonate ions and carbonic acid buffer
the blood:
���Q − + �J ↔ �S��Q
3. Buffers are the most effective at preventing
pH change when the pH of the solution is
similar to the pKa of the buffer
a. Buffering capacity – ability of a
buffer to minimize changes in pH
i. Depends on concentration of
buffer and relationship
between its pKa value and
BSC 300-001 Exam 1 Studyguide (Ch. 1,2,3,4,7)
the pH (expressed by H-H
equation)
4. Three very important buffering systems:
a. Carbonic acid
b. Phosphoric acid
c. Proteins
5. Titration curve of buffer acetic acid
(CH3COOH)
a. pKa = 4.75, so the best range for it
being a buffer is pH = 3.75-5.75
i. Buffers work best in range
1pH unit above and below
pKa
6. Titration curve of phosphoric acid (H3PO4)
(common buffer in biological systems)
a. Have more than one proton capable
of dissociating
b. Biochemical Energetics
i. Kinetic Energy – energy of movement
1. Definitions
a. Thermal energy – flow of energy from higher temp to
lower temp
i. Responsible for homeostatic temp in warm-blooded
animals
b. Radiant energy – kinetic energy of photons or waves of
light
i. Can be converted to thermal energy when light is
absorbed by molecules and the energy is converted
to molecular motion
ii. Can change electronic structure of molecules by
moving electrons into higher-energy orbitals, where
it can later be recovered to perform work
iii. Photosynthesis
c. Mechanical energy – energy of motion
i. Major form of kinetic energy in biology
1. Example: reorganization of cytoskeleton
d. Electric energy – energy of moving electrons or charged
particles
i. Transport of charged ions across cell membrane
ii. Potential Energy – stored energy
1. Chemical potential energy – energy stored in bonds connecting atoms in molecules
a. Example:
i. Energy stored in covalent bonds
ii. Conversion of glucose to ATP and NADH
BSC 300-001 Exam 1 Studyguide (Ch. 1,2,3,4,7)
2. Concentration gradients – potential energy created by storing molecules on one side of a membrane and allowing them to flow across the membrane barrier spontaneously
a. AKA displacement across membranes
3. Electric potential energy – potential energy produced by separating differently charged ions on opposite sides of the membrane
a. AKA membrane potential
iii. First Law of Thermodynamics – law of conservation of energy 1. Energy can neither be created nor destroyed, only transferred or transformed
2. Transduction – conversion of energy from one form to another while being transferred
a. Leads to energy being gained or lost by a system
i. When cells transform energy from potential energy,
some is lost
1. Reactions causing the system to lose energy
– exergonic
2. Reactions causing the system to gain energy
– endergonic
a. Example: ice packs used to treat
injuries – crushing the pack mixes
the reactants
iv. Second Law of Thermodynamics – in the universe, or any closed system, the degree of disorder can only increase
1. Events in the universe proceed from a state of higher order to one of lower order
2. Entropy – measure of disorder of a system
a. Higher entropy = lower order = increased randomness
i. Increasing entropy leads to loss of available energy
in the system
b. In transferring and transforming energy much of it is lost to the system as heat (most disordered form of energy)
i. Cells increase disorder of system to maintain their
own order
3. Free Energy – the internal energy of a system that is available to perform work
a. Total energy - usable energy
v. Spontaneous vs non-spontaneous reactions
1. Spontaneous – favorable and exergonic
a. Occur without external energy
b. Spontaneous DOES NOT MEAN fast
2. Nonspontaneous – not favorable and endergonic
a. Products have more available energy than reactants
vi. Gibbs Free Energy
1. Entropy (S) – measure of randomness or disorder
(Energy/temperature)
BSC 300-001 Exam 1 Studyguide (Ch. 1,2,3,4,7)
2. Free Energy (G) – energy available to perform work
a. ΔG = ΔH - TΔS
i. Determines whether a reaction will occur
spontaneously
1. Negative ΔG = favorable, spontaneous,
exergonic
3. All chemical reactions are reversible
a. Rate is proportional to the product of the reactant
concentrations and a specific rate constant (k) for each
reaction
� + � ↔ � + �
i. Rate of forward reaction = k1[A][B]
ii. Rate of reverse reaction = k2[C][D]
vii. Equilibrium – forward and reverse reaction rates are equal 1. NEED TO KNOW:
a. There is no net change in concentration of any solute
b. There is no more exchange of energy with the surroundings 2. ΔG = 0
a. System cannot do work
b. No energy is entering or leaving the system
3. The greater the difference in free energy between the two sides of the reaction, the more work that can be performed
4. Experimentally, k1[A][B] = k2[C][D]
5. Equilibrium constant Keq can predict the favored direction of a reaction with known solute concentrations
�S= [�][�]
�DT = �U
[�][�]
a. Cells are constantly changing concentration to favor certain directions
b. Keq greater than 1 = forward reaction favored
c. Keq less than 1 = reverse reaction favored
d. At equilibrium, neither the forward nor the reverse reaction is favored
6. Cannot be used to determine if an energetically favorable reaction can potentially be coupled to an unfavorable reaction
a. Standard measure is needed – Standard free-energy change (ΔG°’)
i. Measured under standard conditions of:
1. Starting pressure (1M all solutes)
2. Pressure (1 atm)
3. Temperature (37 degrees Celsius)
ii. Depends only on intrinsic characters of solutes, not their concentrations
iii. Can be determined empirically for any reaction
∆� = ∆�°Y + �� ∗ �� [�][�]
[�][�]
BSC 300-001 Exam 1 Studyguide (Ch. 1,2,3,4,7)
iv. Where:
1. T = temp (K)
2. R = 1.987 mol x K or 8.314 J/mol x K
7. Standard-free energy change and equilibrium constant a. Negative ∆�°Y have Keq greater than 1 (spontaneous_ b. Positive ∆�°Y have Keq less than 1 (non-spontaneous) c. ∆�°Y = 0, then Keq = 1, and the system can do no work d. Why is this important?
i. ∆�°Y allows us to determine ΔG at a specific
concentration
8. Metabolic Reactions
��� + �S� ↔ ��� + �^
a. ∆�°Y for the forward reaction = -7.3 kcal/mol
b. ∆�°Y for the reverse reaction = +7.3 kcal/mol
c. ΔG can be considerably different from ∆�°Y at cellular concentration of reactants and products
i. [ATP] = 10mM, [ADP] = 1mM, and [Pi] = 10mM
∆� = ∆�°Y + 1.4����
��� ∗ log[���][��]/[���]
d. Cells get more energy out of ATP by keeping its
concentration relatively high compared to its hydrolysis product ADP
9. Reaction rate depends on activation energy necessary to energize the reactants
a. Chemical reactions involve the breaking/reformation of covalent bonds
b. Both processes require atoms of reactant molecules to adopt high-energy, transient configurations: transition
states
i. Reactants in this state are transition state
intermediates
c. Activation Energy (ΔG‡) – added energy needed to reach this transition states
i. Even required for spontaneous reactions
ii. NEED TO KNOW - Lowering the activation energy (decreasing free energy of the transition state)
accelerates the overall reaction rate
1. Lowering Ea by 1.36 kcal/mol produces a
10-fold increase in reaction rate, while
lowering it by 2.72 kcal/mol increases the
reaction rate 100-fold
d. From the transition state, a molecule can go backwards and reform the reactants or go forward and generate the
products
e. Increasing transition state intermediates (catalysts)
increases reaction velocity
BSC 300-001 Exam 1 Studyguide (Ch. 1,2,3,4,7)
i. Catalysts DO NOT influence thermodynamics of a
reaction
viii. Life is chemical disequilibrium
1. Cells keep reactions going in specific directions by altering relative
concentrations of reactants and products by
a. Consuming products
b. Altering products
c. Moving products
d. Importing ot producing more reactants
2. This makes multi-step pathways highly favorable over single-step
processes
3. Reactions with positive ∆�°Y can occur because:
a. The ratio of reactants to products are low enough to favor
the forward reaction
i. Products of unfavorable reaction siphoned off by
favorable reaction
ii. Hundreds of reactions occur at the same time
iii. ΔG for a series of sequential reactions = the sum of
ΔG values for each reaction
4. Activated carrier molecules
a. Store energy as a chemical group readily transferred or as
high-energy electrons
Activated Carriers
Group Carried in High Energy Linkage
ATP
Phosphate
NADH, NADPH, FADH2
Electrons and hydrogens
Acetyl CoA
Acetyl group
Carboxylated biotin
Carboxyl group
S-Adenosylmethionine
Methyl group
Uridine diphosphate glucose
Glucose
ix. Oxidation-Reduction Reactions – electrons are transferred between atoms of reacting species
1. May be total transfer – resulting in ionization
2. May be partial transfer – new polar covalent bond formation with
electron pairs pulled more closely to an atom in the new bond
a. When they move closer together, they give up energy that
is used elsewhere in the system
3. OIL RIG
4. Relative oxidation state of an organic molecule can be determined
by the number of hydrogen vs oxygen and nitrogen atoms per
carbon atom
a. More hydrogen = more reduced and more stored energy
x. NAD+ and FAD couple many biological oxidation and reduction reactions 1. NAD+ = nicotinamide adenine dinucleotide
a. Reduced to NADH
BSC 300-001 Exam 1 Studyguide (Ch. 1,2,3,4,7)
2. FAD = flavin adenine dinucleotide
a. Reduced to FADH2 – can transfer protons and electrons to
other molecules, thereby reducing them
3. Intermediate coenzymes that function as electron acceptors and
donors in enzymatically catalyzed biochemical reactions
4. Reduced by accepting 2 electrons and either 1-2 protons from
various substrates and then donating those electrons to other
molecules
a. Makes the receiving molecules more reactive (lowers Ea)
for subsequent reaction
IV. Chapter 3 – Protein Structure and Function, Part 1
a. Intro Notes
i. We need to be able to determine if something is an amino R group, but we do not need to memorize them
b. Overview of protein structure and function
i. Proteins are polymers of amino acids
ii. The human genome has 20,000-23,000 protein-encoding genes
1. Alternative mRNA splicing and post-translational modification
allows for thousands of distinct protein activities
a. There could be millions of possible combinations
iii. Protein hierarchical structure
1. Primary structure – linear sequence of amino acids linked by
peptide bonds
2. Secondary structure – local α-helices or β-sheets
a. Driven by hydrogen bonds between nearby amino acids
b. Beta turn – turn between beta sheets
i. Beta turn is part of a protein, a protein is not part of
a beta turn
3. Tertiary structure – peptide 3D shape
a. May be a functional protein
4. Quaternary structure – association into multipeptide complexes
5. Supramolecular complexes – can be very large, consisting of tens
to hundreds of subunits
c. Protein Structure
i. First level – primary structure
1. Specific linear sequence of amino acids
2. Information for sequence encoded in DNA
3. The function of a protein is derived from 3D structure, or
conformation
a. Determined by amino acid sequence and intramolecular
non-covalent interactions
4. From the ribosome onto mRNA, which reads it in triplet code
a. Includes one new monomer at a time to a growing
polypeptide
b. Goal – fold into shape that has the lowest free energy
associated with it
BSC 300-001 Exam 1 Studyguide (Ch. 1,2,3,4,7)
i. Chaperones makes sure there is no interference and
that proteins can fold properly
5. -Ala-Glu-Val-Thr-Asp-Pro-Gly
a. Carboxylic acid exposed terminus at the end
6. First primary structure discovered was that of insulin
7. Structure of a polypeptide
a. Proteins are unbranched polymers of 20 biological amino acids
b. Side chains determine the distinct properties of individual proteins
c. Peptide bond – formed from dehydration reaction between one amino acid ( - COO-) to another amino acid (NH3+)
i. Carbody terminus
ii. Amino terminus
d. Polypeptide – linear polymer has a free amino end (N
terminus) and a free carboxyl end (C-terminus)
ii. Second level – secondary structure
1. Discrete regional conformation of amino acids into α-helices, β sheets, hinges, turns, loops, or finger-like extensions
a. These stable arrangements of polypeptide regions are held together by hydrogen bonds between backbone amide and
carbonyl groups
b. Driven by hydrogen bonding between the carboxy group of one and the amino group of another
2. 60% of a protein is alpha helix and beta sheets. The rest is irregular structures, coils, and turns
3. The α-helix, a common secondary structure
a. About 3.6 amino acids per turn (0.54 nm)
b. Stabilized using hydrogen bonds between carboxyl oxygen of one amino acid and amino H of another
i. Each amino acid is Hydrogen bonded to the amino
acid 4 residues above it
1. Means that a carboxy group is bonded to the
4th NH group from it, creating a spiral twist
c. Prolines are excluded from the helices because they cannot participate in Hydrogen bonding
i. They have a unique kink to them due to the spiral
d. R groups project outward the surface of the helix
i. This determines the chemical nature of the helix
face, such as whether or not it is hydrophobic or
hydrophilic
ii. Large R groups sterically interfere with helix
formation
4. The β-sheet
a. Parallel polypeptide segments crosslinked by H-bonds
between carboxy and amino
BSC 300-001 Exam 1 Studyguide (Ch. 1,2,3,4,7)
i. Strands can be parallel (all pointing right) or
antiparallel (alternating directions)
b. α carbon bond angle generates a pleated polypeptide
backbone
c. Alternate R groups project above and below the plane of the sheet
i. Strands of a sheet can be close to one another in the
polypeptide or separated, even generating other
secondary structures between strands
5. Structure of a β-turn
a. Four residues that reverse direction of a polypeptide chain i. 180 degrees
ii. U-turn
b. Cα carbons of the 1st and 4th residues are typically less than 0.7 nm apart and are linked by a hydrogen bond
c. These turns facilitate the folding of long polypeptides into compact structures
d. Glycine (smallest R group) and proline (built in bend) are commonly found in β-turns
i. Glycine does not have a real R chain – it is
hydrogen
iii. Third level – tertiary structure
1. The conformation of the entire polymer
2. Stabilized by:
a. Hydrophobic and Van der Waals interactions between
nonpolar side chains
b. Hydrogen bonds – polar side chains and backbone amino and carboxyl groups
3. These are weak forces, so tertiary conformation is not rigidly stabilized and undergoes constant minor fluctuations
4. Shape can also be altered easily by the addition of several post translational modifications
5. The formation of noncovalent bonds results in the release of energy and a more stable structure
a. Lower energy state proteins fold into 3D shapes that
require the least amount of energy to maintain
6. Hydrophobic resides group together in the core (blue)
7. Charged and uncharged polar side chains form stabilizing interactions with surrounding water and ions on the protein surface (yellow)
8. Disulfide bonds (strong covalent bonds) between cysteine residue also play role in stabilizing tertiary structure
a. Very important for secreted proteins
9. Four broad structural categories of proteins
a. Well-ordered proteins
BSC 300-001 Exam 1 Studyguide (Ch. 1,2,3,4,7)
i. Globular proteins – water-soluble and compactly
folded
ii. Fibrous proteins – large, elongated, often stuff
molecules
1. Building blocks
2. Ex: collagen
iii. Integral membrane proteins – embedded within the phospholipid bilayer of membranes
b. Intrinsically disordered proteins – no well-ordered structure in native conformation
i. Polypeptide chains are flexible with no fixed
conformation
1. Constantly changing interaction with the
aquatic environment
ii. Few to no hydrophobic residues
iii. Interact with multiple partner proteins and only fold into defined conformations through such
interactions
iv. Induced fit – the interaction between two molecules results in conformational changes that allow the
molecules to interact with greater affinity for one
another
v. Typically serve as signaling molecules, regulators
of the activities of other molecules, or as scaffolds
for multiple proteins
vi. Can be identified experimentally using tests of
sensitivity to protease digestion and spectroscopy
10. Four ways to present protein structure
a. Cα backbone trace – depicts how the polypeptide is tightly packed into a small volume
b. Ball-and-stick representation – reveals locations of all atoms
i. Most complex representation
c. Ribbon diagram – emphasizes secondary structures and their positions within the protein
d. Water-accessible surface model – reveals protein surface topology with positive charge (blue) and negative charge regions (red)
i. Represents a protein as it is “seen” by another
molecule
e. Hybrid models combine two of the approaches
11. Establishing function through building unique conformation a. Structural motifs – regular combinations of secondary structure usually with an associated specific function
b. Three common motifs (NEED TO KNOW PICTURE AND FUNCTION)
BSC 300-001 Exam 1 Studyguide (Ch. 1,2,3,4,7)
i. Coiled-coil motif – two α helices wound around
each other
1. Stabilized by hydrophobic interactions
2. Used for protein-protein interactions
ii. EF hand motif – one type of helix-loop-helix motifs
1. Loop chelates calcium to stabilize their
motif structure
2. Used for protein-protein or protein-DNA
interactions
3. Used for sensing calcium levels
iii. Zinc-finger motif – one or more Zn2+ coordinate an
alpha helix and a small Beta sheet
1. Present in many RNA and DNA-binding
proteins
12. Protein domains – part of tertiary structure that function and evolve independently of the rest of the protein
a. Each domain forms a compact 3D structure that folds and functions independently of others
b. Many proteins contain several distinct domains
c. Can often function independently when separated from the rest of the protein
d. Proteins with similar domains grouped into families
reflecting evolutionary relationships – protein families
evolve through gene duplication and rearrangements
e. Three classes of protein domains
i. Functional domains – exhibit specific activity,
independent from other regions of the protein
1. Enzymatic activity
ii. Structural domains – region of 40+ residues
arranged as a single, stable, distinct structure often
comprised of one or more secondary structures
1. There are over 1000 unique structural
domains
2. Establish functional shape
iii. Topological domain – regions of proteins defined
by spatial relationship to the rest of the protein
1. Membrane spanning proteins have
extracellular, membrane embedded and
cytoplasmic domains
a. Each may comprise multiple
iv. Fourth level – quaternary structure
1. Not all proteins are functional as soon as they fold into a 3D structure
2. They combine with other polypeptides as subunits of larger protein complexes that have a collective and complex function
a. Ex: initiation complex
BSC 300-001 Exam 1 Studyguide (Ch. 1,2,3,4,7)
b. This is quaternary structure
i. Mostly achieved through non-covalent interactions
ii. Some require covalent bonds
c. Complexing may enhance the efficiency of enzymatic
reactions or promote conformations of subunits that confer
activity
i. Metabolic coupling
3. Proteins that share domains are evolutionarily related
a. Gene duplication and exon shuffling (accidental
translocation of exon from one gene to another) drive such
evolution
b. Proteins with this common ancestry are called homologs
and are grouped together in families and superfamilies
d. Protein folding
i. The primary determinant of 3D conformation, thereby affecting function is the peptide amino acid sequence
ii. Most domains fold spontaneously after translation, but some protein regions require help to prevent inappropriate associations
1. ATP-dependent molecular chaperones assist protein folding in vivo
2. Misfolded/denatured proteins can form highly-ordered amyloid fibril aggregates that can cause diseases including Alzheimer’s and Parkinson’s
iii. Native conformation/state – normal configuration that a polypeptide will fold into under standard physiological conditions
1. If they do not accept native conformation, they are considered misfolded
iv. Hypothetical protein-folding pathway
1. Proteins assume native conformation through:
a. Single polypeptide folding hierarchy
i. Primary to secondary to structural motifs to
domains to tertiary structure
ii. Domain folding is usually independent of the other
regions of the protein
2. If you take a stretch of amino acids and put them in water, they automatically adopt a shape
a. If you boiled them, they would denature and unfold
b. If you added urea, they would unfold
c. If you cool it, it will fold back into the same shape
3. In cells, other biomolecules (other proteins not yet folded) can interfere with a polypeptide adopting its native conformation
a. Unfolded/partially folded proteins tend to aggregate
together into large water-insoluble masses
b. Chaperones – proteins that bind unfolded/denatured
proteins and facilitate proper folding by preventing
association with other molecules
BSC 300-001 Exam 1 Studyguide (Ch. 1,2,3,4,7)
i. Uses a lot of ATP
ii. Two families, distributed into all organelles and
compartments of eukaryotic cells
1. Molecular chaperones – bind short segments
of a nascent protein and stabilize unfolded
or partly folded regions, preventing
aggregation or degradation
a. Heat Shock Protein 70 (HSP70)
and HSP90 family proteins bind
emerging proteins and prevent
inappropriate interactions
i. We need to be
knowledgeable about
HSP70
ii. HSP 70 is in the cytosol
iii. Major chaperones in all
organisms that use an ATP
dependent cycle to fold their
substrates
iv. When bound to ATP, HSP70
assumes an open
conformation, in which an
exposed hydrophobic
substrate-binding pocket
binds to exposed
hydrophobic regions of an
incompletely folded or
partially denatured target
protein and then rapidly
releases this substrate
v. Hydrolysis of bound ATP
causes molecular chaperone
to assemble closed form that
binds substrate protein more
tightly-appears to facilitate
target protein’s folding
vi. Exchange of ATP for the
chaperone-bound ADP
causes conformational
change in chaperone that
releases target protein and
regenerates empty, ATP
bound HSP70 reads to fold
another protein
b. ATP-dependent proteins
BSC 300-001 Exam 1 Studyguide (Ch. 1,2,3,4,7)
c. Bound to ATP chaperone
configuration has exposed
hydrophobic residues that associate
with hydrophobic resides in unfolded
polypeptides
i. HSO protein is a complex
d. ATP hydrolysis alters chaperone
conformation
i. Cleaves terminal phosphate
from ATP
ii. Bound to ADP, it has a
tighter interaction with
nascent protein, promoting
proper folding by preventing
interactions with other
molecules
e. BAG1 promotes exchange of ADP
for ATP, which loosens association
allowing HSP 70 to slide along the
nascent protein, or bind a new target
2. Chaperonins – form folding chambers into
which all or part of a nascent protein can be
sequestered without interference from other
molecules (aka HSP 60)
a. ATP dependent
b. They are responsible for processing
up to 15% of the cells’ proteins
c. Basically, a chamber into which
another protein is going to be fed
i. AKA garbage can
d. ATP hydrolysis closes the lids,
providing an environment free of
interacting molecules
i. Release of ATP opens
chamber and allows folded
protein to exit
V. Chapter 3 – Protein Structure and Function, Part 2
a. Intro
i. Ligand – molecule to which a protein binds
1. Often induces conformational change in the protein and alters its
function
2. Enzymes may be bound by multiple ligands that can change their
function or binding ability
ii. Substrate molecules – the molecules that enzymes act upon
1. Need to know the difference between a ligand and a substrate
BSC 300-001 Exam 1 Studyguide (Ch. 1,2,3,4,7)
iii. Enzymes lower the activation energy and stabilizing transition-state intermediates, accelerating the rate of a cellular reaction
1. Act on substrate molecules
2. Use acid-base catalysis mediated by one or more amino acid side chains
3. Cofactors often associate with enzymes and aid in catalysis
b. Ligand binding regulates the function of many proteins
i. Specificity – ability of a protein to bind one molecule or small group of molecules in reference to all other molecules
ii. Affinity – tightness or strength of binding between protein and ligand measured by Kd
1. Low Kd = high affinity
2. Strength driven by non-covalent interactions
iii. Molecular complementarity required for specificity and affinity – complementary shapes and numerous non-covalent interactions drive both iv. Ligand binding by vertebrate specific antibodies – great example of specificity and affinity
1. Antibodies recognize specific antigens (macromolecules present on non-self structures, like the surface of a bacterium)
a. Antigen – something that has infected
2. Each antibody recognizes a very molecule substructure – its
epitope
a. One antigen can have hundreds of regions that are epitopes
to different antibodies
3. Structure
a. Each antibody is a quaternary complex of 2 heavy chains
and two light chains
b. Each chain composed of multiple domains (Immunoglobin
Ig domains)
c. The four chains are linked by di-sulfide bonds
d. Genetic recombination at heavy and light genes produces
variants in each B cell that produces antibodies
i. Amino acid sequences I the terminal Ig domains of
each heavy and light chain differs slightly between
each B cell
e. These tow regions of heavy and light chains constitute
complementarity-determining region (CDR)
f. Antibody has high affinity for a very narrow range of
potential epitopes
g. A single modification can be sufficient to inhibit antibody
antigen interaction
i. This is specificity
c. Enzymes as Biological Catalysts
i. 99% are protein catalysts
ii. Each enzyme has a unique 3D conformation that allows it to interact only with specific substrate molecules
BSC 300-001 Exam 1 Studyguide (Ch. 1,2,3,4,7)
1. They associate with substrate molecules and promote specific chemical reactions
iii. Properties of enzymes
1. Work at cell-specific temp and pH
2. Highly specific to their substrates
3. Catalyze reactions in an orderly manner to prevent production of by-products that could negatively impact the cell
a. Usually an enzymatic pathway
4. Like all catalysts, enzymes:
a. Are required in small amounts
b. Are not consumed or altered irreversibly by the reaction
i. They do sometimes form covalent bonds with a
substrate, but they are returned back to the original
state
c. Have no effect on the thermodynamics of the reaction
i. Cannot make a positive delta G negative
d. Can be regulated to meet the needs of a cell
5. Can increase reaction rates by 105 – 1020
iv. Many enzymes are non-covalently conjugated to non-protein components that facilitate chemical catalysis
1. May change pKa to speed up reaction
v. Cofactors – inorganic atoms or molecules, mostly metal ions 1. Participate directly in catalysis through ionic interactions with the substrate
vi. Coenzymes – organic enzyme conjugates, usually derived from vitamins, that function as intermediate carriers of electrons, specific atoms or functional groups that are transferred in the catalyzed reaction 1. Basically move molecules from one to another
2. Directly participate with substrate by stealing electrons by it, bonding and passing molecules onto other reactions
3. NAD and Coenzyme A are some of the most important
vii. Overcoming the activation energy barrier
1. Activation energy – barrier that inhibits formation of
thermodynamically unstable intermediate, the transition state a. Enzymes decrease Ea by binding more tightly to the
transition state than to the substrate or product, stabilizing
the intermediate
viii. The Active Site
1. Enzyme-substrate (ES) complex – formed when an enzyme interacts with its substrate
a. Substrate bound by enzyme at the active site
i. Made up of the substrate binding site (specificity
and affinity) and the catalytic site (including the
formation of that transition state, responsible for the
chemistry behind it)
BSC 300-001 Exam 1 Studyguide (Ch. 1,2,3,4,7)
ii. Active site and substrate have complementary
shapes/conformations that promote substrate
specificity through hydrophobic and electrostatic
interactions
1. Clamp down effect onto substrate – places
site exactly where it needs to be
2. Substrate binds to binding site – causes
conformation change to allow catalytic site
iii. Alteration of just a couple of atoms is sufficient to
disrupt association
ix. Mechanisms of Enzyme Catalysis
1. Any enzyme can use any of these three to speed up a reaction: a. Changing Substrate Reactivity – substrate influenced by amino acid side chains at active site
i. Alter chemical properties of substrate
ii. Change ability of substrate to undergo reaction
b. Inducing Strain in the Substrate – enzyme changes
conformation of substrate to bring closer to conformation
of transition state
i. Places strain and makes chemical bond to be
weakened
c. Substrate Orientation – multiple substrates brought together in correct orientation to catalyze reaction
x. Induced fit and strain
1. Induced fit caused by interaction with substrate
2. Induced fit – many electrostatic interactions cause enzyme to undergo conformational change, a tighter fit that places stress on substrate
a. Result of complementary state
b. Change in conformation of enzymes as it clamps down on substrate
3. May strain covalent bonds of the substrate
a. Changes affinity – makes enzyme have greater affinity for substrate to have transition state
4. Or bring critical residues of the catalytic site into position 5. Transition state stabilized because enzyme binds transition state more tightly than substrate or product
a. Enzymes have highest affinity for transition state (not
stable) – when it is achieved it is released
i. So it can release and bind to substrate again
xi. Enzyme Kinetics
1. Kinetics – study of rates of enzymatic reactions under various experimental conditions
a. If you had 20 test tubes with the same buffer and enzyme concentration and add increasing concentrations of
substrate to each tube and immediately stop the reaction
BSC 300-001 Exam 1 Studyguide (Ch. 1,2,3,4,7)
Vmax
V = [P]/s
[S]
i. Initially linear but asymptotes to plateau
ii. Linear: at low [S], few enzymes encounter
substrate, therefore substrate is rate limiting
iii. Asymptote: change in velocity slows because most
enzyme molecules are occupied, therefore enzyme
is rate limiting
1. Enzyme is becoming saturated – all enzymes
are occupied by substrate
2. If you add more enzyme, you can change
Vmax (Vmax is concentration dependent)
a. Maximal velocity – can be used to
determine the turnover number (aka
catalytic constant kcat)
i. kcat – number of reactions
catalyzed by a single enzyme
per second when operating at
saturation
�;9h = �j96
[�]h?h9l
xii. Michaelis-Menten Kinetics
1. Vmax changes with enzyme concentration, so it can’t be used to characterize specific aspects of enzyme kinetics
2. Michaelis and Menten studied enzyme invertase, which catalyzes hydrolysis of sucrose into glucose and fructose
a. 1914 they showed slope of such an enzymatic reaction is characterized by:
�m = �j96[�]
[�] + �o
BSC 300-001 Exam 1 Studyguide (Ch. 1,2,3,4,7)
Vmax
)
v
(
y
t
i
c
o
l
e
v
n
o
i
Vmax / 2
t
c
a
Non-enzyme-catalyzed reaction
e
r
l
a
i
t
i
n
I
Km
Substrate concentration [S]
i. The velocity at any point is equal to Vmax times substrate concentration at velocity over Michaelis constant plus concentration at that velocity
1. Km is a measure of the affinity for the substrate and is equal to [S] at exactly ½
Vmax – it is a fixed amount
a. If Km is large, the enzyme requires a
high concentration of substrate to
reach half Vmax and therefore has
low affinity for the substrate
b. Higher Km = lower affinity
3. Km – measurement of relative affinity of enzyme for its substrate a. Typically ranges from 10-1 to 10-7
b. Inversely proportional to substrate affinity
c. Why is Km important?
i. Can help researchers studying enzymatic pathways determine potential order of interactions
ii. Can help determine mechanism by which an
enzymatic inhibitor functions
iii. May help determine potential bottlenecks in
pathways – where within a pathway a new drug
might aide in speeding up or slowing down
production of a specific metabolite
BSC 300-001 Exam 1 Studyguide (Ch. 1,2,3,4,7)
iv. Explains much of the physiological diversity seen in
populations
v. Ex: hangovers
��Q��S�� + ���J → ��Q��� + ����
1. Alcohol dehydrogenase
2. Produces acetyl aldehyde, which is a nasty
toxin that gives you headaches, nausea, and
facial flushing
��Q��� + ���J → ��Q���K + ����
3. Acetaldehyde dehydrogenase
4. Two forms of acetaldehyde dehydrogenase:
a. Mitochondrial – low Km (promotes
rapid turnover of acetaldehyde)
b. Cytosolic – high Km (oxidizes
acetaldehyde very slowly)
5. Some people inherit a mutation in the
mitochondrial acetaldehyde dehydrogenase
gene that significantly increases the Km
leading to excess acetaldehyde
a. Have mitochondrial dehydrogenase
with high Km – cannot turnover
acetaldehyde as quickly
b. Other inherit an alcohol
dehydrogenase mutation that
significantly lowers the Km of that
enzyme, also leading to excess
acetaldehyde
xiii. Serine proteases example of active site
1. Serine proteases – large family of enzymes that catalyze cleavage of specific peptide bonds via hydrolysis reactions
a. Hydroxyl group added to carbon – carboxylic acid and
nitrogen added, making it an amino group
2. Each protease has specificity for peptide bonds that involve particular amino acids
3. Trypsin preferentially cleaves the peptide bond C-terminal to residues with positively charged side chains (arginine and lysine) a. 2 key non-covalent interactions promote specificity:
i. Side chain specificity binding pocket has correct
size, depth and acidic asparagine residue to accept
and hydrogen bond to arginine/lysine side chains
1. Specificity – pocket has the right size and
charge to hold
2. Affinity – multiple H bonds created in Beta
sheet region
ii. Substrate residues C-terminal to arginine form Beta
sheet-like association in the active site
BSC 300-001 Exam 1 Studyguide (Ch. 1,2,3,4,7)
b. This locks the substrate into place and prevents the peptide bond to be cleaved to the catalytic site
c. Activity of trypsin
i. In the catalytic site a catalytic triad of three
precisely positioned residues cooperate to attack the
peptide bond of the substrate
1. Peptide bond presented to catalytic site
(clamped down because of induced fit)
2. Histidine is not fully protonated at
physiological pH
a. It is partly protonated – can go either
way
3. All 3 are H-bonded to one another (Asp to
His and His to Ser)
4. As a result, histidine pulls on serine’s
hydroxyl hydrogen which enhances serine’s
nucleophile capacity
5. Serine’s hydroxyl oxygen attacks carbonyl
carbon in the peptide bond
6. Triad:
a. Raises pKa when Asp bonds with
His
b. Because Ser is so close it attacks O
of Ser, trying to steal hydrogen
i. Makes Ser a powerful
nucleophile
ii. Very unstable transition state
iii. His stole H from Ser – Ser
makes transient bond with C
c. Bond snaps – Ser remains covalently
transiently attached to C – releases
Carboxy terminus
ii. This forms a tetrahedral intermediate – the
transition state – an unstable geometry that is
stabilized by H bonding through residues in the
oxyanion hole
1. This very unstable arrangement
instantaneously collapses:
a. Forming covalent acyl bond between
serine and the carboxyl carbon
b. The amide accepts the H back from
histidine and the peptide C terminus
is released
xiv. Key features of enzyme catalysis
1. Catalytic sites have evolved to promote transition state stability, which lowers Ea and accelerates reaction
BSC 300-001 Exam 1 Studyguide (Ch. 1,2,3,4,7)
2. Uniquely positioned residues interact with the substrate, often through a multistep process
3. Acid-base catalysis is often employed to break and form new bonds, therefore pH plays a high role in enzyme activity
xv. pH and temperature dependence of enzyme activity
1. Acid-base catalysis is pH dependent
a. Catalysis requires a particular ionization state of one or
more amino acid side chains in the catalytic site
i. Found in lower intestine, where there is a relatively
high pH
b. For example, the pKa of histidine is 6.8, at or below this histidine is protonated and cannot activate serine in the
active site of chymotrypsin
c. Other proteases, like lysosomal hydrolases, have evolved sequences that maintain catalytic activity at low pH (like
that in lysosomes)
d. Enzyme activity is also influenced by temperature
i. Temp can introduce energy to the system and
increase kinetic activity – this could increase the
rate of enzyme catalyzed reaction
ii. We change the chemistry of reactions since we
cannot change the body temp
1. H
e. High temp can also affect 3D structure of enzyme since
high temps can disrupt ordered structures in proteins
xvi. Enzyme inhibitors
1. Slow the rates for enzymatic reactions
a. Irreversible inhibitors bind tightly to the enzyme
i. You don’t want to produce irreversible enzyme
inhibitors – basically means death of cell
b. Reversible inhibitors bind loosely to the enzyme
i. Cells take advantage of reversible inhibitors
ii. Competitive inhibitors bind enzyme active sites and
therefore inhibit the normal substrate from being
acted upon
1. Resemble substrate in structure
2. Bind enzyme active site
3. Vmax is not affected
4. Km increased, decreased affinity for
substrate
5. Can be overcome with high
substrate/inhibitor ratio
iii. Noncompetitive inhibitors
1. Bind enzyme at location other than active
site, altering enzyme conformation and
ability to bind substrate
BSC 300-001 Exam 1 Studyguide (Ch. 1,2,3,4,7)
2. Closes active site to prevent interacting with
substrate
3. Vmax cannot be reached, it’s like reducing
enzyme concentration
4. Km unchanged
5. Cannot be overcome with high
substrate/inhibitor ratios
d. Protein Regulation
i. Covalent modifications – covalently attached molecules can alter proteins’ chemical composition
1. Examples: phosphorylation, glycosylation, uniquination,
methylation, acetylation
ii. Allosteric modification – non-covalent, physical association with another molecule can produce modest to extreme changes in protein conformation 1. Allosteric – other shape
2. Examples: interaction with metal co-factors, coenzymes or
nucleotides
iii. Three general mechanisms for regulating protein/enzyme activity:
1. Change concentration by altering rate of synthesis or rate of
degradation
a. Protein degradation – regulates life spans of intracellular
proteins – varies from as short as a few minutes to the age
of an organism for proteins in the lens of the eye
i. Removes damaged proteins that may be toxic to
cells
ii. Cells possess several mechanisms for the controlled
destruction of protein
iii. Ex: lysosomes are organelles containing diverse
acid hydrolases that degrade proteins
2. Change conformation or activity through covalent or non-covalent association with other molecules
3. Change protein subcellular localization
VI. Chapter 4 Lecture 6 – Culturing and Visualizing Cells
i. Culturing animal cells requires nutrient rich media (culture medium) and special solid surfaces
1. Media requirements (solution of nutrients that makes growth
possible):
a. Culture medium kept in incubator that controls
temperature, atmosphere, etc
i. Antibiotics are often added to reduce bacterial or
fungal contamination
1. Worked with in sterile cabinets to avoid
contamination by microorganisms in the air
b. 9 essential amino acids and 3 amino acids not produced by
many cell types
c. Vitamins, salts, fatty acids, and glucose
BSC 300-001 Exam 1 Studyguide (Ch. 1,2,3,4,7)
d. Blood serum – fluid remaining after blood cells clot
i. Contains:
1. Insulin – stimulates cells to absorb glucose
2. Transferrin (enzyme) – supplies iron in a
useable form
3. Growth factors (bind to receptors on cell
surface that tell cell to divide) that stimulate
proliferation
2. Surface requirements:
a. Most animal cells must be attached to a solid surface to grow (adherent cultures)
i. Have to stick to a substrate to grow – anchorage
dependency
b. Petri dishes coated with cell-adhesion molecules (CAMs) and protein components of the extra cellular matric such as collagen, fibronectins, and lamins (don’t worry about this
list of things)
i. Some cells (red blood cells and cancer) lack this
requirement and can be grown in suspension
c. Harvesting primary cells to be cultured from tissue requires breaking association with the ECM, usually by trypsin
digestion
i. Trypsin seed to digest cells from tissue and plate
them onto petri dishes
d. Fibroblasts – cells of connective tissue
i. Hardiest and easiest cells to propagate
ii. Produce EMA components such as collagen that
bind to CAMs, anchoring them to a surface
ii. Primary culture and cell strains have a finite lifespan
1. Primary cells – cells isolated directly from tissues
a. Preparing individual tissue cells for a primary culture
i. Cell-cell and cell-matrix interactions must be
broken
1. Tissue fragments are treated with a combo
of a protease (trypsin, collagen-hydrolyzing
enzyme collagenase, or both)
2. Most normal cells divide 50 times before entering senescence – exiting the cell cycle
a. Phase I – seeded cells grow to confluence (fill the plate) b. Phase II – cells can be removed, diluted, and re-plated and will continue to grow
c. Phase III – after 50 generations, cells exit the cell cycle and become senescent due to shortening of telomeres following each round of DNA replication
i. Exception – embryonic stem cells and some
immortalized lines
BSC 300-001 Exam 1 Studyguide (Ch. 1,2,3,4,7)
1. Embryonic stem cells can be cultured
indefinitely under the appropriate conditions
3. Some cells can avoid senescence by acquiring oncogenic mutations that maintain telomere length
a. They are transformed and have played important roles in cell function studies
b. Colony of cells that has become immortalizes – oncogenic mutations – turn on telomerase that lengthens telomeres
and allow them to live forever
4. Cell lines – cultured cells with indefinite life spans
a. Harbor major alteration to chromosome structure and
number, which limits their utility for certain types of study
i. Usually have more chromosomes than the normal
cell from which they arose
1. Aneuploid – cells with an abnormal number
of chromosomes
ii. They are very unnatural
iii. Often times scientists rely on fibroblasts rather than
establishing cell lines
iii. Flow Cytometry used to separate and sort cell types
1. Cells may be forced to express fluorescent proteins by genetic engineering
a. Antibodies conjugated to fluorescent molecules may be
used to coat surface of target cells
b. Such cells only fluoresce when a specific wavelength of light excites them
i. Fluorescence activated cell sorter (FACS) – flow
cytometer that streams cell single column through a
laser beam that excited the fluorophore
1. Do not need to know the mechanisms of
FACS – just be familiar with this type of
tool
2. Mimicking in vivo by growth in 2 and 3 dimensions
a. Adherent cultures are not very representative of in vivo
conditions
i. Epithelia – sheets of tightly linked cells that line
and surround tissues and organs – function to
transport nutrients from one side of an organ to
another
ii. Have polarity, apical surfaces facing the interior or
an intestine
1. Ex: lateral surfaces joined to other cells and
basal surfaces adhered to a complex ECM
iii. More complex dishes mimic conditions and allow
researchers to study such functions
BSC 300-001 Exam 1 Studyguide (Ch. 1,2,3,4,7)
iv. More recent advances allow researchers to mimic
developmental processes and culture organlike
structures with cells that have differentiated to
perform complex tasks
1. Ex: canine kidney cells (MDCK cells)
cultured on a supportive ECM establish a
tubular structure similar to a kidney nephron
2. 3D printed cartilage structures are being
used to generate replacement parts, like ears
and noses by culturing patient’s cells on
these frameworks
a. Spontaneously form tubules and
function as they would have inside of
a dog’s kidney
b. Move material from outside and
pump it into the endothelium
v. Researchers have also been able to coax stem cells
to differentiate in culture and from organoids,
masses of cells that differentiate in tissue specific
manner and construct a miniature organ that can be
used to study aspects of human development and
disease
1. Used growth factors to push cells down a
neural pathway – began to differentiate as
different parts of the brain
iv. Hybridomas and monoclonal antibodies
1. Antibodies are powerful research and therapeutic tools
a. Core of acquired immune system
b. Produced by white blood cells (B cells) – bind on bacteria and tell immune system to attack
c. Antibodies against specific epitopes (determinant on an
antigen – generally a small area containing amino acids)
can be used in immunohistochemistry experiments to
visualize the location and abundance of specific proteins in
tissues
d. Antibodies raised against specific proteins that promote
cancer formation and progression are being used as new
tools that bind and allosterically inhibit the functions of
those proteins
e. Produced by B cells – specific white blood cells – of the immune system
i. Unstimulated B cells produce membrane bound
antibodies (B cell receptors) – each B cell produces
antibodies with different CDRs and specificity for
different epitopes
BSC 300-001 Exam 1 Studyguide (Ch. 1,2,3,4,7)
ii. B cell proliferates and some of its descendants take
up residue in the spleen – memory mechanism in
case the offending epitope is ever encountered again
iii. Culturing B cells to isolate antibodies
1. Host organism injected with concentrated
antigen
2. Incubation period
3. Period B cells harvested from spleen – large
number of B cells secreting antibodies
against epitopes on the antigen
4. B cells fused with immortalized myeloid cell
line (cancer of white blood cells),
establishing hybridoma cell lines
5. Hybridomas cultured in small wells
a. Each line will release concentrated
antibodies
6. Test each culture for reactivity to the
specific antigen
a. Any clone that responds is producing
an antibody with specificity to the
antigen
b. Microscope
i. Compound light microscopes
1. Lenses
a. Condenser lens – focuses light
i. Takes beam of light and focuses as a cone and
carries inverse image
b. Objective lens – magnifies and establishes resolution
i. Captures image and magnifies it – also establishes
resolution (how close they can be without being
seen as 1 object)
c. Ocular lens – magnifies, but does not enhance resolution
i. Magnifying image, not specimen
ii. Empty magnification – does not do anything for
resolution
1. The ability to distinguish between two very
closely positioned objects
2. Most important property of any microscope
d. Total magnification = product of magnification of objective
and ocular
ii. Magnification is nothing without resolution
1. Objective lens composition, size, and shape determine degree of magnification and resolution
a. Resolution – shortest distance between two objects by
which they can be distinguished as separate entities
BSC 300-001 Exam 1 Studyguide (Ch. 1,2,3,4,7)
i. Resolution power of any objective is a function of
wave length of light and a value called numerical
aperture (NA) – specific for each lens
1. NA depends on lens size, curvature, and
focal length – distance from center of lens to
focal point
a. Smaller radius of curvature = shorter
focal length = better resolving power
α
F
� = 0.61 � �
��
�� = � � ����
2. d = smallest distance between 2 points that
can be resolved
3. λ = wavelength of visible light (527 nm
average)
4. N = refractive index – degree to which the
media between object and lens bends the
light
a. Ranges from 1.0 (air) to 1.5 (oil)
5. α = half angle of the cone of light that can
enter or exit the lens
a. Shorter focal distance = greater α
and larger denominator = increased
resolution
6. Do not have to apply equation – do need to
know how parts of the equation apply
iii. Contrast: enhancing visibility
1. Bright-field microscopy – simplest form of optical microscopy a. Sample illuminated by transmitted white light from below i. Contrast results from absorbance of some
transmitted light by denser areas of the sample
ii. Works best with fixed, stained samples (cells have
to be killed) and incubated with dyes that
preferentially bind subcomponents of the cell
BSC 300-001 Exam 1 Studyguide (Ch. 1,2,3,4,7)
2. Fixation – treating cells/tissues with chemicals (like formaldehyde) that chemically cross-links molecules so they remain in place within cells
iv. Phase-contrast: live cell imaging
1. Phase-contrast microscopes increase contrast in transparent objects by converting differences in light refraction (bending of light) of some part of the specimen into differences in light intensity
a. Can be used in living non-stained cells
2. Takes advantage of path light travels through microscope a. Has a few extra filters than light microscope – annular
diaphragm separates light into ring that passes through
condenser that condenses it to cone and passes through the
sample
b. More dense areas diffract light so the light beams become out of phase and cross each other out, creating dark spots in microscope
3. How it works
a. Phase ring (annular diaphragm) projects ring of light onto condenser which focuses a cone of light onto specimen
b. Light travels undeviated through regions of sample with little intensity
c. Light refracted by regions of sample with high density
(organelles)
d. Deviated light is out of phase (wave is shifted backward compared to undeviated light)
e. A second ring, phase plate, increases this shift up to 180 degrees
f. Projection lens focuses light onto image plane (ocular lens) g. Shifted and unshifted light interfere with one another,
cancelling each other out
i. Reduces amplitude of light and creates darker
image where those regions are located
4. Best for thin samples or single cells
a. Useful for examining location and movement of larger
organelles in live cells
v. Differential interference contrast (DIC) (aka Nomarski) – gives 3D quality to image
1. Based on splitting the light into two perpendicular components before passing them through the specimen and then recombining them to observe their interference pattern
2. Complex system of filters and prisms that alters both the phase and polarity of light in order to generate contrast
3. Also works in living cells
4. Light that gets shifted also gets rotated
5. Still can’t see proteins and resolve nucleus
6. Useful for visualizing extremely small details and thick objects
BSC 300-001 Exam 1 Studyguide (Ch. 1,2,3,4,7)
7. Objects appear to cast a shadow to one side
vi. Fluorescent microscopy
1. Fluorescent staining is perhaps the most versatile and powerful technique for localizing molecules within a cell by light
microscopy
2. Fluorophore/fluorochrome – molecule that absorbs a specific wavelength of light and emits another, lower wavelength of light a. The fluorescence of fluorochromes is dependent on its
concentration of Ca2+ and H+ ions
i. Fura-2 is a fluorochrome sensitive to Ca2+ -
increases fluorescence
3. Fluorescent microscopes split white light into discrete wavelengths or use lasers to emit specific wavelengths
a. Sample treated with a fluorophore – like an antibody
conjugated to a specific fluorophore – will emit light
wherever the fluorophore is located
b. Dichroic mirrors allow only specific wavelength of light to excite the sample and only the emitted wavelength of light
to pass through to the ocular lens
4. Immunofluorescence microscopy (most widely used method of detecting specific proteins) can detect specific proteins in fixed cell a. Tissue or cells are fixed – incubated, usually in
formaldehyde which covalently crosslinks molecules,
locking them in place in the cells
b. Samples incubated with primary antibody, which have
affinity for the antigen/epitope of interest (rabbit anti
tubulin)
i. Antibody binds to epitope (high specificity)
c. Sample then incubated with secondary antibody, with
affinity for any antibody produced by the host species of
the primary antibody (goat anti-rabbit)
i. Covalently conjugated to a fluorophore
ii. This is called indirect immunofluorescence
microscopy
1. Using them to see where protein of interest
is
2. Advantages:
a. Single primary antibody will be
bound by many secondary antibodies
– signal amplified
i. One protein could be bound
by 20 fluorescent proteins
b. Allow double, triple, or even more
antibody labeling
BSC 300-001 Exam 1 Studyguide (Ch. 1,2,3,4,7)
i. Primary antibodies from
different species target
different antigens
ii. Secondary antibodies
conjugated to different
fluorophores tag each antigen
with different wavelength
c. Epitope tags – short sequence of
amino acids
i. An antibody may not be
available for an antigen of
interest
ii. Many cell types and
organisms can be made
transgenic – an exogenous
piece of DNA can be inserted
into genome
iii. DNA can be used to express
protein of interest
iv. Gene encoding protein of
interest can be modified to
include sequences that
encode short peptide
sequence attached to the
protein of interest, called
epitope tags (myc and flag
are common tags)
v. Primary antibodies against
myc or flag are then used to
visualize the recombinant
protein
d. Drawback to antibody and epitope
tags
i. Samples must be fixed =
killed
ii. So cannot visualize dynamic
changes in localization,
change in expression levels,
etc
5. Fluorescent proteins
a. First cloned and used fluorescent protein called GFP (green fluorescent protein) from jellyfish Aqueora Victoria
b. Used similarly to epitope tags – combining them with protein of interest or placing them adjacent to the DNA regulatory sequences in a transgenic construct
i. Allows scientists to track its distribution
BSC 300-001 Exam 1 Studyguide (Ch. 1,2,3,4,7)
ii. Can look at two or more proteins simultaneously if they are each tagged with a different-colored
fluorescent protein
c. Since the utilization of GFP, many additional fluorescent proteins have been isolated or created by mutation to
existing proteins
d. GFP and other transgenic fluorophores can be visualized in living cells
e. Challenge to fluorescence microscopy of thick samples: i. When illuminated, the cells/molecules in the focal
plane fluorescence and above and below the plane
illuminate
ii. Techniques:
1. Collect light only from focal plane
2. Collect multiple images from the top to the
bottom of the sample (a Z-stack)
3. Stitch these images together to generate a
complete, high-resolution image of the
sample
iii. Methods for doing this:
1. Deconvolution microscopy
a. Purely computational
b. Can tell it where your sample is and
it can filter out light that is not the
focal point
c. Software algorithms used to calibrate
a microscope in order to determine
which fluorescence is out of focus
and which is derive from the focal
plane
d. Once calibrated when a sample Z
stack is generated, the algorithm
processes the images and removes
pixels from each image that
represent out of focus fluorescence
2. Confocal microscopy
a. Optical methods exclude light from
above and below focal plane
i. Obtains images from a
specific local plane and
excludes light from other
planes
b. Laser of specific wavelength is
focused onto focal plane
c. Transmitted light from specimen
travels through a pin hole aperture
BSC 300-001 Exam 1 Studyguide (Ch. 1,2,3,4,7)
that excludes light transmitted from
above and below focal plane
d. A number of advances to confocal
microscopy have been developed
over the last ten years that:
i. Increase resolution below
200nm (super-resolution
microscopy)
ii. Image into deep tissue (2
photon confocal)
iii. Imaging a very thin focal
plane (TIRF)
iv. Rapid Imaging in living
tissues (light sheet
microscopy)
v. Measuring the distance
between two fluorophores
(FRET)
vi. These techniques will not be
on the test!!!
vii. Electron microscopy: High-resolution imaging
1. Bombards samples with beams of accelerated electrons
a. Major difference between electron microscopy and light
microscopy is that in electron microscopes, electromagnetic
lenses focus a high-velocity electron beam instead of the
visible light used by optical lenses
2. Takes advantage of electron wavelength being very long or short 3. Electrons pass through a sample (transmission EM) or are reflected by the sample (scanning EM)
a. Electrons captured by a detector and used to generate an
image of the sample
b. The short wavelength of traveling electrons can enhance
resolution by as much as 2,000 times compared to a light
microscope
i. Limit of resolution = 0.005 ∝m theoretically
ii. Practically, in animal cells = 0.010 ∝m
4. Limitation – EM cannot be performed on live samples
a. Samples must be completely dehydrated and either fixed or embedded with heavy metals
viii. Transmission electron microscopy
1. Electrons are emitted from a filament and accelerated in an electric field
a. Condenser lens focuses electron beam onto sample
b. Living material cannot be imaged by electron microscopy – everything has to be under an ultrahigh vacuum
2. Specimen must be very thin since electrons pass through it
BSC 300-001 Exam 1 Studyguide (Ch. 1,2,3,4,7)
a. A cell suspension of thin slices of tissue
3. Specimen mounted on small grid coated with plastic and carbon,
through which electrons can easily pass
4. In negative staining, samples are bathed in a heavy metal solution
that coats the grid but is excluded from the sample
a. Electrons are absorbed by the grid, but are transmitted
through the sample
i. So sample appears light against a dark background
ii. Dark – area where electrons are absorbed
ix. Scanning Electron microscopy
1. Specimen may be very small and thin or quite large, allowing
detailed resolution of large areas
2. Samples are coated with heavy metals which
a. Absorb some of the electrons
b. Deflect some of the electrons
i. Deflected electrons captured by a detector and the
image generated, also using rotation to produce a
3D image
3. By imaging consecutive slices of very thin samples, 3D
reconstructions called tomographics can be generated
a. How to cut cells and tissues into thin sections:
i. Chemically fix sample, dehydrate it, impregnate it
with a liquid plastic (similar to Plexiglas), and cut
sections of about 5-100 nm in thickness
4. And antibodies linked to heavy metals can be used to visualize
minute details of cell morphology and protein localization
a. Thin slivers and used antibody bound to Golgi apparatus
and created 3D structure of Golgi apparatus
VII. Chapter 7 Lecture 7 – Biomembrane Structure
a. The Lipid Bilayer: Composition and Structural Organization
i. Membranes function as barriers between aqueous compartments
1. Proteins are the gate keepers that only allow certain substances out of the membrane
ii. Amphipathic phospholipids spontaneously form bilayers with hydrophilic faces and a hydrophobic core
iii. Biological membranes
1. Cary in lipid composition
2. Have a viscous consistency with fluid-like properties
3. Lipids are associated with diverse integral and peripheral proteins
4. Impermeable to water-soluble molecules and ions
iv. Plasma Membrane – outer boundary of the cell is a thin, fragile structure about 5-10 nm thick
1. Not detectable with light microscope – you have to have an
electron microscope
2. The 2 dark-staining layers in this electron micrographs correspond primarily to the inner and outer polar surfaces of the bilayer
BSC 300-001 Exam 1 Studyguide (Ch. 1,2,3,4,7)
3. All membranes examined from plants, animals, or microorganisms have the same ultrastructure
v. Fluid Mosaic Model of Biomembranes
1. Fluidity – each layer, or leaflet, behaves like a 2D fluid
a. The lipids can freely move laterally and spin in place
i. They are not locked in place
b. Allows cells to assume specific shapes, but also act
dynamically
i. Ex: during budding, fusion, or dividing
2. Mosaic – composed of a diverse array of lipids and proteins scattered throughout the bilayer
vi. Plasma Membrane Structure
1. Barrier – hydrophobic core prevents unassisted movement of water-soluble substances across the bilayer
2. Proteins – membrane proteins provide each cellular membrane its unique set of functions
a. Integral membrane proteins (transmembrane proteins) –
span bilayer and often form dimers and higher-order
oligomers
b. Lipid-anchored proteins – tethered to one leaflet by a
covalently attached hydrocarbon chain
c. Peripheral proteins – associated primarily by specific
noncovalent interactions with integral membrane proteins
or membrane lipids (including cytoskeletal proteins)
vii. Bilayer structure of Biomembranes
1. Membrane lipids (primarily phospholipids and cholesterol): a. Amphipathic molecules – ends have different chemical
properties
i. Hydrophobic tail
ii. Hydrophilic polar head group
1. Most energetically favorable orientation for
polar head groups is facing the aqueous
compartments outside of the bilayer
a. Two sides of the bilayer – leaflets
iii. All lipids in biological membranes are amphipathic
2. Bilayer forms spontaneously, driven by hydrophilic and hydrophobic regions:
a. Nonpolar tails – close packing stabilized by van der Waals and hydrophobic interactions between hydrocarbon chains
b. Polar head groups – Ionic and hydrogen bonds stabilize
interactions with each other and with water
c. Micelles – single layer of lipids, single fatty acid tail, can pack tightly
i. Synthetic – if you purified a lipid and put it in
water, it would form this or a liposome structure
d. Liposomes – sphere with hollow center
BSC 300-001 Exam 1 Studyguide (Ch. 1,2,3,4,7)
e. Bilayer
f. Which structure forms depends on concentration,
temperature, length, and saturation of tails
g. Studying membranes in the lab:
i. Phospholipids can be purified form biological
membranes by treatment with organic solvents such
as chloroform
1. Precipitates proteins and carbohydrates
ii. When reintroduced to an aqueous environment, the
phospholipids spontaneously form liposomes which
have proven useful in drug delivery
iii. Or can form bilayers within holes of partitions in
order to study biochemistry of various
phospholipids
viii. Faces of cellular membranes
1. Plasma Membrane – single bilayer that encloses cell
a. Cytosolic and exoplasmic leaflets of the bilayer
b. Sidedness – cytosolic side to inside and exoplasmic side to outside
2. Vesicle and some organelles:
a. Bounded by single membranes
b. Lumen – internal aqueous space
3. Nucleus, mitochondrion, and chloroplast
a. Enclosed by two membranes separated by a small
intermembrane space
b. Exoplasmic face of the inner and outer membranes borders the intermembrane space
c. Chloroplast has 3 different membrane systems
4. Faces of cellular membranes are conserved during membrane budding and fusion
a. Endocytosis – plasma membrane segment buds inward
toward cytosol and pinches off as a separate vesicle
i. Cytosolic face – remains facing the cytosol
ii. Exoplasmic face – faces vesicle lumen
b. Exocytosis – intracellular vesicle fuses with the plasma
membrane
i. Vesicle lumen released to extracellular medium
ii. Cytoplasmic face remains facing cytoplasm
c. Membrane-spinning proteins retain asymmetric orientation during vesicle budding and fusion
i. Same protein segment always faces the cytosol
ix. Chemical composition of membranes
1. Lipid and protein compartments are held together by non-covalent interactions
2. Membranes also contain carbohydrates – primarily covalently attached to lipids and proteins
BSC 300-001 Exam 1 Studyguide (Ch. 1,2,3,4,7)
3. Protein/lipid ratios vary among membrane types
a. As does protein and lipid composition
b. Ex: most animal cells have a sphingosine: phospholipid: cholesterol ration of 0.5: 1/5: 1
i. However cells within the intestinal lumen (where
pH and mechanical stress is extreme) have a ratio of
1: 1: 1 – more sphingosine due to its protective
benefits
4. Three types of amphipathic membrane lipids be familiar with names, definitions, and be able to recognize them structurally a. Phosphoglycerides – diacylglycerides with small functional head groups linked to the glycerol backbone by
phosphoester bonds
i. Glycerol backbone (a 3 Carbon alcohol)
ii. Tails – two esterified fatty acid chains
(hydrophobic)
1. Vary in length (16 or 18C)
2. Vary in saturation
a. There are no trans carbon-carbon
double bonds in biological
membranes, only cis
3. Usually one saturated and one unsaturated
tail
iii. Head – polar group esterified to the phosphate
1. 4 major head groups (just need to know their
names- don’t have to recognize structure)
a. Phosphatidylcholine (PC)
b. Phosphatidylethanolamine (PE)
c. Phosphatidylserine (PS)
d. Phosphatidylinositol (PI)
e. Each contributes distinct chemical
properties to membranes
f. Found in different ratios in inner and
outer leaflets and organelles
g. Unique roles in different cell
activities
iv. Plasmalogens – one fatty acyl chain esterified to
glycerol
1. Another fatty acyl attached by a stronger
ether linkage
2. Have the same head groups as
phosphoglycerides
a. One fatty acid tail held by ether bond
instead of ester bond
b. Less chemical reactivity – less harms
to it
BSC 300-001 Exam 1 Studyguide (Ch. 1,2,3,4,7)
3. Found principally in heart and brain cells
where they are thought to protect cells from
damaging effects of reactive oxygen species
produce by high rates of metabolism
b. Sphingolipids – ceramides formed by the attachment of sphingosine to fatty acids
i. Sphingosine derivative (amino alcohol with long
hydrocarbon chain)
ii. Varied fatty acyl chains connected by an amide
bond
iii. A polar head group usually attached by a phosphate
group
iv. Sphingomyelins (SM) (most abundant) –
phosphocholine head group
1. Provides rigidity and structure to cells found
within neural protective cells
v. Some are glycolipids – head group is a single sugar
or branched oligosaccharide
vi. Carbohydrate not linked by phosphate, so not
technically a phospholipid
1. Ex: glucosylcerebroside (ClcCer) has a
glucose head group
vii. Gangliosides – glycolipids with highly branched
sugar chains
1. Great protective and insulative abilities,
therefore predominately found in neural
cells
c. Sterols – smaller and less amphipathic lipid that is only found in animals we only need to know cholesterol
i. Membrane components – animals (cholesterol),
fungi (ergosterol), and plants (stigmasterol)
1. Up to 50% of animal membrane lipids are
cholesterol
ii. Amphipathic structure:
1. Head group – single polar hydroxyl
2. Tail – four conjugated hydrocarbon rings
and short hydrocarbon chain
iii. Cholesterol
1. Not as amphipathic as other sphingolipids
2. Small amount of polarity
3. Animal specific
4. Be able to recognize structure
x. Membrane lipids and membrane fluidity
1. Lateral diffusion – several micrometers per second
a. Viscosity like olive oil
2. Fluidity is critical for membrane dynamics
BSC 300-001 Exam 1 Studyguide (Ch. 1,2,3,4,7)
3. Without such fluidity, cells could not divide, change shape, bud, traffic
a. Gel-like consistency is bad
b. Too fluid is also bad – they can melt and the cell will burst 4. When the temperature falls below 37 degrees Celsius artificial membranes undergo phase transition, from a liquid crystal (fluid) state to a gel-like (semisolid) state – membrane dynamics are lost a. Biological membranes can also experience phase shift when transition temp is reached
i. Transition temp depends on
1. Length of FA tails (longer promotes more
van der Waals interactions and hydrophobic
effect, therefore decreases fluidity)
2. Saturation (unsaturated tails have kinks that
prevent regular packing, therefore increase
fluidity)
5. Maintaining membrane fluidity
a. In animals and birds cholesterol buffers membrane fluidity i. Low temp – prevents tight packing of lipids and
therefore promotes a more fluid state
ii. High temp – lowers diffusion rate of lipids and
therefore promotes a less fluid state
1. Kinked shape prevents lipids from packing
too tightly; decreases fluidity at high temp
because OH is not really repelled by other
groups – cholesterol stays in the same place
and prevents bonding
6. Enzymes called flippases use ATP hydrolysis to mobilize specific lipids from one side of the membrane to the other
7. Lipid composition influences membrane thickness, which can alter protein distribution, promote membrane curvature, and thereby promote or inhibit budding, formation of cilia
8. Lipid composition varies among membrane types and leaflets a. Each contributes unique functional character traits to the membrane:
i. SM is a great insulator and found at high
concentration in neuron myelin sheaths
ii. PI and PS are negatively charged and primarily in
the inner/cytosolic leaflet
1. Membrane spanning domain of integral
proteins is usually adjacent to a stretch of
positively charged residues, which
spontaneously orients the TM domain in the
membrane
9. Lipid rafts just understand basic definition
a. Specialized plasma membrane outer leaflet regions
BSC 300-001 Exam 1 Studyguide (Ch. 1,2,3,4,7)
b. Ordered microdomain that floats within the more fluid and
disordered bilayer
c. Tail structure of sphingomyelin may interact across the
bilayer, allowing cytoskeleton to anchor them in place
d. Sequester proteins involved in cell-cell recognition and
signaling
10. Cells store excess lipids in lipid droplets
a. Single layer of lipids derived from outer membrane of ER
and stores them for later use
b. Small ER derived cytoplasmic vesicles that store
concentrated triglycerides and cholesterol esters
(cholesterol joined by ester bond to a long chain fatty acid)
for cell use
i. Primarily found in adipose/fat tissue
c. May also store proteins bound for degradation
d. In non-adipose cells lipid droplets protect cells from
lipotoxicity, accumulation of lipid intermediates that
promote cell dysfunction and death
b. Membrane Proteins: Structure and Basic Functions
i. Proteins interact with membranes in three ways:
1. Integral membrane proteins (transmembrane proteins) span the bilayer and have 3 domains:
a. Cytoplasmic and exoplasmic domains have hydrophilic
exterior surfaces – resemble water soluble proteins in this
respect
b. Transmembrane domain – exposed hydrophobic resides –
usually as the exterior of one or more alpha helix or a beta
sheet rolled into a barrel shape (B-barrel)
i. B-barrels: outer membrane channels found in many
bacteria, mitochondria, and chloroplasts
1. Porins create open channel sized for specific
molecules to pass through
a. Small hydrophobic molecules such
as nutrients and waste
2. Outer membrane protects from harmful
agents such as antibiotics and proteases
3. 16 B-strands twist into a barrel with
hydrophobic residues facing outside and
hydrophilic residues lining the interior of the
barrel
c. Most transmembrane proteins have membrane spanning
alpha helices
i. Nonpolar residues promote hydrophobic and Van
der Waals interactions with the fatty acid tails of
phospholipid and cholesterols
BSC 300-001 Exam 1 Studyguide (Ch. 1,2,3,4,7)
ii. Adjacent positively charged residues aid in
orienting TM domain within the membrane
iii. Alpha helix TM domain typically 20-25
hydrophobic residues
iv. Can form dimer – as in Glycophorin A examples
v. As well multimers – aka multipass TM proteins – a critical class we’ll discuss later are the 7 TM
proteins G protein coupled receptors
vi. Because TM domains are characterized by
hydrophobic domains, we can product such
domains with hydropathy plots
1. Looking at residues along length of residues
and asking if it is hydrophilic or phobic
2. If it has 20-25 you can be pretty sure it is an
alpha helices
d. Charged sidechains of TM proteins
i. Charged residues in otherwise nonpolar TM
domains can guide assembly of higher-order
structures
ii. Multimeric T-cell receptor responsible for antigen
recognition in our immune system forms a
multimeric complex from several subunits
2. Peripheral membrane proteins – associated via noncovalent interactions
a. Dynamic association
3. Lipid anchored proteins – covalent attachment to polar head group of different classes of lipids
a. Hydrocarbon tail of lipid anchor is embedded in bilayer b. Proteins are covalently linked to specific lipid types c. 3 types of lipid anchored proteins
i. GPI anchors
1. Exoplasmic only
a. Faces exterior of cell
2. C-terminus of protein covalently attached to
sugar of glycolipid
3. Glycosylphosphatidylinositol (GPI)
a. Sugar portion varies, lipid is always
phosphatidylinositol
b. Protein can be cleaved from sugar
base and released as a signal
c. Typically signaling molecule
ii. Acylation
1. Cytosolic only
2. N-terminal glycine of protein covalently
attached to a fatty acyl group
a. Long fatty acid
BSC 300-001 Exam 1 Studyguide (Ch. 1,2,3,4,7)
3. Critical for many cellular activities like
signal transduction
4. Ex: a critical oncogene called v-Src is only
oncogenic when myristylated
iii. Prenylation
1. 15-20 carbon molecule
2. Proteins anchored by C-terminus instead of
N
3. Do not have to know names or examples –
just basic definition
4. Anchorage makes them functional
a. Anchored by thioester bond
c. Phospholipids, Sphingolipids, and Cholesterol: Synthesis and Intracellular Movement
i. Fatty Acid Synthesis
1. Fatty acids in phospholipids contain 14, 16, 18, or 20 Carbon atoms – both saturated and unsaturated chains
2. Synthesized from the two-carbon building block acetate
3. A complex set of enzyme catalyzed reactions esterifies acetate to the large water-soluble molecule Coenzyme A (CoA)
4. Sequential addition of acetate produces a 16-carbon unsaturated FA called Palmitoyl CoA in the cytoplasm
a. Palmitoyl-CoA is imported into the ER where enzymes can
elongate and desaturate it to generate various FAs
b. The CoA group added to FAs:
i. Makes them hydrophilic and soluble
ii. Makes them highly reactive (charges it), allowing
the addition of more carbons to the chain
c. Intro to CoA
i. Derived from essential vitamin B5
ii. Coenzyme for several biochemical processes,
notable FA synthesis (anabolism), FA and glucose
oxidation (catabolism)
iii. Covalently liked through thiol group to carboxylic
acid groups to generate an unstable (high free
energy) thioester linkage
1. Unstable – easy to transfer by creating a
more stable bond, which is the function of
many coenzymes
2. Promotes ready breakage of thioester bond
and transfer or the attached molecule to
another more stable linkage
5. Activated carrier – bond is a thioester bond which is high in energy and is highly unstable
a. Linkage is outside of the cytoplasm
BSC 300-001 Exam 1 Studyguide (Ch. 1,2,3,4,7)
6. Binding of a fatty acid to the hydrophobic pocket of a fatty acid binding protein (FABP)
a. In order to transport FAs in the cell, unesterified FAs
associate with Fatty Acid Binding Proteins
b. A hydrophobic pocket linked by Beta sheets
c. Produced in cells, like muscles, that need to transport large volumes of FAs for ATP synthesis and fat storage
d. Tightly regulated, expressed only when needed
ii. Phospholipid synthesis in the ER membrane
1. New membranes synthesized by expansion of existing membranes 2. Membrane-associated enzymes catalyze the last steps of phospholipid synthesis at the interface between the smooth ER membrane and cytosol Need to know the 3 steps
a. Two FAs from FA-CoA are esterified to glycerol phosphate by acyl transferases – spontaneously insert into cytosolic
leaflet due to hydrophobicity
i. Product: Phosphatidic acid
b. Phosphatase removes the phosphate group generating
diglycerol
c. A phospholipid specific phosphotransferase catalyzes
transfer of the polar head group from a diphosphocytidine
precursor
i. Generates new phospholipid
ii. Note: All new phospholipids are first placed in the
cytosolic leaflet
iii. Enzymes called flippases catalyze ATP dependent
movement of specific enzymes to the exoplasmic
face (those phospholipids with sidedness)
1. Puts the right lipid in the right leaflet
iv. Other enzymes called scramblases randomly
scramble phospholipids that do not need to be in
specific leaflets in order to maintain equal numbers
of lipids in the two leaflets
1. Evenly distributing ration from one side of
membrane to another
2. Same number of lipids on one leaflet as the
other so the shape is not dramatically altered
d. Once synthesized in the ER, new phospholipids are
transported to other organelles and the plasma membrane
e. Mitochondria generate some of their own specific
phospholipids such as cardiolipin
f. Sphingolipid assembly begins in the ER and the polar head group is added in the Golgi
iii. Cholesterol biosynthetic pathway
1. Cholesterol synthesized by enzymes in cytosol (first) and ER membrane (later), mainly in the liver
BSC 300-001 Exam 1 Studyguide (Ch. 1,2,3,4,7)
a. Begins with esterification of a carbon chain to CoA
i. CoA addition creates high energy bond that allows
carbon chains to be joined (very spontaneous)
1. Catalyzed by ER transmembrane protein
HMG-CoA-Reductase which removes CoA
releasing mevalonate into ER
2. If cholesterol levels are high in the ER membrane they force the enzyme to interact with two addition integral membrane proteins a. Targets HMG-CoA-Reductase for ubiquination and degradation
i. Shuts off cholesterol synthesis
b. Statin (drug) (AKA HMG-CoA-Reductase inhibitors) binds to HMG and inhibits cholesterol synthesis
i. Most successful at lowering cholesterol
ii. Causes degradation to stop producing cholesterol
3. Mechanisms of transport of cholesterol and phospholipids between membranes Be able to say which answer choice is not a way of transport
a. Vesicles bud off one membrane and fuse with a target membrane to transfer lipids between membranes
b. Lipids transferred directly by membrane-embedded proteins between contacting membranes
c. Small, soluble lipid-transfer proteins mediate faster