Biochem Exam 1 Study Guide
Biochem Exam 1 Study Guide BIO 412-01
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This 10 page Study Guide was uploaded by Kiara Lynch on Tuesday February 2, 2016. The Study Guide belongs to BIO 412-01 at La Salle University taught by Stefan Samulewicz in Summer 2015. Since its upload, it has received 29 views. For similar materials see Biochemistry in Biology at La Salle University.
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Date Created: 02/02/16
Exam 1 format 15 matching about amino acids 4-5 short definition 3 15pt essays CHAPTER 1 Biochemistry- study of the chemistry of life processes TATA box- transcription binding protein Domains- Eukarya (multicellular), prokaryotes (no nucleus)- bacteria and archaea Angstrom- 1x10^-10 m Bonds- covalent o Noncovalent- electrostatic (Coulomb’s law), hydrogen, van der Waals (weakest but most numerous), hydrophobic Determine whether reactions can take place (reversible, easily formed/broken) o Ionic- strongest but fewest Repulsion b/w adjacent phosphate groups of DNA, allows for double helix DNA has unfavorable electrostatic interactions from negative phosphates so Na+ and Mg2+ neutralize the negative charge; H bonds between base pairs; distance between planes- Van der Waals contact distance H bonds are strongest in linear conformation- 2 angstroms apart; reason for double stranded DNA o Each strand of DNA can act as a template for new strands Electron clouds are influenced by each other o Optimal distance- lowest energy o There is repulsion until optimal distance (partial positive and negative charges have a slight attraction) o Repulsion again closer to optimal distance- outer electron clouds overlap Properties of water o Gecko adhesive system- macro, meso, micro, nanostaructures A lot of nonpolar molecules When angles are adjusted, the number of van der waals interactions are adjusted o Polar, cohesive, hydrophobic o Ultimate H bonder, bonds with itself o Hydrophobic interactions- molecules “afraid” of water Natural reaction when nonpolar object goes into water (ex: oil) o Polar molecules in water are soluble (ex: sugar) o Hydrophilic- “love” of water CHAPTER 2 Functions of proteins o Catalyze reactions (enzymes) o Key components of cells and membranes o Transport molecules (ex: O2 in Fe in hemoglobin) o In muscles (actin, myosin) o Communication b/w and within cells o Structure (ex: collagen) o Antibodies o Growth and development (ex: regulatory proteins) Structures of proteins o Primary- just looks at the sequence, not shape or size o Secondary- folding, local shape, interactions of amino acids close to each other o Tertiary- shape at a distance within the same protein, such as one end interacting with the other end o Quaternary- different protein interactions; form multi-subunit complexes o Dimer- quaternary, 2 identical subunits o L isomer (S) and D isomer (R) of proteins Mirror images L- forms parts of proteins D- very rare o Alpha C in center o 4 groups covalently bound-------------] Primary amino group---------]-----backbone of polypeptide chain Carboxylic acid group---------] H R group- side chain (20) pH- characteristic of the environment (solution) o [ ] of H+ ions; more H+, lower pH; less H+, higher pH o Zwitterionic form (in acid)- amino group is protonated and carboxyl group is deprotonated o Ionization state varies with pH o If pH goes up, COOH is the first to give up a H o Affects amino acids because of ionizable groups o K- equilibrium constant o pKa- characteristic of the molecule o buffer- prevents change in pH picked based on pKa Amino acids- 4 groups o Hydrophobic- nonpolar R groups Glycine Gly G smallest Alanine Ala A Proline Pro P kinky, terminates alpha helices, cis or trans Valine Val V Leucine Leu L Isoleucine Ile I Methionine Met M Tryptophan Trp W optically active Phenylalanine Phe F o Polar- neutral R group, uneven charge Serine Ser S Threonine Thr T Tyrosine Tyr Y aromatic, optically active Aspargine Asn N Glutamine Glu Q Cysteine Cys C o Positively charged- positive R group Lysine Lys K pKa= 10.8 basic Arginine Arg R pKa= 12.5 basic Histidine His H pka= 6.0 basic o Negatively charged- negative R group Aspartate Asp D pKa= 4.1 acidic Glutamate Glu E pKa= 4.1 acidic Covalent disulfide bonds between amino acids o Internal- within the same chain o Interchain- between two chains o Disulfide bonds must be intact for protein to be functional Peptide bonds (C—N)- rigid and planar o Single bond but free flow of electrons through it gives characteristics of a double bond Trans and cis o Trans- adjacent amino acid side chains o Cis- steric hindrance o Proline can be cis or trans- is bad both ways Ramachandran plot o Free rotation o Shows what orientations occur in nature Motifs o Alpha helix Right handed Terminated by proline o Beta sheet 1 strand 7 Angstroms is as far as it can stretch 2 variations Antiparallel- carboxy terminis amino terminis, amino terminis carboxy terminis Parallel- both strands run the same way (AT to CT); diagonal bonds o Amino acids must loop back around and connect to the beginning o Beta turn Surface of proteins, interacts with other proteins/molecules Alpha helical coiled helix- 2 alpha helices wrapped around each other o Uses a lot of energy instead uses hydrophobic and hydrophilic interactions Ex: leucine zipper- hydrophobic interactions hold strands together o Collagen- 3 subunits wrapped around each other; most abundant Globular proteins o Cavity in myoglobin for Heme group, iron atom, O2 o Hydrophobic inside and hydrophilic outside o Reverse distribution- has pores in membranes, in hydrophobic environment Water filled hydrophilic channel, largely hydrophobic exterior Fatty acid binding protein- helps move fatty acids from 1 membrane to another Domains- secondary or tertiary structure that folds independently, is a functional unit, and tend to appear over and over again in different proteins (same function) o compact globular units; motifs o Helix-turn-helix- secondary structure Prosthetic group- “chaperone proteins,” helps protein do its job (ex: heme helps myoglobin carry Oxygen) Hemoglobin o 4 heme groups o Alpha helices o 2 identical subunits similar to other 2 identical subunits Coat of Rhinovirus o 60 copies of each of four subunits Native conformation (lowest energy state) and Denatured (unfolding, random coil) o Every protein has 1 native conformation- lowest energy; functional o Ex: bovine ribonuclease (ase- enzyme that breaks up RNA molecules that get out of cells); ability to unfold and refold o chemicals to denature proteins added to buffer, disrupts interactions beta- mercaptoethanol reduces disulfide bonds disulfide bonds break and protein unfolds ribonuclease can be reformed in presence of beta- mercaptoethanol some proteins will never get back to native structure o folding funnel top- all possible denatured conformations dips- semistable intermediates, can help or hinder native structure bottom- native structure, lowest energy Conformation limits o Genome, cis and trans, double bond nature, free rotation Post-translational modifications o Glycosylation- addition of sugar groups o Kinase- adds phosphate, turning on o Hydroxyproline- adds hydroxyl; stabilizes fibers of collagen o Carboxyglutamate- clotting protein o Carbohydrate-aspargine adduct- adds sugar, makes hydrophilic o Adding fatty acid to amino group or cysteine sulfhydryl group, makes hydrophobic CHAPTER 3 Assay- test Differential centrifugation o Cells disrupted o Homogenate centrifuged Denser material forms pellet at lower force Supernatant o Increase force Isolated fragments used for further purification Aliquot- small portion Precipitate- ppt Supernatant- sup Salting Out of Proteins o Useful for concentrating dilute solutions of proteins o Salt concentrations at which a protein precipitates differs among proteins o Can use dialysis to remove salt if necessary o Hydrogen bonds in solution (water) and solubilized protein Takes water away from proteins o 10% cut Ammonium sulfate (100 mL + 10 g AS) o Centrifuge precipitate resuspend test o The more soluble the longer it takes to come out Dialysis o Based on molecular weightthMW) Daltons unit (1/16 oxygen atom) Average- 5000-120000 da 5-120 kilodaltons o Dialysis bag (semipermeable membrane), concentrated solution, buffer o Smaller molecules diffuse through pores o Reaches equilibrium Chromatography- separates different types of molecules by passing them in a mobile phase or through a stationary phase o Columns Mobile buffer Stationary beads Gel Filtration Chromatography o Size exclusion protein mixture at top, buffer flows through column Collect buffer in test tubes (fractions) Proteins start moving through aqueous solution largest proteins go between the beads and flow more rapidly intermediate size only fit through some channels of the beads smallest size fit into every bead so they come out last spectrophotometer- shines wavelengths of light through sample tryptophan and tyrosine are optically active- 280 nm Ion Exchange Chromatography o Size does not matter protein must have a charge o If protein is still in the column, the pH must be changed to get rid of the charge If positive, increase pH Net positive charge at pH 7- binds Negative charge won’t bind o Proteins with low density of positive net charge emerge first o Positively charged proteins bind to negatively charged bead; negatively charged protein flows through Affinity Chromatography o Means of isolating transcription factors o Stationary phase- glucose attached protein with binding site sticks o Nonspecific proteins wash through o Add loose glucose protein goes out of column High Pressure Liquid Chromatography (HPLC) o Beads- ion exchange, column is sealed on both sides o Does not use gravity to move proteins through column uses pressure o Can regulate which buffers and what amounts are added on computer o Separated by tiny differences in size and charge differences induced by buffers o More interaction sites greater resolving power; sharp peaks of absorbance PGE (SDS PAGE) o Stationary phase- jelly like substance o The smaller the protein, the faster it moves o Stimulate the movement with an electric field Proteins move toward positive electrode o SDS detergent (sodium dodecyl sulfate) Charged head penetrates hydrophobic core of a protein Changes environment Unfolds protein B-mercaptoethanol breaks disulfide bonds and denatures o Migration depends on Electric field strength Net charge of proteins Frictional coefficient- depends on mass and shape o Harder for larger molecules to move Polyacrylamide Gel Electrophoresis o Porous gel- separates proteins by size Smallest move most rapidly o 10 wells- micropipette into wells o Cover o Apply voltage o Negatively charged SDS protein complexes migrate in direction of anode at bottom of gel o Staining after electrophoresis- shows how much a tissue uses a protein Left side- standards Middle- unknowns Right side- measurement for each band; Rf values Farthest point down on gel- low MW blue dye o Rf vs log(MW) (Apparent MW) mass in kd vs. Relative mobility Electrophoresis can determine mass Isoelectric focusing o pI- pH at which net charge is zero o pH gradient- different micro environment throughout gel o SDS PAGE o Separated on basis of relative contents of acidic and basic residues o Each protein moves until it reaches a position in the gel at which the pH is equal to the pI of the protein o Resolves patterns that differ in pI by as little as 0.1 o Low pH +, high pH – 2-Dimensional gels o 1D isoelectric focusing- Run gel, separate based on pI o Cut gel- lay on top of SDS PAGE gel across o SDS PAGE- separate by size; in second dimension perpendicular to first dimension o Isoelectric focusing map- moves to the right based on pI and down based on mass Can zero in on small sections and look at small clusters (ex: normal colon mucosa vs colorectal tumor tissue) o Quantification How much total protein? Measures total activity Ex: converts this much substrate to product per min Specific activity- total activity/total protein When % yield goes down it’s good Purification level goes up more pure than at beginning o Take aliquot of sample and run it on SDS PAGE Band for the protein of interest becomes more prominent relative to other bands More and more proteins disappear they move to the right; 1 band gets darker which means it is being purified Repeating motifs in a protein chain arise by gene duplication o Repeats can reveal the history of an individual protein; duplication then diversification o Calmodulin- storage/transport/signaling; inflammation, apoptosis, memory issues Amino Acid Analysis o Vacuum pump o Heating block o HCl o Separated by volume of buffer needed to remove the amino acid from the column Buffers of increasing pH are used o Ion exchange chromatography o Acidic proteins are first to elute o Process Put protein in glass vile with concentrated HCl Suck out air with vacuum, seal it Put in heating block O/N Goal- cleave every peptide bond Open vile and inject protein into HPLC- separate molecules with similar characteristics Ninhydrin o Purple dye o Covalently attaches to each amino acid Edman Degradation o Label amino terminal amino acid with phenyl isocyanate o Cleave it from peptide without disrupting other bonds o Removes one amino acid at a time o Process Pluck off AA from chain Bind label to amino terminus Adjust conditions in chamber so only the first bond is broken Shoot amino acid down HPLC column Repeat If there is a limited window break/cleave into smaller pieces o Unknown amino acids can be identified by its elution position relative to the known ones o After a certain point it’s hard to tell what is residual or not why it does not work well for long chains and why they should be cleaved only works with 50 residues o Cleaving process Cleave Separate with HPLC Sequence one at a time Take full protein and cut it with something else Look for overlap to find order Specific cleavage of polypeptides Chemical cleavage Cleavage site Cyanogen bromide Carboxyl side of methionine residues O-Isodosobenzoate Carboxyl side of tryptophan residues Hydroxylamine Asparagine-glycine bonds 2-Nitro-5- Amino side of cysteine residues thiocyanobenzoate Enzymatic cleavage Trypsin Carboxyl side of lysine and arginine residues Clostripain Carboxyl side of arginine residues Staphylococcal protease Carboxyl side of aspartate and glutamate residues Thrombin Carboxyl side of arginine Chymotrypsin Carboxyl side of tyrosine, tryptophan, phenylalanine, leucine, and methionine Carboxypeptidase A Amino side of C-terminal amino acid (not arginine, lysine, or proline) Genomic and proteomic methods are complementary approaches to find the structural basis DNA sequence amino acid sequence post translational modifications o Cloning is easier Immunoglobin G (IgG)- ribbon structure o Tetramer (2 dimers); 4 chains of antibody connected by disulfide bonds Two heavy chains and two light chains The ends are the same for every antibody o Epitope- binding site o Antibody has epitope (antigen binding site) Primary antibody binds to antigen Secondary antibody with a tag binds o Enzyme and substrate added for detection (color) Polyclonal antibodies- most antigens have several epitopes Monoclonal antibodies- 1 specific epitope, yields more reliable results Process of isolating and finding proteins in a rat o Inject proteins o Rat produces antibodies (polyclonal) o Isolate 1 clone (monoclonal) o Grow in culture secrete into buffer o Limited life span fuse with cancer cells because they don’t die o Grow in culture put in wells; induce tumors in rat produces antibodies o Can pick one that makes the antibody we want o Can use it to isolate and find proteins Enzyme Linked Immunosorbent Assay (ELISA) o Enzyme reacts with colorless substrate to produce color product o Detects presence of antibody o Indirect ELISA Used to test for HIV Coat well with HIV antigen; take blood sample and put into well If person has HIV, they have antibodies that will bind (primary antibodies) Secondary antibody (produced by rabbit that recognizes and binds with the human antibody constant region) carries label Give enzyme and substrate color Rate of color formation is proportional to the amount of antibody Can detect less than a nanogram of protein o Sandwich ELISA Monoclonal antibody coated well Antigen binds to antibody Secondary antibody linked to enzyme binds to immobilized antigen Substrate added and converted by enzyme into colored product Rate of color formation is proportional to the amount of antigen Western Blot o Stack of antibodies o Separation of proteins by gel electrophoresis (immunoassay) o Process Proteins onto gel sheet Add antibodies Rinse w/ secondary antibodies to detect w/ fluorescent labeling or enzyme o Used for monitoring protein purification, cloning genes, and testing for Hep C 3-D protein structure o X-Ray Crystallography and NMR Spectroscopy X-ray source, crystal (protein), detector Wavelengths of x-ray correspond to that of a covalent bond Atoms cause x ray beams to scatter based on structure of crystal What you know from this process What amino acids are present Primary structure All of atoms present How it folds Electron density map Has reflections (dots where beams were scattered) The darker the spot, the more beams that hit it Try to fit known amino acid structure to pattern The quality of the crystal affects data Immunohistochemistry (immune- antibody, chemistry- reaction) o Actin- helps form cytoskeleton o Every antibody we make has a constant region o Secondary antibody named by the animal it came from Ex: rabbit anti rat
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