General Biochemistry Exam 1 study guide
General Biochemistry Exam 1 study guide 4115
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This 6 page Study Guide was uploaded by Thomas Salazar on Sunday September 18, 2016. The Study Guide belongs to 4115 at Virginia Polytechnic Institute and State University taught by Dr. Richard Helm in Fall 2016. Since its upload, it has received 19 views. For similar materials see General Biochemistry in Biochemistry at Virginia Polytechnic Institute and State University.
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Date Created: 09/18/16
Study Guide for Exam 1 General Biochemistry PROTEIN STRUCTURE BASICS Proteins become folded in specific ways to form their final functioning structure. When proteins are at normal physiological temps. they have a greater free energy state: this means that they can “do work for free” and will fold spontaneously to achieve a thermodynamically stable structure o Adding heat lowers the free energy state of a protein, to where bonds will break, the structure “unfolds” or becomes loose and disorganized, and possibly denatures the protein altogether, destroying it permanently o The equilibrium expression for conversion of [folded proteins] [unfolded proteins] is equal to a constant K eqthat dictates the ratio of folded | unfolded at any given temperature. WHY ATP HAS LOTS OF ENERGY ATP contains phosphor-anhydride energy bonds, basically 3 (previously) inorganic phosphate groups bonded to one another in a chain of 3 by their oxygens. o Though ATP is marginally stable (or else it would be decaying in your body constantly), the breaking of the phosphate bonds is energetically favorable, and LARGE ~ -30.5kJ/mol o Despite not being THE most energy filled molecule in your body, ATP is simple enough and dependable enough to act as the body’s energy currency o Cleaving the phosphate groups from the ATP molecule releases energy, which is most often used in coupling reactions to initiate other reactions, or begin cascades of signals in the body ATP can be cleaved one P atia time, or have two cleaved off the end at once, or all three, or one, and then two, or two and then one! Mostly though, reactions cleave a single P oif the ATP molecule because it provides adequate energy, and is easier to recycle ATP + H O2 ADP + P i PROTEIN SEQUENCE DETERMINATION Protein peptide bonds have restricted rotation Sequence for Determination of Primary AA Sequence 1. *Purify to homogeneity 2. Cleave disulfide bonds (cysteine), red./ox. thiols, then alkylate to prevent reformation 3. Separate polypeptide chains 4. Determine AA composition by hydrolysis Study Guide for Exam 1 General Biochemistry 5. Determine N-terminus AA and C-terminus AA 6. Selectively cleave polypep. into smaller ones to determine sequence; overlapping sequences of peptides can be used to determine the sequence *More on purifying later Cleaving disulfide bonds between cysteine residues is done either by reduction or oxidation (depends on the reagent used). Cysteines are then “blocked” by adding an alkylating agent (several kinds) to the peptide to prevent reformation of the disulfide. Amino acid polypeptides are separated and can be hydrolyzed to break the amide bonds. After hydrolysis AAs can be separated by chromatography, and analyzed/measured by various methods of mass spectrometry. o During hydrolysis, Asn and Gln are hydrolyzed to Asp and Glu respectively, so one must take this into account use other methods to determine original compositions of each Though there are other methods, determining N-terminus amino acids is most commonly done through Edman Degradation: through cycles of treatment with Edman reagent and acid, one AA at a time is removed from the N-terminus, allowing easy sequencing of the terminal AAs. Endoproteinases: Aminopeptidases and Carboxypeptidases cleave from their respective terminal ends, and make “cuts” until they hit specific amino acids. o However, if the AA next to the terminal AA is Proline, this treatment will not work o Trypsin: hydrolyzes peptides bonds to the right side (C-terminal) of lysine and arginine, unless proline is to the C-term side of the K or R. o Chymotrypsin: hydrolyzes peptide bonds to the right side (C-terminal) of aromatic AAs, Y/F/W, unless proline follows. Will also hydrolyze to L and M if given time Cyanogen Bromide: cleaves to the C-term side of Methionine, converting it to a lactone In general, proteases with the formula [AA three letter abbreviation] followed by “C” or “N,” cleave FROM the C or N terminus, UNTIL they reach the named AA. MASS SPECTROMETRY Molecule must be charged, and in the gas phase for mass spec to work. (peptides are better than entire proteins) LC-MS Mass is a stand in for structure: since amino acids have characteristic mass based on structure, chromatography can differentiate; however the problem comes with post translational modifications (PTMs) that alter the AA residues Study Guide for Exam 1 General Biochemistry After rough differentiation, algorithms try to match peptide masses with corresponding fragment ions in a database POST TRANSLATIONAL MODIFICATIONS Phosphorylation of Ser, Thr, and Tyr: kinase puts P oni phosphatase takes off Acetylation of Lys: the enzymes that add and remove acetyl groups to lysine are no fully understood yet “O-GlcNAc” on Ser and Thr: related to the energy state of the cell, one enzyme in charge of putting on, and another for taking off N-terminal modifications primarily determine the stability of a protein; how long that protein will be around before needing to be degraded PROTEIN STRUCURES 3D structure o Side chains limit the allowable 3D space a protein can take up o Some proteins aren’t inherently structured, they are loosely folded but can induce a structure when in contact with other molecules or proteins o The final structure of a protein is when it is at its lowest free energy state at physiological temperature Secondary structure o Proteins form sheet, helices, and turns (turns are just…turns) Tertiary o Peptides form complex structures with disulfide bonds, hydrogen bonds, and produce the overall structure of the chain o This is the “folding” of a protein Quaternary o The entire 3D structure of the multi-unit functional protein assembly, units are held together by hydrophobic interactions mainly, also H bonds and salt bridges What is the Advantage of Subunits over Giant Proteins? 1) catalysis and metabolic channels are easier organization is easier 2) cooperativity is possible between subunits (binding on one unit synergizes activity of another unit) 3) allows for flexibility/alteration 4) synthesis mistakes are easier to replace 5) Easier to fold (maybe…) Study Guide for Exam 1 General Biochemistry PROTEIN ISOLATION and CHARACTERIZATION Proteins can be salted out of a sample with ammonium sulfate CHROMATOGRAPHY: “Running a mixture of protein(s) through a column with the intent of differentiating by various means o Different types are defined by if they isolate proteins using particle size, fractionation speed, pressure o Size exclusion chromatography is based on molecule size o Ion exchange, ect… SDS-PAGE: inactivates proteins, lose quaternary structure! “coats” all proteins with a negative charge heat proteins to inactivate and loosen bonds alkylate disulfide bonds block off disulfide cysteines with bulkier compounds run analysis based solely on molecular mass (proteins are now all negative so charge is irrelevant). o Band Identification of SDS-PAGE 1. Cut bands out, put in test tube 2. Remove the stain from the gel 3. Block thiols and dehydrate 4. Rehydrate with trypsin Endoproteinases to hydrolyze peptide bonds 5. Peptides release from gel, run mass spec against a database of peptides ISOELECTRIC FOCUSING o Proteins are loaded on a gel that has an immobilized pH gradient built in o Proteins migrate based on pI, then stop o Hydrophobic membrane proteins will precipitate out when reaching their pI o 2-D analysis: Isoelectric trial + SDS-PAGE = charge + mass analysis CHROMATOGRAPHY TYPES o Size-exclusion: apparatus includes pump column, UV detector, fraction collector Maintains quaternary structure! Column has specifically sized pores Larger molecules don’t fit in pores go through column faster Smaller molecules interact with pores go through column slower o Ion-exchange Column material is charged +/- Cation exchange columns interact with positively-charged proteins Anion exchange columns interact with negative-charged proteins o ions “stick” to the column, so based on the pKa of the protein, you can customize the chromatography to bind only certain proteins at certain pH’s o After the target protein(s) are isolated in the column, adding a salt solution “washes out” all ions in the column. Study Guide for Exam 1 General Biochemistry o Affinity Chromatography Ligand is attached to column beads binds selectively to specific proteins Process: add proteinwashelute with imidazole Most common type is the HIS tag: Adding several Histidine to the terminus of a protein sequence WESTERN BLOTTING: o SDS-PAGE o Transfer proteins to nitrocellulose membrane o Block the rest of membrane with non-antibody proteins wash o Add antibody #1 to bind target proteinswash o Add antibody #2; binds to #1, and allows for chemical rxns. on #2 that can act as chemical “handles.” Quantitative western blotting uses pixelated digital analysis of western blots, based on intensity of bands normalized against “unchanging” proteins such a beta-actin or beta-tubulin and standards for comparison. HEMOGLOBIN Proteins have dynamic shapes They have microenvironments that can affect pKa and ligand binding activity Non-covalent interactions can influence proteins function and form The effects of allosteric binding can be modeled using technology Hemoglobin is used in mammalian O deliv2ry, CO cycling2 and other molecular transport o Erythrocytes = red blood cells = ~25% of the cells in a human body o Hemoglobin is not an enzyme, it is a transporter protein can bind a total of 4 O2molecules also carries CO 2 NO, and some other molecules has a “sigmoidal” affinity curve, due to allosteric effects and varies based on the amount of cooperativity occurring in the molecule o Myoglobin stores O in2muscular environments has a mono-peptide structure, and holds only 1 O m2lecule however, it has a stronger affinity for oxygen than hemoglobin Cooperativity of Heme groups: O bi2ding to a heme group moves the Iron atom closer into the ring, which pulls on HIS 87. The resultant flexing of the molecule puts the protein from a “tense” T-state, into a “relaxed” R-state. The flexing of the heme group gives subsequent heme groups higher affinity for O 2 Study Guide for Exam 1 General Biochemistry Oxy-hemoglobin (Hb-O2), is a much stronger acid than deoxy-hemoglobin (deoxy- Hb) o The following rxn. occurs in the presence of excess acid Hb-O 2 H deoxy-Hb + O 2 SO the things that favor deoxy form: o Acidic environments reduce Hb’s affinity for binding oxygen. This is known as the Bohr effect + - o CO H 2,Cl , 2,3-BPG 2,3-BPG binds in the cavity inside the tetramer of Hb This binding makes it harder for oxygen to bind to the heme group CO 2eacts to form carbonic acid, creating more acidic environment o When it encounters an energy-consuming environment (working muscles), Hb-O f2vors the release of O at2the site HEMOGLOBIN VARIATIONS o Sickle cell anemia Abnormal shaped crescent blood cells Comes from a single amino acid mutation GV Impinges circulation, but creates a higher resistance to malaria o Nitric Oxide (NO) Neurotransmitter, derived from arginine Extremely high affinity for Hb; however, it reacts with CYS92 thiol group in Hb before it reacts with heme Mechanism for transport not well understood Binds to form other S-nitrothiol groups o A 1C Formed by glycating a heme group Acts as a primary indicator/marker for diabetes
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