General Biochemistry - week 1-3 notes
General Biochemistry - week 1-3 notes 4115
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This 3 page Class Notes was uploaded by Thomas Salazar on Sunday September 11, 2016. The Class Notes belongs to 4115 at Virginia Polytechnic Institute and State University taught by Dr. Richard Helm in Fall 2016. Since its upload, it has received 66 views. For similar materials see General Biochemistry in Biochemistry at Virginia Polytechnic Institute and State University.
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Date Created: 09/11/16
Note set #1 – week 3 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 Keq that 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 at i 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 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 Note set #1 – week 3 General Biochemistry 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 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 Note set #1 – week 3 General Biochemistry 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 nd 2 ary 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…)
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