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167 Cards in this Set
- Front
- Back
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What are 4 reasons amino acid sequence is dynamic?
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It is dynamic due to splicing, post-translational modifications, proteolytic processing, and covalent cross-bridging
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What does amino acid sequence in a protein determine for the protein?
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It determines the protein's physiological function
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All amino acid sequences in the human genome are due to what?
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The Human Genome Project
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What is a mechanism to find the functional encoding gene in a protein?
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Experimentally, specific functional isolated proteins are isolated and analyzed by spectormetric methods
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What does allelic variation account for?
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It accounts for differences in amino acid sequence between individuals
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Do these allelic variations produce diseases, and if they do, what is an example of such a thing?
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They can produce molecular disease, and an example is the change in the alpha and beta chains of hemoglobin, which causes hemoglobin apathies
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How can peptides be reproduced biologically?
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They can be produced by inserting an amino acid sequence into a plasmid to produce a recombinant protein in large amounts, which can be tagged for testing
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How are peptides reproduced chemically?
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Via solidphase peptide synthesis
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Who developed this?
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Merrifield in the 1960s
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What happens during this method?
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The desired peptide chain is created by protecting the N (at alpha amino group) and C (at the alpha carboxyl group) termini, and then selecting removing the protecting group from the alpha-amino group to add an amino acid in a cyclical fashion until the desired polypeptide is formed
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What does it mean if during this method the polypeptide chains are considered to be pure?
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The C-terminus of the desired polypeptide chain is attached to a polymer resin bead and all bi-products of the reaction remain in solution
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What do quaternary structures come together to form?
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They come together to form even higher complexes to make structures like organelles (ribosomes, etc)
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Why are peptide bonds polar?
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The nitrogen takes on a partially positive charge in the resonance form, and the oxygen takes on a partially negative charge in the resonance form
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Why are peptide bonds polar?
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The resonance form of the bond is lower in energy than its extremes, so its atoms resonate
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Why are peptide bonds planar?
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The resonance form of the bond is lower in energy than its extremes, so its atoms resonante
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What is necessary for the atoms of the peptide bonds to be in order for resonation to occur?
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They have to be in the same plane in order for resonation to be possible
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What is the ratio of charge : uncharged forms?
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50:50
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Are peptide bonds in trans or cis configuration?
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They are in trans configuration
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Why do they like trans over cis?
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Steric interactions between the N termial and C terminal substituents of the peptide bond prohibit the cis configuration
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Which has lower energy, cis or trans?
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Cis
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If peptide bonds are planar, how can the chain fold?
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Through rotation about two bonds
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What rotates in a phi bond?
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The alpha carbon to nitrogen
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What rotates in psi bond?
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The alpha carbon to carbonyl bond
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What is defined by the phi and psi angles of each individual amino acid?
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The complete structure of the polypeptide
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Does folding of peptide bonds assume any particular confirmation usually?
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It assumes the conformation of lowest energy
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In an alpha helix, where do hydrogen bonds form?
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They form between the carbonyl oxygens and hydrogens within the same polypeptide chain
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Which axis do the hydrogen bonds align on?
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They align on the long axis
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What does aligning along the long axis allow?
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It allows the packing of amino acids over a short distance
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Is there a hole in the middle of the alpha helix?
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No, there is no hole
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Where do polar side chains exist in an alpha helix?
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They exist on the side chain in contact with the aqueous environment
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Where do nonpolar side chains exist on the alpha helix?
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They exist on the side chain in contact with proteins
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Are alpha helices amphipathic in nature as a result?
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Yes, they are
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What is a beta sheet characterized by?
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It is characterized by polypeptide chains running in opposite directions attached by hydrogen bonds between different polypeptide molecules
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Does the beta sheet appear 3-dimensional when viewed at an edge?
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No, largely it appears flat when viewed on edge
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How do polar and nonpolar sidechains orient themselves on different sides of the Beta sheet, and does this relate to an alpha helix style of orientation?
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Polar and nonpolar side chains are on different sides of the beta sheet, and yes, this is just like the alpha helix
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Are beta sheets amphipathic by nature?
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Yes, they are
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What do beta-turns encompass?
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They encompass three residues and stabilization by hydrogen bonding
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What do beta-turns do?
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They reverse the direction of the polypeptide chain
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When looking at tertiary structure of proteins, is there continuity between folding patterns from protein to protein?
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Yes. Similar folding patterns are seen in proteins that are not homologous in sequence
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What can be used to predict folding patterns to some extent?
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Algorithms
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Patterns can be better predicted by what?
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The nature of amino acid residues
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Where do hydrophobic residues lie?
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They lie in the center of the protein
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Where do hydrophilic residues lie?
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They lie on the exterior
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Can uncharged residues be found on the exterior? What about charged on the interior?
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Uncharged residues can be found on the exterior, but charged residues are not found on the interior
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Are secondary structures closely packed or far packed?
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They are closely packed to maximize the surface area to volume ratio
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What are aggregated secondary structures known as?
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They are known as super-secondary structures
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Are super-secondary structures common?
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Yes
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What are 3 examples of super-secondary structures?
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Beta-Beta, Alpha-Alpha, and Beta-Alpha-Beta
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What are Beta-Beta?
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They are a beta-turn connecting two inverted beta sheets
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What are alpha-alphas?
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They are two alpha helices coupled by a turn
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What are beta-alpha-betas?
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They are characterized by parallel Beta sheets coupled by an alpha helix
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What do these structures come together to form?
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Tertiary structures
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What are 4 examples of tertiary structures?
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Saddle-shaped type
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What are saddle shaped?
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They are parallel beta sheets that configure into a spiral shape in 3d space
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What are two examples of saddle-shaped?
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Flavodoxin and Carboxypeptidase
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What are beta barrel type?
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They are parallel beta sheets folded over on themselves to form a barrel structure
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What are 2 examples of beta barrel type?
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Triose phosphate isomerase, and pyruvate kinase domain I
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What are anti-parallel beta sheets?
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Beta sheets come together to make a sandwich
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Can the anti-parallel beta sheet be seen anywhere of note?
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They can be seen in antibodies
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What are the beta sheets in anti-parallel beta sheet structures held together by?
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They are held together by a disulfide bridge
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Four alpha-helix motif structures are what?
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They are alpha-alpha structures that comes together to form bundles
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Where is four alpha-helix motif seen?
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It is seen in cytochromes and some virus proteins
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If we have 2 alpha helices and another 2 alpha helices coming togehter, do we still call them alpha alpha?
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Yes. The way it folds up is that two alpha structures come together to form a cylinder
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What are integral membrane proteins?
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They span the membrane with hydrophobic properties on the interior
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What do integral membrane proteins require to solubilize?
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Detergents
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What are membrane assisted proteins?
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They interact with lipid head groups or integral membrane proteins
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What does it take to solubilize membrane associated proteins?
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It requires an increased salt concentration
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What are 2 examples of membrane proteins?
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Bacteriorhodopsin
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What are bacteriorhodopsin proteins?
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They are light harvesting and embedded in the membrane by a set of parallel polypeptide chains in alpha helical conformation
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Where do bacteriorhodopsins have charged residues?
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They have charged residues ont he interior
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What are bacteriorhodopsins embedded in the membrane by?
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A set of paralle polypeptide chains in alpha-helical conformation
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Why are there no charged residues on the interior of the membrane?
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This is because of hydrophibicity
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What is a bacterial porin?
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It forms a pore in the membrane
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The pore of a bacterial porin is filled with what?
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Solvent-filled
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What is the pore delineated by?
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Beta sheets build its perimeter
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Does each arrow show a single row of a beta sheet?
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Yes
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What is something post-translationally to insert a membrane protein into the membrane?
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They can have hydrocarbon additions
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Historically, what was 3d structure determined by?
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It was determined by single crystal x-ray crystallography
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What does this technique give?
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It gives precise information about the 3d structure of protein
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Is it high resolution or low resolution?
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High resolution
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Does it give a dynamic or static picture of a 3d protein?
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It gives a static picture of 3d protein
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Why are proteins not really static though?
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They arent static because structures are, to some degree, always dynamic
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Does a polypeptide appear on an electron density map?
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No, because it is moving around so much and has no defined structure
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Why must proteins fold during their lifetimes?
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They must fold so that they can be functional
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Can proteins have more than one defined 3d structure shape?
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Yes. They can flip between 2 or more structure
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Why is it important that proteins can assume more than one shape?
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It is important because it allows cooperativity of proteins
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What is an example of structural changes like this?
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Hemoglobin relies on conformational chages and flips from a high affinity for oxygen to form a low affinity for oxygen form
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Do some proteins have entirely or largely disordered structures?
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Yes
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What are these proteins called?
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Intrinsically unstructured proteins
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What does computation suggest about the number of this kind of protein?
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One-third of proteins may be this kind
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Where do intrinsically structured proteins have polypeptides exposed to?
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They have polypeptides exposed to solvent
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How are the amino acid compositions in these disordered proteins?
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They are very rich in positively / negatively charged residues
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Are these proteins hydrophic or hydrophilic?
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They are very hydrophilic
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What is an example of an intrinsically structured protein?
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p-27 protein is one
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The cell cycle is regulated by what?
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It is regulated by cyclin protein, which is the master timekeeper of the cell cycle
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What does cyclin protein determine?
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It determines when the cell changes from each stage in the cell cycle
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What does cyclin combine with?
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It combines with kinase called cyclin-dependent kinase
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What does this form?
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A dimeric protein with two subunits (cyclin and kinase)
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Does this activate or inactivate the kinase?
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It activates the kinase, which is an enzyme that phosphorylates other proteins, which are then responsible for the remodeling that takes place when a cell divides
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These kinases also trigger something else in the cell cycle, what is this?
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They trigger DNA synthesis
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The whole cycle is then regulated by couplings of what two things?
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Cyclin and cyclin-dependent kinases
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Is this process self-limited, or must it be stopped by something else?
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It must be stopped by something else
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So how does p-27 enter into this?
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It is a cyclin-dependent kinase inhibitor, which binds to this pair of subunits and stops them from working
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When looking at a molecular graphic simulation of p-27, what is notable about the N and C terminals of the structure?
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THey are floppy pieces of polypeptide linked together by an alpha helix
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Which domain of p-27 binds to Cyclin A?
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Domain 1
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Where does p-27 bind to?
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It binds to the cyclin at terminus
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What does this do?
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It causes conformational changes
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What is this finally followed by?
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It is followed by a folding of domain 2 to adopy a correct conformation to interact with cyclin dependent kinase
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What does this boil down to, when considering unstructures proteins?
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A protein that starts out without a structure assumes a structure when binding to another protein.
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Do many proteins interact this way?
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Yes
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The folding pathway is analogous to what?
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A metabolic pathway with a defined sequence of events that take place in order to get from a fully unstructured protein to a correct structural one
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Does more occur during this process than just rotation of phi and psi angles?
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Yes
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What happens when a protein is folding up, and its structural stabilization by disulfide bridges is wrong?
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If it locks into this position, it is dead, so protein disulfide isomerase fix it.
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How do they work specifically?
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They shuffle disulfide bridges to give dead proteins another change to fold to the correct conformation
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What is the amino acid that is not in the trans configuration?
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Proline
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So does this mean peptide bonds can be formed in the cis configuration?
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Some proline forms do form cis configuration peptide bonds
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How does this happen?
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During the folding, they shift from trans to cis
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What is the enzyme that allows them to do this?
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Peptidyl proline isomerase
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Is there anything to catalyze the folding process in the lab?
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They have folding machines
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When newly unfolded polypeptide chains are made, what is the danger for them?
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Hydrophobic-hydrophobic interactions will cause them to aggregate and become dead junk
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What is the goal then to prevent this?
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The goal is to sequester unfolded proteins away from interactions with other unfolded proteins
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What does this?
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Chaperones
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How does it work?
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Unfolded proteins can bind to chaperones, and they can fold up on their arms without having destructive interactions with other proteins
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What else can chaperone proteins assist in?
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They also assist in shock responses like heat shock
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What is a histone?
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Histones are responsible for the organization of chromatin
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Does the introduction of chaperones fix prion disease?
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Its unrealistic from a therapeutic standpoint
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Is there any evidence that proteins fold while being translated?
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There is evidence of this
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Polypeptides are so long that they have what that fold into globular structures?
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They have subregions that fold in globular regions
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So, when the polypeptide is finished, what does it look like?
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It looks like beads on a string
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What are these beads on a string called?
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They are called domains
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What are domains?
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They are independently folding subsections of a single polypeptide strand
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What does a characteristic domain structure of an antibody protein look like?
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It is composed of 4 chains, with 2 light chains and 2 heavy chains
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Light chains are like 2 balls, and each ball is what?
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A domain
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Are these domains usually genetically related to one another?
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They usually are related to one another
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What do these domains arise from?
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They arose by duplication of mutational divergence of a primordial immunoglobulin folding gene
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What is a primordial immunoglobulin fold gene?
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It's a gene that encodes the protein and adopts the 3d structure of an immunoglobulin domain
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How much heavier is the heavy chain than the light chain?
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It is about twice as long
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How many domains does a heavy chain have?
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It has 4 domains
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What is the structure held together by?
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It is held together by disulfide bridges between second domain of light chain and second domain of heavy chain, and between the two heavy chains in the hinge region
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What is the hinge region?
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It allows two arms to rotate relative to one another
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What are the ends of the structure?
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They are the antigen-combining site of the antibody molecule, which the infectious agent recognizes as foreign and interacts with
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Immunoglobulin G has how many polypeptide chains hooked together?
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It has 4 polypeptide chains hooked together by disulfide bridges
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What is the result functionally of some protein aggregates having a very low affinity?
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They are too transitory to be isolated
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What is an example of this?
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Enzymes of the Kreb's cycle
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How so?
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The enzyme sof the Kreb's cycle in the test tube are too slow in reacting
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The enzymes of Kreb's cycle in mitochondria are faster or slower than in a test tube?
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Must faster
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Why is this?
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Proteins collect into 2-3 big complexes
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What is an example of this, and how does it work?
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Metablomes are like this, and they are where the substrate on one enzyme is the transformed product of the prior reaction.
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Diseases of 3d structural proteins arise from?
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Misfolding / abnormal reactions between proteins
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What happens in cystic fibrosis?
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There is a deficiency of chloride ion conductance channel on the cell surface
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What causes this?
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Amino acid substitution causes the protein to flow more slowly than it should
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What does this lead to?
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Aggregation
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What happens to this aggregation?
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The ER tries to get rid of protein, which diminishes the number of active proteins
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What about sickel cell anemia?
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The deoxygenated form of hemoglobin is normally completely soluble, but a hydrophobic patch on hemoglobin due to amino acid substitution interacts with its neighbors, so hemoglobin precipitates
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Does aggregation happen in Huntington Disease, Parkinsons Disease, and Alzheimer's Disease?
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Yes, due to the abnormal interactions between proteins
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When thinking about protein-protein interaction, how does it relate to insulin taking diabetics and glucose in the blood?
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It takes some time for the insulin to build up to a point where it will prevent destabilization of glucose in the blood
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Why is this?
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Injected insulin is a 6 protein aggregate, but it needs to dissociate into a dimer and then a monomer to be active
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How does this relate to pharmacology?
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The rate at which a medicine is effective is limited by the rate the complexes dissociate from one another
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What is Lispro?
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It is a fast acting insulin that stops aggregation from taking place and helps facilitate dissociation to form more active monomers
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In botulism shots for cosmetic reasons, why is it necessary for the protein to be monomer?
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If it aggregates or dimers form, the medicines efficacy vastly increases, which can be lethal
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Quality control of medicine to nail down aggregate state is important or not important then?
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Extremely important
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Review classical saturation binding curve, in Section V, Figure 1
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Kd increases when?
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When there is a large amount of dissociation
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Kd decreases when?
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When a small amount is dissocated
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What does r refer to in the saturation binding equations?
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It refers tot he fraction of protein molecules bound to ligand
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What does [A] refer to?
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Unbound ligand at equilibrium
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Kd is effective in large amounts?
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No. It's more effective in smaller amounts.
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