Lecture 7. Tuesday, September 19. Anfinsen Experiment. Robustness of Protein Structure to Mutation

Lecture 7. Tuesday, September 19. Anfinsen Experiment. Robustness of Protein Structure to Mutation

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Unformatted text preview: Restricted: Lecture 7. For students enrolled in Chem130/MCB100A, UC Berkeley, Fall 2006 ONLY PROTEIN SEQUENCE DETERMINES STRUCTURE The sequence of a protein allows it to adopt its unique fold s pontaneously: n o t emplates o r g uides a re needed. How do we know this? By studying the folding and unfolding of protein molecules experimentally. Folded proteins can be unfolded by temperatures, or by denaturing agents such as urea or guanidinium chloride: 1 Restricted: Lecture 7. For students enrolled in Chem130/MCB100A, UC Berkeley, Fall 2006 ONLY These denaturing agents can be added to protein solutions and their effects studied experimentally: The sharp transition arises because the hydrophobic core collapses all at once. 2 Restricted: Lecture 7. For students enrolled in Chem130/MCB100A, UC Berkeley, Fall 2006 ONLY Anfinsen used experiments like this to show that all the information required to form a folded protein structure is present in the amino acid sequence. He used a small enzyme called RIBONUCLEASE. Enzymes are active as catalysts only when they are folded precisely into the correct shapes: Ribonuclease c ontains c ovalent l inks b etween cysteine residues called disulfide bonds: 3 Restricted: Lecture 7. For students enrolled in Chem130/MCB100A, UC Berkeley, Fall 2006 ONLY Disulfide b onds a re t ypically f ound i n s ecreted proteins, to give them additional stability in the extracellular environment. Ribonuclease contains 8 cysteine residues, linked into precisely 4 specific disulfide bonds: Assumption: This particular pairing of Cys Residues can only occur when the protein is properly folded. 4 Restricted: Lecture 7. For students enrolled in Chem130/MCB100A, UC Berkeley, Fall 2006 ONLY How d o w e k now t hat t he a ddition o f 8 M u rea completely unfolds ribonuclease? 5 Restricted: Lecture 7. For students enrolled in Chem130/MCB100A, UC Berkeley, Fall 2006 ONLY Comparing t he r esults o f t he f ollowing t wo experiments indicates that ribonuclease is completely unfolded by urea, but regains its proper structure when the urea is removed: The loss of activity in the second experiment is consistent with the formation of disulfide bonds in urea such that the pairing between Cys residues is truly random chain is completely flexible and unfolded. SEQUENCE DETERMINES STRUCTURE 6 Restricted: Lecture 7. For students enrolled in Chem130/MCB100A, UC Berkeley, Fall 2006 ONLY How many ways can disulfide bonds form between 8 cysteine residues? Number of ways of choosing Cys residue = 8! = 40,320 Correct for equivalence of order of cysteines: 40,320 = 2520 2x2x2x2 Correct for equivalence of vertical order: 2520 4 x 3x 2 x1 = 105 There are 105 ways of forming 4 disulfide bonds. 7 Restricted: Lecture 7. For students enrolled in Chem130/MCB100A, UC Berkeley, Fall 2006 ONLY 2. Structural changes in distantly related proteins: number of folds are limited If you align the sequences of two unrelated proteins you will get alignments with ~10% sequence identity quite often by chance. Hence, the sequence differences between plant globins and mammalian globins appear random if looked at purely at the level of sequence identity. If we look at BLOSUM ( to be discussed later) scores the similarities become easier to discern. Despite the low levels of sequence identities the structures are quite similar. 8 Restricted: Lecture 7. For students enrolled in Chem130/MCB100A, UC Berkeley, Fall 2006 ONLY Structure of myoglobin We define a dihedral angle, Ω, and a distance between pairs of helices: For a given helical pair, the range of values of Ω are within ~20° of each other when comparing different globins. The values of r are within ~2 Å of each other. 9 Restricted: Lecture 7. For students enrolled in Chem130/MCB100A, UC Berkeley, Fall 2006 ONLY The range of observed structural variation in the globins can be illustrated in the following graph: Note that each helix pair is in a particular “click state”, give by the geometry of ridges into grooves packing of α-helices (see Chapter 3). Thus, as sidechains become larger or smaller the structure changes, but the structure is not arbitrarily variable. Presumably, if the changes become too large then the protein unfolds and is lost to the population. The fact that there are limits on the structural variation of stable domains may account for the observation that the number of distinct folds in nature may be limited, even though the number of proteins is, in principle, unlimited. 10 Restricted: Lecture 7. For students enrolled in Chem130/MCB100A, UC Berkeley, Fall 2006 ONLY Microbial organism No. of proteins in genome 480 8.4 1,522 1,715 1,760 3,200 4,289 6,530 A B C D E F E. coli S. cerevisiae (yeast) Universal population No. of structurally characterized domains 70 122 154 74 62 220 353 234 ~1000 Predicted number of folds 250 – 350 350 – 500 400 – 550 300 – 400 300 – 450 450 – 650 500 – 720 500 – 720 ~1000 The protein structure is extremely tolerant of mutations as long as the hydrophobic nature of the core is maintained. Discuss Lambda repressor. Conclusion: It is likely that nature uses a limited number of protein folds to generate an essentially unbounded number of proteins. 11 Restricted: Lecture 7. For students enrolled in Chem130/MCB100A, UC Berkeley, Fall 2006 ONLY SEQUENCE IDENTITY & STRUCTURAL VARIATION The level of sequence identity between two sequences still remains a useful metric when comparing proteins because it is simple to understand what is meant by it. However, as we have seen, it can be a poor predictor of structural similarity. When two proteins have diverged from a common ancestor, they retain a common structural core: 12 Restricted: Lecture 7. For students enrolled in Chem130/MCB100A, UC Berkeley, Fall 2006 ONLY When can we be certain that two proteins have similar structure, based on sequence identity alone? 13 Evolutionary Change in Protein Structure John Kuriyan UC Berkley Chem130/MCB100A, Fall 2007 Lecture 7 To run the animations, you will need to use the PYMOL demos that can be downloaded from the course web page • How do proteins tolerate changes in sequence without falling apart? An experiment using bacterial genetics shows that proteins can tolerate many substitutions in their hydrophobic cores Lambda repressor is a dimeric protein that binds to a specific DNA sequence and controls transcription of certain genes in bacteria. The three-dimensional structure of the lambda repressor protein is important for DNA recognition. This structure is, in turn, dependent on residues in the hydrophobic core of the protein, some of which are shown here. - make genes with random changes at these 7 positions - insert these genes into E. coli that are dependent on this gene for life - select bacteria that live - sequence the gene for lambda repressor in bacteria that live - what changes at these 7 positions are compatible with function? Lim and Sauer Many substitutions in the hydrophobic core resulted in functional variants of the protein. This demonstrates that the hydrophobic core can tolerate many mutations, as long as they conser ve hydrophobicity. ...
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This note was uploaded on 01/12/2010 for the course MCB 100A taught by Professor Kuryian during the Fall '09 term at University of California, Berkeley.

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