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1. Draw the dissociation reactions for the non-standard amino acid drawn below. Using D and E as a models to estimate the pKas for all the possible dissociations. Justify your estimates by discussing structural factors that raise and lower pKas. O NH3 + O - O - O 2. Assume you are working in lab that uses E. coli to produce peptides to examine structure function relationships. You are to work on a peptide that is known to bind to DNA with the sequence AACGG. Your peptides should have the following sequence: WTEEEDRIIYQAHKRLGNRWAEIAKEEPGRTDNAIKNHWNSTMRRHV However some of the other students believe that one of the Rs in this sequence has been mutated to an S and hence it is no longer functioning as it has in the past. What chromatographic technique would you use to determine if this hypothesis is correct? Explain how the technique will separate the wild-type peptide from the proposed mutant version. 3. PKU is one of the most common inborn errors of metabolism. There are nearly 300 amino acid substitution mutations in the enzyme phenylalanine hydrolase (PheOH) that result in varying severity of PKU in humans. Many of these mutations map to the catalytic domain but 12 have been shown to be on the structure at the interface between the tetramerization domain and the catalytic domain or within the tetramerization domain itself. a. Using the BLOSUM62 table (see Table 5.18 on page 125 of G&G) hypothesize which of the following you would have predicted to have a profound effect and which not. Explain your reasoning in 1-2 sentences. i. R408W ii. R408Q iii. A259T iv. F299C b. R408 has been shown to be involved in H-bond interactions with the backbone of the protein at position 309, 309 and 311. Using this structural information which mutant, R408W or R408Q, would you predict to have a less severe phenotype and why? 4. The human PheOH structure has been solved and several papers examining different mutants have been published. a. Use the PDB and file 1PAH to show the interactions described in 3b. i. Show screen shots of the analysis ( 4-8 screen shots each showing another view of the interaction) ii. Explain how each picture furthered your understanding of the interactions 5. A protein c-Myb is a transcription factor that binds to DNA. It binds to DNA through a small domain of three repeat regions. Each of the repeats folds into a helix turn helix domain which is stabilized by a hydrophobic core. Interestingly the mutation of an isoleucine residue to leucine residue has been shown to result in a less stable structure. A quote for the article that sums up the result is: “ Upon the I118L mutation, the degree of decreased enthalpy change for unfolding was larger than that of decreased entropy change, resulting in decreased stability.” a. Deduce the sequence of the first 15 amino acids in c.Myb from the following experiments: i. The N-terminal amino acid was determined to be M. ii. Trypsin cleavage and subsequent sequencing of the resulting 3 peptides resulted in: 1. EEDQR, GPWTK, MLIK and there was a V released alone 2. Chymotrypsin cleavage resulted in two fragments with the compositions: D, Q, E, V, R, T, K and P, W, M, G, K, I, L. b. Write a paragraph describing how a funnel represents the protein folding process. Please include both the ways this accurately represent the process and at least one limitation of the model. c. The cMyb mutant describe here has multiple conformations which are in thermodynamic equilibrium. How would you modify the funnel model to match these results? Write a paragraph describing the modified model for mutant cMyb folding. d. There are three non-covalent interactions that contribute to the enthalpic contribution to folding stability: H-bonds, Salt-bridges, and van der Waals interactions (induced dipole interactions). Which of these is most likely disrupted by the I118L mutation and why?

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1. The pKa values for D (Aspartic Acid) and E (Glutamic Acid) are compared in table 4.1 to estimate the pKa values for the non-standard amino acid shown above. By observing the values for E and D in the table, the side chain for the aspartic acid is one carbon closer (-CH₂) compared to glutamic acid
(-CH₂-CH₂), which is further and the value changes from 3.86 to 4.07. Therefore, if an additional –CH₂ group is added to the structure, the pKa value should change to approximately 4.28....
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