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.
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
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.
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
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
a. Deduce the sequence of the first 15 amino acids in c.Myb from the
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
2. Chymotrypsin cleavage resulted in two fragments with the
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....