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Biological Chemistry II: Problem Set 1 1. (a) Tropomyosin, a 70 kDa muscle protein, is a double-stranded, α helical coiled coil. How long is the molecule? (b) A 40 amino acid peptide fragment forms a two-stranded, antiparallel β-sheet with a hairpin loop consisting of 4 residues. What is the largest dimension of this motif? (c) Although β-hairpin structures are often unstable in solution, incorporation of D-ProXaa sequences has been found to promote autonomous hairpin formation in aqueous solution. The corresponding L-Pro-containing sequences are completely disordered. Why? 2. Poly-L-leucine is α helical in organic solvents like dioxane, whereas poly-L-isoleucine adopts another conformation. Why do these amino acids, which possess the same type and number of atoms, exhibit completely different helical propensities? 3. The conformation of poly-L-lysine is pH dependent. At relatively high pH values, the polymer adopts a predominantly helical structure, but at acidic and neutral pH it exists as a random coil. Furthermore, at high temperatures the helix converts to an aggregated antiparallel β sheet. Explain these observations. 4. (a) Define identity, similarity and homology. (b) What percent sequence identity do you expect for two non-homologous proteins of identical length, if you assume that all 20 proteinogenic amino acids occur with equal frequency and are distributed uniformly over the length of the protein? (c) BLAST searches are performed to identify proteins having similar amino acid sequences. A BLAST tutorial can be found at http://www.ncbi.nlm.nih.gov/BLAST/, which describes how the program works. Explain the difference between the following BLAST subroutines: blastp, blastn, blastx, tblastn, tblastx (d) Find the sequence for the Bacillus subtilis chorismate mutase (BsCM) in the “Protein knowledgebase“ on the ExPASy Molecular Biology Server (www.expasy.org). Determine the number of subunits BsCM has and their length. (e) At the very bottom of the ExPASy data sheet for BsCM there is a link to BLAST submission on ExPASy/SIB. Use this link and the blastp subroutine to search for similar proteins. What is the sequence identity between BsCM and the chorismate mutase from Bacillus halodurans? Is the sequence identity high? What conclusions can you reach regarding the structural similarity of the two proteins? (f) Alignments can be made to facilitate comparisons of similar amino acid sequences. Click on the box in front of the first ten sequences found in (e) and initiate “Clustal W alignment” above the box by hitting “submit query”. You have to click “Run Clustal W“ again on the newly opened page. The alignment can be viewed in the results window (clustalw aln). Asterisks indicate conserved amino acid residues. Why is it significant that certain residues are conserved? Biological Chemistry II: LITERATURE Lecture 1: Protein Structure & Stability General Voet, Voet & Pratt: Chapter 6 Stryer: Chapter 2 Branden & Tooze: Chapters 1 & 2 Creighton, Chapters 4 & 5 Secondary Structure Prediction Chou & Fassman, Annu. Rev. Biochem.1978, 47, 258 Blaber et al., Science 1993, 260, 1637 Protein stability Lim & Sauer, Nature 1989, 339, 31 Eriksson et al., Science 1992, 255, 178