Problem Set 2 for BIBC 100 Winter 2008 1a) What do the pK values in the chart below tell you? Compound pK1 pK2 pKR pI Glycine 2.34 9.60 5.97 Glutamate 2.19 9.67 4.25 3.22 Arginine 2.17 9.04 12.48 10.76 The pK1 is the pH at which the terminal carboxyl groups (–COOH) are half-ionized, i.e. –COO- and –COOH exist at equimolar concentrations. The pK2 is the pH at which the terminal amino groups (–NH3+) are half-ionized, i.e. –NH3+ and –NH2 exist at equimolar concentrations. The pKR is the pH at which the side chain is half-ionized. The pI is the pH at which the net electric charge is zero. b) Will the following amino acids have an overall positive, negative or zero charge at pH 3.22, pH 5.97, pH 10.76? Fill in the chart. pH 3.22 pH 5.97 pH 10.76 Glycine + Zero - Glutamate Zero - - Arginine + + Zero Look at the pI of the amino acid relative to the pH. Review section 3.1 in your book. 2. Calculate the net charge on the following tripeptides at pH7. a) Asp-Glu-Ser -2 b) Ser- Gly-Thr 0 c) Gly–Lys-Arg +2 3. Refer to Table 3-2 in the textbook for this question: a) How many proteins in the table exhibit quaternary structure? How can you tell? 5 proteins consist of more than one polypeptide chain, therefore they must have a quaternary structure. b) How many proteins exhibit supersecondary structures? How can you tell? Sort of a trick question. Supersecondary structure is defined as “a recognizable folding pattern involving two or more elements of secondary structure and the connection(s) between them.” This is synonymous with “fold” or “motif”. See Chapter 4.3. So, these proteins are all large enough to probably contain supersecondary structural elements.4. How would the following agents/procedures interfere with/disrupt the different levels of protein architecture? Why? a) addition of SDS Unfolds the protein, and thus would disrupt secondary, tertiary and quaternary levels of protein architecture. SDS doesn’t cleave covalent bonds. b) addition of protease Cleaves peptide linkages, and thus would disrupt the primary structure. This might disrupt secondary and the higher levels of structure, depending on how heavily the protein was digested with protease, or light digestion might kept domains intact. For example, light digestion with papain (a protease) cleaves IgG into Fab and Fc fragments, but the fragments retain proper domain folds. 5. Histones are proteins found in eukaryotic cell nuclei, tightly bound to DNA. Remember that DNA has many phosphate groups. The pI of histones is about 10.8. a) What amino acid residues must be present in relatively large numbers in histones? Strongly basic amino acids, like Lysine (K) and Arginine (R). b) In what way do these amino acid residues contribute to the strong binding of histones to DNA? They give the histones a net positive charge, which helps the histone bind to the negatively charged DNA backbone. 6. Following are 3 different protein sequences. A) IVMMIALFMIILVP B) IRWHMYGPQNKLQ C) REDQYTWWKRRS i) Which one would most likely be found traversing the hydrophobic interior of a lipid bilayer? A – it consists entirely of hydrophobic amino acids ii) Which would most likely be found on a loop region of a protein? B – it contains some hydrophilic amino acids, and most importantly, a GP pair in the middle of the sequence that will introduce a sharp kink iii) Which one would most likely be found lying on a protein surface? C – it consists almost entirely of hydrophilic amino acids 7. The side chains of which of the following amino acid are able to form H-bonds in water?Y R V Q G 8. Two amino acids of the standard 20 contain sulfur atoms. They are: methionine (M) and cysteine (C) 9. For amino acids with neutral R groups, at any pH below the pI of the amino acid, the population of amino acids in solution will have: A) a net negative charge B) a net positive charge C) no charged groups D) no net charge E) positive and negative changes in equal concentration 10. By adding SDS (sodium dodecyl sulfate) during the electrophoresis of proteins, it is possible to: A) determine a protein’s isoelectric point B) determine an enzyme’s specific activity C) determine the amino acid composition of the protein D) preserve a protein’s native structure and biological activity E) separate proteins on the basis of molecular weight 11. About how many different kinds of proteins are found in a typical human cell? A) hundreds B) thousands C) more than a hundred thousand, less than a million D) millions E) billions 12. In an α helix, the R groups on the amino acid residues: A) alternate between the outside and the inside of the helix. B) are found on the outside of the helix spiral. C) are each 5.4 Angstroms apart. D) generate the hydrogen bonds that form the helix. E) stack within the interior of the helix.13. The interactions of ligands with proteins: A) are relatively nonspecific. B) are relatively rare in biological systems. C) are usually irreversible. D) are usually transient. E) usually result in the inactivation of the proteins. 14. Before running protein samples on an SDS-PAGE gel, they must first be treated with two substances. What are these substances and why are they important? SDS (sodium dodecyl sulfate), a detergent, which unfolds the proteins and coats them with a uniform negative charge. See Chapter 3.3. Reducing agent (typically beta mercapto-ethanol or BME) which breaks disulfide linkages, causing disulfide linked chains (in the native protein) to run separately from one another on the SDS-PAGE gel. You have a semi-purified protein sample that contains just three proteins: Protein A (100 kD), Protein B (45 kD), and Protein C (60 kD). You run this sample on a SDS-PAGE gel and stain it with coomassie blue (a protein dye). Draw your gel below. Be sure to label each protein, the top and bottom of the gel, placement of electrodes, and the direction of travel. How do you know the identity of each protein on the gel?You have a semi-purified protein mixture containing two proteins that have nearly identical molecular weights. Will SDS-PAGE be sufficient to visualize both proteins? Why or why not? If not, what other technique would you use? SDS-PAGE separates entirely on the basis of molecular weights, so this would not be a good technique to use to separate your proteins.
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