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UCSD BIBC 100 - Problem Set 2

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Problem Set 2 for BIBC 100 Spring 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. The side chains of which of the following amino acid are able to form H-bonds in water? Y R V Q G 7. Two amino acids of the standard 20 contain sulfur atoms. They are: methionine (M) and cysteine (C)8. 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? 9. 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. You might want to use isoelectric focusing to separate your proteins on the basis of charge, which is very likely to be different for proteins with different amino acid compositions (i.e. different proteins).10. Go to the PDB website (Protein Data Bank), and retrieve PDB ID “1ucb”. Note that under the “Primary Citation” heading you can retrieve a PDF file which describes the research that led to this structure being deposited in the PDB. You will have to be on campus, or connected to the campus network in order to retrieve this PDF file; it is worth looking over although most of it will not make a lot of sense to you. Note the title. What does this tell you? This structure is a composite file, composed of two different structure files (1CLY, 1CLZ). One is the antibody Fab fragment bound to antigen, one is without bound antigen. This file has been made to highlight the structural changes that occur when an antibody binds to its antigen. What is this protein? What does it do? IgG Fab fragment; this particular antibody binds to a tumor antigen. Is this protein made up of a single amino acid chain, or multiple chains? How long are the chain(s)? This structure shows 2 different polypeptide chains, both of which happen to be 219 a.a. in length according to the sequence descriptions (“sequence details” tab), although if you look at the sequences as listed (again, under the “sequence details” tab), you will see that one chain (Chain H, the heavy chain fragment) is 227 a.a. in length – and the Chain L (light chain) is 214 a.a. Note that the first 3 a.a. of Chain L are greyed out; this means that even though they were present in the protein that was crystallized, they do not contribute to the structure as displayed. This is probably because those 3 a.a. do not form a single stable conformation, and thus don’t contribute to the Xray scatter data (i.e. they are disordered). Describe the secondary structural composition and any major folds/motifs you see in this protein. largely beta sheet; these are all Ig domains How many domains does the protein contain? 4 total; 2 Ig variable domains and 2 Ig constant domainsIn the light chain, the NH from T164 (main chain) is hydrogen bonded to another main chain atom/ functional group. Which residue/atom/functional group is it hydrogen bonded to? It is hydrogen bonded to the oxygen in the main chain C=O (carbonyl) group of Ser174 (Ser174 is also in the


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