Chemistry Problem Set: Protein Characterization and Quaternary Structure Analysis, Assignments of Biochemistry

Download Chemistry Problem Set: Protein Characterization and Quaternary Structure Analysis and more Assignments Biochemistry in PDF only on Docsity! CHEM 641 Problem Set #4, assigned Tuesday 9/18/07, answers posted 9/25/07 1) Suppose you are interested in a particular protein to study due to a connection to human disease. You manage to express the His-tagged protein at 10 mg/L of E. coli expression. a. Following purification across a Ni-column and thrombin cleavage of the His-tag, you are now faced with the need to characterize the purity and homogeneity of the sample. What analysis would you do to assess both the purity and homogeneity of your protein? b. Suppose it is determined above that your protein is “pure”, meaning the solution does not include any impurity E. coli proteins. However the protein sample does include a heterogeneous mixture of oligomeric states (monomer, dimer, trimer, etc…). What could you do to solve this problem and have a homogeneous sample with only one oligomeric state? 2) In humans (37 °C) the enzyme alcohol dehydrogenase (ADH) is a homodimer with a large hydrophobic inter-subunit contact region. The formation of a quaternary structure for ADH is a reversible process. Suppose the enzyme dissociates according to the equation: dimer 2 monomer ∆Gº' = 12 Kcal/mol a. Calculate the concentration of monomer and dimer present at equilibrium if the initial concentration of ADH dimer was 1 x 10-6 M prior to any dissociation to single subunits. b. Suppose a phenylalanine (Phe) was changed to a serine (Ser) residue by site- directed mutagenesis into the inter-subunit contact region of ADH. What do you think would be the effect of this mutation on the ∆Gº' reported above? 3) In question-2 above, alcohol dehydrogenase from humans is described as a homodimer, where the quaternary structure is composed of identical subunits. Suppose you wanted to make the same site-directed as described in question-2b (Phe→Ser). However, you wanted to study the effect of changing Phe→Ser in only one subunit of a homodimer. From what we covered in class talking about protein techniques, device an expression and purification scheme that could give you a homodimer ADH protein with one subunit wild type (no changes) and the other subunit of the same homodimer mutated (Phe→Ser). You’ll need to be creative here. 4) Suppose as you embark to study a particular protein in greater detail (question-1), you decide it would be good to have a structural model to work with. Discuss in general the advantages/disadvantages of Crystallography, NMR and Homology Modeling to obtain this structural information.