The overall aim of the project is to elucidate which elements in the primary amino acid sequence of polypeptides determine the secondary and tertiary conformation of these polypeptides and the folding mechanism by which this is accomplished. This will add to our understanding of the mechanisms by which proteins function in the body and how they malfunction in some disease states. It is also key to our future ability to design and genetically engineer proteins for desired purposes. An initial goal is to identify amino acid sequences of 20 or fewer residues that show substantial Alpha-helix formation in aqueous solution. This will enable us to make quantitative measurements of the helix dipole effect. We propose to use nuclear magnetic resonance to measure the effect of the helix dipole on the C2H resonance of a single histidine residue placed at various points along an (Ala-HO-propyl-L-Gln)N sequence with blocking end groups. The strength of the interaction between the histidine side chain and the helix dipole should be directly reflected in the pK shift of the histidine. Phase 2 will utilize staphylococcal nuclease which is particularly attractive for the study of tertiary conformation because of its lack of disulfide bonds. We plan to build upon work of C. B. Anfinsen and associates who used polyclonal sera to probe the conformational states of S. nuclease fragments. We will, however, use monoclonal antibodies, thus eliminating problems associated with polyclonal sera. We will also have available a library of S. nuclease mutants produced by site-directed mutagenesis and will be able to generate mutants to further probe antibody specificity and the protein folding mechanism.
| Strehlow, K G; Robertson, A D; Baldwin, R L (1991) Proline for alanine substitutions in the C-peptide helix of ribonuclease A. Biochemistry 30:5810-4 |
| Strehlow, K G; Baldwin, R L (1989) Effect of the substitution Ala----Gly at each of five residue positions in the C-peptide helix. Biochemistry 28:2130-3 |
