The broad, long-term objectives for the research described in this proposal are aimed at the elucidation of the mechanistic pathways for the folding of small globular proteins from the denatured state to the native conformation. The secondary and complementary objective is to determine the specific influence of the precise amino acid sequence on these folding pathways. The analysis will focus initially on the folding of ribonuclease T1, a small enzyme consisting of 104 amino acid residues. In the native conformation ribonuclease T1 contains two disulfide bonds, alpha-helix and beta-sheet secondary structures and amide bonds to two proline residues that are in the cis-configuration. The protein folding pathways will be probed by measurement of the time courses for the formation of individual hydrogen bonds during the transformation from the unfolded state to the native conformation. The time courses for individual H-bond formation will be measured using two-dimensional nuclear magnetic resonance spectroscopy. With this methodology the protein will be unfolded in D2O to label all of the amide nitrogen atoms with deuterium. The denaturant will be diluted below the critical concentration and the protein allowed to refold for specific (and variable) lengths (5 ms - 10 s) of time. the pH will then be raised for a short period (50 ms) in the presence of H2O to label, with hydrogen, all of the amide groups that have not formed hydrogen bonds during the refolding period. The pH will then be lowered with acid to quench any further hydrogen/deuterium amide exchange and the protein allowed to complete the folding process. The occupancy (H vs. D) at individual amide sites will be subsequently determined by 2D-COSY NMR analysis at 500 MHz. The influence of the state of the unfolded protein on the time courses for hydrogen bond formation will be addressed by specific alterations in the primary sequence and various folding conditions. these studies will focus on the oxidation state of the four cysteine residues involved in disulfide bond formation and the two proline residues known to be in the cis-conformation. Site-directed mutant proteins will be constructed at these sites with specific amino acid residues that cannot form disulfide bonds or exist in the cis- conformation. Circularly permutized variants of the native protein sequence of ribonuclease T1 will be constructed in order to determine the effects of transposition of peptide segments on the specific folding pathway. These circularly permuted proteins will, in effect, have the original amino- and carboxy-terminal ends covalently closed by amide bond formation and new termini created within the various loop structures in the native structure of the wild-type protein.
| Garrett, James B; Mullins, Leisha S; Raushel, Frank M (2003) Effect of linker sequence on the stability of circularly permuted variants of ribonuclease T1. Bioorg Chem 31:412-24 |
| Mullins, L S; Pace, C N; Raushel, F M (1997) Conformational stability of ribonuclease T1 determined by hydrogen-deuterium exchange. Protein Sci 6:1387-95 |
| Johnson, J L; Raushel, F M (1996) Influence of primary sequence transpositions on the folding pathways of ribonuclease T1. Biochemistry 35:10223-33 |
| Garrett, J B; Mullins, L S; Raushel, F M (1996) Are turns required for the folding of ribonuclease T1? Protein Sci 5:204-11 |
| Mullins, L S; Raushel, F M (1995) Positional isotope exchange as probe of enzyme action. Methods Enzymol 249:398-425 |
