During the past ten years, we have undertaken to determine the role of the propeptide in the folding of subtilisin, an alkaline serine protease. The 77-residue propeptide is an N-terminal extension which act as single turn- over catalyst or an intramolecular chaperone and is essential for the correct folding of the 275 residue protease-domain of subtilisin. Upon completing the folding process, the propeptide which are defective in subtilisin folding have provided a unique system to study the mechanism of protein folding, since a mutated propeptide is not part of subtilisin. Recently we found that subtilisin can fold to a stable conformer, which is different from the wild-type subtilisin and memorizes a structural alteration in the mutated propeptide. This phenomenon is termed protein memory. Moreover we have determined the three-dimensional structure of the propeptide-(S221C) subtilisin complex at the resolution of 2_ in collaboration with Dr. H. Berman's laboratory at Rutgers University. On the basis of data thus far obtained from molecular genetic, biochemical and biophysical approaches in our laboratory, in addition to the X-ray structure in this application, we will focus on the following specific aims. (1) Mutational analysis of the role of the propeptide. We isolate and characterize mutations in the propeptide that are defective in producing fully active subtilisin, mutations in the residues involved in the hydrophobic core formation in the propeptide and mutations which stabilize or destabilize the interaction between the propeptide and subtilisin. We also attempt to isolate second-site suppressor mutations to some defective mutations. (2) Identification and characterization of folding intermediates. Our results indicate that before the cleavage of the propeptide, pro-subtilisin is much more unstable and hydrophobic surfaces are exposed. We attempt to crystallize pro-subtilisin whose propeptide is designed not be cleaved by specific point mutations. We also characterize propeptide mutations from Specific Aim 1 which cause accumulation of intermediates trapped during the folding process. (3) Characterization of altered folding caused by propeptide mutations. We attempt to elucidate how mutations in the propeptide imprint their structural information on the subtilisin domain, and how their stability of protein memory is maintained in the final enzyme. We will focus on mutations at -48 position of the propeptide for the formation of conformers, and subsequently on mutations at other sites from Specific Aim 1. We will characterize these conformers in terms of enzymatic specificity, thermostability, and secondary and tertiary structures.
| Chen, Yu-Jen; Inouye, Masayori (2008) The intramolecular chaperone-mediated protein folding. Curr Opin Struct Biol 18:765-70 |
| Falzon, Liliana; Patel, Smita; Chen, Yu-Jen et al. (2007) Autotomic behavior of the propeptide in propeptide-mediated folding of prosubtilisin E. J Mol Biol 366:494-503 |
| Fu, X; Inouye, M; Shinde, U (2000) Folding pathway mediated by an intramolecular chaperone. The inhibitory and chaperone functions of the subtilisin propeptide are not obligatorily linked. J Biol Chem 275:16871-8 |
