Subtilisin had been regarded by many researchers as a protein incapable of folding in its mature form. The in vivo production of native subtilisin is dependent on a 77 amino acid propeptide, which is eventually cleaved from the N-terminus of subtilisin to create the 275 amino acid mature form of the enzyme. This proposal is based on three experimental findings from our initial project. 1) The mature form of subtilisin is an unusual example of a monomeric protein with a high activation energy to folding and unfolding. 2) The highly purified 77 amino acid propeptide can catalyze subtilisin folding in vitro. 3) Certain mutants of subtilisin fold rapidly and quantitatively without the participation of the propeptide. The application of spectroscopic and microcalorimetric techniques coupled with x-ray diffraction methods would allow us to pursue three aims 1) Determination of the nature of the kinetic barrier in the uncatalyzed folding reaction. Learning how to fold heterologously expressed proteins is one of the most vexing problems in biotechnology. The demonstration that the folding rate of subtilisin can be dramatically accelerated by mutation, offers hope that other difficult protein folding problems might be similarly addressed, if the kinetic barriers to folding such proteins can be understood. 2) Determination of how the propeptide acts to case of n may a stable common features of extracellular microbial proteases probably because of their large contribution to both thermodynamic and kinetic stability. Unfortunately, the major industrial uses of subtilisins are in environments containing high concentrations of metal chelators, which strip calcium from subtilisin and compromise its stability. It would be of great practical significance to create a highly stable subtilisin which is independent of calcium.

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National Institute of General Medical Sciences (NIGMS)
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Molecular and Cellular Biophysics Study Section (BBCA)
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Ruan, Biao; London, Viktoriya; Fisher, Kathryn E et al. (2008) Engineering substrate preference in subtilisin: structural and kinetic analysis of a specificity mutant. Biochemistry 47:6628-36
Sari, Nese; Fisher, Kathryn E; Bryan, Philip N et al. (2007) Main chain NMR assignments of subtilisin Sbt70 in its prodomain-bound state. Biomol NMR Assign 1:209-11
Sari, Nese; Ruan, Biao; Fisher, Kathryn E et al. (2007) Hydrogen-deuterium exchange in free and prodomain-complexed subtilisin. Biochemistry 46:652-8
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Abdulaev, Najmoutin G; Zhang, Cheng; Dinh, Andy et al. (2005) Bacterial expression and one-step purification of an isotope-labeled heterotrimeric G-protein alpha-subunit. J Biomol NMR 32:31-40
Ruan, Biao; Fisher, Kathryn E; Alexander, Patrick A et al. (2004) Engineering subtilisin into a fluoride-triggered processing protease useful for one-step protein purification. Biochemistry 43:14539-46
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Tangrea, M A; Alexander, P; Bryan, P N et al. (2001) Stability and global fold of the mouse prohormone convertase 1 pro-domain. Biochemistry 40:5488-95

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