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Deciphering the Role of Intramolecular Chaperone in Subtilisin Folding through NMR Dynamics Analysis

Yu-Jen Chen
BSc, National Taiwan University - 1998

Thesis Advisor: Masayori Inouye, Ph.D.
Graduate Program in Biochemistry
& Molecular Biology

CABM Room 341

Thursday, February 10, 2011
2:00 p.m.


Subtilisin, a serine protease, folds with the assistance of a specific propeptide coded as an N-terminal sequence extension, and is thus termed ¡§intramolecular chaperone¡¨. It, however, plays no role in the enzymatic activity and needs to be removed through autoproteolysis after folding. Earlier studies suggested that subtilisin folds through an intermediate and requires the propeptide to climb over the kinetic barrier of the transition state so the native conformation can be formed.
In the present study, we sought to further investigate the folding of subtilisin at the late stage through characterizing the structural dynamics of the free subtilisin and the propeptide-subtilisin complex at multiple time scales using NMR spectroscopy. We also hypothesize that the propeptide starts chaperoning subtilisin at an earlier stage during translation due to its nature of locating at the N-terminus.
To analyze the NMR dynamic parameters, the backbone assignments of the free subtilisin were completed for over 97%. The 1H-15N assignments of the propeptide-bound subtilisin were also completed for 86% through selective labeling strategies. Our results showed that the mature subtilisin is relatively very rigid with limited local fluctuations at both ms~ƒİs and ns~ps time scales. Binding of the propeptide drastically perturbs the dynamics of subtilisin at both time scales. This perturbation is not restricted to the binding interface or the active pocket, which indicates a global structural rearrangement guided by the propeptide.
In addition, we aimed to characterize the N-terminal fragments of the subtilisin precursor, which were designed to mimic the translational intermediate. We found that helical content increased as the fragment length extended. A partially folded molten globule-like structure was observed when the helices interacting with the propeptide were present. Notably, deletion of the propeptide caused significant precipitation for all the fragments. These results suggest that the propeptide can start chaperoning subtilisin and prevent the misfolding of the nascent polypeptide chain during translation.

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