Comparative NMR study on the impact of point mutations on protein stability of Pseudomonas mendocina lipase.

TitleComparative NMR study on the impact of point mutations on protein stability of Pseudomonas mendocina lipase.
Publication TypeJournal Article
Year of Publication2006
AuthorsSibille, N., A. Favier, A. I. Azuaga, G. Ganshaw, R. Bott, A. M. J. J. Bonvin, R. Boelens, and N. A. J. van Nuland
JournalProtein Sci
Volume15
Issue8
Pagination1915-27
Date Published2006 Aug
ISSN0961-8368
KeywordsAmino Acid Sequence, Deuterium Exchange Measurement, Enzyme Stability, Hot Temperature, Hydrogen Bonding, Lipase, Nuclear Magnetic Resonance, Biomolecular, Point Mutation, Protein Conformation, Protein Denaturation, Protein Structure, Secondary, Pseudomonas mendocina, Urea
Abstract

In this work we compare the dynamics and conformational stability of Pseudomonas mendocina lipase enzyme and its F180P/S205G mutant that shows higher activity and stability for use in washing powders. Our NMR analyses indicate virtually identical structures but reveal remarkable differences in local dynamics, with striking correspondence between experimental data (i.e., (15)N relaxation and H/D exchange rates) and data from Molecular Dynamics simulations. While overall the cores of both proteins are very rigid on the pico- to nanosecond timescale and are largely protected from H/D exchange, the two point mutations stabilize helices alpha1, alpha4, and alpha5 and locally destabilize the H-bond network of the beta-sheet (beta7-beta9). In particular, it emerges that helix alpha5, undergoing some fast destabilizing motions (on the pico- to nanosecond timescale) in wild-type lipase, is substantially rigidified by the mutation of Phe180 for a proline at its N terminus. This observation could be explained by the release of some penalizing strain, as proline does not require any "N-capping" hydrogen bond acceptor in the i+3 position. The combined experimental and simulated data thus indicate that reduced molecular flexibility of the F180P/S205G mutant lipase underlies its increased stability, and thus reveals a correlation between microscopic dynamics and macroscopic thermodynamic properties. This could contribute to the observed altered enzyme activity, as may be inferred from recent studies linking enzyme kinetics to their local molecular dynamics.

DOI10.1110/ps.062213706
Alternate JournalProtein Sci.
PubMed ID16823035
PubMed Central IDPMC2242590