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

¹ Bijvoet Center for Biomolecular Research, Department of NMR Spectroscopy, 3584 CH Utrecht, The Netherlands
² Departamento de Química Física, Facultad de Ciencias, Universidad de Granada, 18071 Granada, Spain
³ Genencor International, Inc., Palo Alto, California 94304, USA

(RECEIVED March 13, 2006; FINAL REVISION April 26, 2006; ACCEPTED April 29, 2006)

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., ¹⁵N 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 1, 4, and 5 and locally destabilize the H-bond network of the -sheet (7–9). In particular, it emerges that helix 5, 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.

Keywords: NMR; protein stability; protein dynamics; H/D exchange; ¹⁵N relaxation; mutation effects

⁵ Present address: Institut de Biologie Structurale Jean-Pierre Ebel, Laboratoire de RMN, 38027 Grenoble Cedex 1, France.

Supplemental material: see www.proteinscience.org

Reprint requests to: Nico A.J. van Nuland, Departamento de Química Física e Instituto de Biotecnología, Facultad de Ciencias, Universidad de Granada, Campus Fuentenueva s/n, 18071 Granada, Spain; e-mail: najvan{at}ugr.es; fax: +34-958-272879.

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.062213706.

Abbreviations: CD, circular dichroism; CSI, chemical shift index; DSC, differential scanning calorimetry; HN, amide proton; H/D, proton-deuterium; HSQC, heteronuclear single-quantum coherence spectroscopy; MD, molecular dynamics; NMR, nuclear magnetic resonance; NOESY, nuclear Overhauser enhancement spectroscopy; PG, F180P/S205G mutant lipase.

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