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


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. Study holds ProTherm entries: 22273, 22274, 22275, 22276, 22277, 22278 Extra Details: in the presence of diethyl p-nitrophenyl phosphate NMR; protein stability; protein dynamics; H/D exchange; 15N relaxation; mutation effects

Submission Details

ID: vsdkn82p3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:53 p.m.

Version: 1

Publication Details
Sibille N;Favier A;Azuaga AI;Ganshaw G;Bott R;Bonvin AM;Boelens R;van Nuland NA,Protein Sci. (2006) Comparative NMR study on the impact of point mutations on protein stability of Pseudomonas mendocina lipase. PMID:16823035
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)