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.
Sibille
Nathalie
N
Bijvoet Center for Biomolecular Research, Department of NMR Spectroscopy, Utrecht, The Netherlands.
Favier
Adrien
A
Azuaga
Ana I
AI
Ganshaw
Grant
G
Bott
Richard
R
Bonvin
Alexandre M J J
AM
Boelens
Rolf
R
van Nuland
Nico A J
NA
D000595:Amino Acid Sequence; D041961:Deuterium Exchange Measurement; D004795:Enzyme Stability; D006358:Hot Temperature; D006860:Hydrogen Bonding; D008049:Lipase / Q000737:chemistry* / Q000235:genetics*; D019906:Nuclear Magnetic Resonance, Biomolecular; D017354:Point Mutation; D011487:Protein Conformation; D011489:Protein Denaturation / Q000187:drug effects; D017433:Protein Structure, Secondary / Q000187:drug effects; D044203:Pseudomonas mendocina / Q000201:enzymology*; D014508:Urea / Q000494:pharmacology
D014508:Urea; D008049:Lipase