Prediction of New Stabilizing Mutations Based on Mechanistic Insights from Markov State Models.

Autor:	Zimmerman MI; Department of Biochemistry & Molecular Biophysics, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, Missouri 63110, United States., Hart KM; Department of Biochemistry & Molecular Biophysics, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, Missouri 63110, United States., Sibbald CA; Department of Biochemistry & Molecular Biophysics, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, Missouri 63110, United States., Frederick TE; Department of Biochemistry & Molecular Biophysics, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, Missouri 63110, United States., Jimah JR; Department of Molecular Microbiology, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, Missouri 63110, United States., Knoverek CR; Department of Biochemistry & Molecular Biophysics, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, Missouri 63110, United States., Tolia NH; Department of Biochemistry & Molecular Biophysics, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, Missouri 63110, United States.; Department of Molecular Microbiology, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, Missouri 63110, United States., Bowman GR; Department of Biochemistry & Molecular Biophysics, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, Missouri 63110, United States.; Department of Biomedical Engineering and Center for Biological Systems Engineering, Washington University in St. Louis, One Brookings Drive, St. Louis, Missouri 63130, United States.
Jazyk:	angličtina
Zdroj:	ACS central science [ACS Cent Sci] 2017 Dec 27; Vol. 3 (12), pp. 1311-1321. Date of Electronic Publication: 2017 Nov 21.
DOI:	10.1021/acscentsci.7b00465
Abstrakt:	Protein stabilization is fundamental to enzyme function and evolution, yet understanding the determinants of a protein's stability remains a challenge. This is largely due to a shortage of atomically detailed models for the ensemble of relevant protein conformations and their relative populations. For example, the M182T substitution in TEM β-lactamase, an enzyme that confers antibiotic resistance to bacteria, is stabilizing but the precise mechanism remains unclear. Here, we employ Markov state models (MSMs) to uncover how M182T shifts the distribution of different structures that TEM adopts. We find that M182T stabilizes a helix that is a key component of a domain interface. We then predict the effects of other mutations, including a novel stabilizing mutation, and experimentally test our predictions using a combination of stability measurements, crystallography, NMR, and in vivo measurements of bacterial fitness. We expect our insights and methodology to provide a valuable foundation for protein design. Competing Interests: The authors declare no competing financial interest.
Databáze:	MEDLINE
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