Revealing enzyme functional architecture via high-throughput microfluidic enzyme kinetics-Reference-Cited by-同舟云学术

Revealing enzyme functional architecture via high-throughput microfluidic enzyme kinetics

Published:2021-07-23 Issue:6553 Volume:373 Page:
ISSN:0036-8075
Container-title:Science
language:en
Short-container-title:Science

Author:

Markin C. J.¹^ORCID,Mokhtari D. A.¹^ORCID,Sunden F.¹^ORCID,Appel M. J.¹^ORCID,Akiva E.²,Longwell S. A.³^ORCID,Sabatti C.⁴⁵^ORCID,Herschlag D.¹⁶⁷^ORCID,Fordyce P. M.³⁷⁸⁹^ORCID

Affiliation:

1. Department of Biochemistry, Stanford University, Stanford, CA 94305, USA.

2. Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, CA 94158, USA.

3. Department of Bioengineering, Stanford University, Stanford, CA 94305, USA.

4. Department of Biomedical Data Science, Stanford University, Stanford, CA 94305, USA.

5. Department of Statistics, Stanford University, Stanford, CA 94305, USA.

6. Department of Chemical Engineering, Stanford University, Stanford, CA 94305, USA.

7. ChEM-H Institute, Stanford University, Stanford, CA 94305, USA.

8. Department of Genetics, Stanford University, Stanford, CA 94305, USA.

9. Chan Zuckerberg Biohub; San Francisco, CA 94110, USA.

Abstract

Go big or you'll get lost Rational mutagenesis is a common approach to investigating or engineering enzyme function in vitro, but the ease with which one can manipulate protein sequences belies many pitfalls in connecting sparse activity data to an enzyme's true functional landscape. Using a high-throughput platform, Markin et al. expressed, purified, and performed an array of kinetic measurements on a target esterase, collecting data from >1000 mutations spanning the entire protein (see the Perspective by Baumer and Whitehead). Protein misfolding into an inactive state, rather than decreased equilibrium stability, was a crucial factor in negatively affected variants spread throughout the protein. When combined with prior mechanistic understanding and structures, four “functional components” help to rationalize the otherwise complex spatial pattern of effects of mutations on different aspects of enzyme function, all of which would be invisible from mutagenesis of just a few residues. Science , abf8761, this issue p. eabf8761 ; see also abj8346, p. 391

Funder

National Institutes of Health

Alfred P. Sloan Foundation

Gordon and Betty Moore Foundation

Chan Zuckerberg Initiative

National Energy Research Scientific Computing Center

Canadian Institutes of Health Research

Ono Pharma Foundation

Joint Initiative for Metrology in Biology

Stanford Bio-X Interdisciplinary Initiative

Stanford Interdisciplinary Graduate Fellowship

Stanford Medical Scientist Training Program