Trade-offs between enzyme fitness and solubility illuminated by deep mutational scanning

Justin R. Klesmith, John Paul Bacik, Emily E. Wrenbeck, Ryszard Michalczyk, Timothy A. Whitehead

Research output: Contribution to journalArticlepeer-review

48 Scopus citations

Abstract

Proteins are marginally stable, and an understanding of the sequence determinants for improved protein solubility is highly desired. For enzymes, it is well known that many mutations that increase protein solubility decrease catalytic activity. These competing effects frustrate efforts to design and engineer stable, active enzymes without laborious high-throughput activity screens. To address the trade-off between enzyme solubility and activity, we performed deep mutational scanning using two different screens/selections that purport to gauge protein solubility for two fulllength enzymes. We assayed a TEM-1 beta-lactamase variant and levoglucosan kinase (LGK) using yeast surface display (YSD) screening and a twin-arginine translocation pathway selection. We then compared these scans with published experimental fitness landscapes. Results from the YSD screen could explain 37% of the variance in the fitness landscapes for one enzyme. Five percent to 10% of all single missense mutations improve solubility, matching theoretical predictions of global protein stability. For a given solubilityenhancing mutation, the probability that it would retain wild-type fitness was correlated with evolutionary conservation and distance to active site, and anticorrelated with contact number. Hybrid classification models were developed that could predict solubilityenhancing mutations that maintain wild-type fitness with an accuracy of 90%. The downside of using such classification models is the removal of rare mutations that improve both fitness and solubility. To reveal the biophysical basis of enhanced protein solubility and function, we determined the crystallographic structure of one such LGK mutant. Beyond fundamental insights into trade-offs between stability and activity, these results have potential biotechnological applications.

Original languageEnglish (US)
Pages (from-to)2265-2270
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume114
Issue number9
DOIs
StatePublished - Feb 28 2017

Bibliographical note

Funding Information:
We thank B. Hackel for helpful comments, M. Ostermeier for helpful suggestions on an earlier version of this manuscript and the gift of the TEM-1 BLA mutagenic primer set, and S. Thorwall and V. Kelly for their help in the laboratory. Use of the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, is supported by the US Department of Energy (DOE), Office of Science, Office of Basic Energy Sciences under Contract DE-AC02-76SF00515. The Stanford Synchrotron Radiation Lightsource Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research and by the NIH, National Institute of General Medical Sciences (including Grant P41GM103393). This work was supported by US Department of Agriculture National Institute of Food and Agriculture (NIFA) Award 2016-67011-24701 (to J.R.K.) and US National Science Foundation Career Award 1254238 CBET (to T.A.W.). J.-P.B. was partially funded through the Protein Crystallography Station from the DOE Office of Biological and Environmental Research.

Keywords

  • Deep mutational scanning
  • Fitness landscapes
  • High-throughput screening
  • Protein solubility
  • Yeast surface display

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