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


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 full-length 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 solubility-enhancing 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 solubility-enhancing 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.

Submission Details

ID: FpUWknYT

Submitter: Connie Wang

Submission Date: Oct. 26, 2018, 2:33 p.m.

Version: 1

Publication Details
Klesmith JR;Bacik JP;Wrenbeck EE;Michalczyk R;Whitehead TA,Proc Natl Acad Sci U S A (2017) Trade-offs between enzyme fitness and solubility illuminated by deep mutational scanning. PMID:28196882
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)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1AXB 1997-10-14T00:00:00+0000 2.0 TEM-1 BETA-LACTAMASE FROM ESCHERICHIA COLI INHIBITED WITH AN ACYLATION TRANSITION STATE ANALOG
1BT5 1998-09-02T00:00:00+0000 1.8 CRYSTAL STRUCTURE OF THE IMIPENEM INHIBITED TEM-1 BETA-LACTAMASE FROM ESCHERICHIA COLI
1BTL 1993-11-01T00:00:00+0000 1.8 CRYSTAL STRUCTURE OF ESCHERICHIA COLI TEM1 BETA-LACTAMASE AT 1.8 ANGSTROMS RESOLUTION
1CK3 1999-04-27T00:00:00+0000 2.28 N276D MUTANT OF ESCHERICHIA COLI TEM-1 BETA-LACTAMASE
1ERM 2000-04-06T00:00:00+0000 1.7 X-RAY CRYSTAL STRUCTURE OF TEM-1 BETA LACTAMASE IN COMPLEX WITH A DESIGNED BORONIC ACID INHIBITOR (1R)-1-ACETAMIDO-2-(3-CARBOXYPHENYL)ETHANE BORONIC ACID
1ERO 2000-04-06T00:00:00+0000 2.1 X-RAY CRYSTAL STRUCTURE OF TEM-1 BETA LACTAMASE IN COMPLEX WITH A DESIGNED BORONIC ACID INHIBITOR (1R)-2-PHENYLACETAMIDO-2-(3-CARBOXYPHENYL)ETHYL BORONIC ACID
1ERQ 2000-04-06T00:00:00+0000 1.9 X-RAY CRYSTAL STRUCTURE OF TEM-1 BETA LACTAMASE IN COMPLEX WITH A DESIGNED BORONIC ACID INHIBITOR (1R)-1-ACETAMIDO-2-(3-CARBOXY-2-HYDROXYPHENYL)ETHYL BORONIC ACID
1ESU 2000-04-11T00:00:00+0000 2.0 S235A MUTANT OF TEM1 BETA-LACTAMASE
1FQG 2000-09-05T00:00:00+0000 1.7 MOLECULAR STRUCTURE OF THE ACYL-ENZYME INTERMEDIATE IN TEM-1 BETA-LACTAMASE
1JTD 2001-08-20T00:00:00+0000 2.3 Crystal structure of beta-lactamase inhibitor protein-II in complex with TEM-1 beta-lactamase

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
99.7 Beta-lactamase Q48406 BLAT_KLEOX
100.0 Beta-lactamase P62594 BLAT_SALTI
100.0 Beta-lactamase P62593 BLAT_ECOLX