Work

Comprehensive mutational scanning of a kinase in vivo reveals substrate-dependent fitness landscapes

Year: 2014

Type: article

Abstract: Deep mutational scanning has emerged as a promising tool for mapping sequence-activity relationships in proteins, ribonucleic acid and deoxyribonucleic acid. In this approach, diverse variants of a sequence of interest are first ranked according to their activities in a relevant assay, and this ranking is then used to infer the shape of the fitness landscape around the wild-type sequence. Little is currently known, however, about the degree to which such fitness landscapes are dependent on the specific assay conditions from which they are inferred. To explore this issue, we performed comprehensive single-substitution mutational scanning of APH(3')II, a Tn5 transposon-derived kinase that confers resistance to aminoglycoside antibiotics, in Escherichia coli under selection with each of six structurally diverse antibiotics at a range of inhibitory concentrations. We found that the resulting local fitness landscapes showed significant dependence on both antibiotic structure and concentration, and that this dependence can be exploited to guide protein engineering. Specifically, we found that differential analysis of fitness landscapes allowed us to generate synthetic APH(3')II variants with orthogonal substrate specificities.

Source: Nucleic Acids Research

DOI: 10.1093/nar/gku511

Authors: Alexandre Melnikov, Peter Rogov, Li Wang, Andreas Gnirke, Tarjei S. Mikkelsen

Institution: Broad Institute

Language: English

Topic: Evolution and Genetic Dynamics

Subfield: Genetics

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: gold

Estimated APC paid: $3,992

Funder: Broad Institute