Comprehensive mutational scanning of a kinase in vivo reveals substrate-dependent fitness landscapes
By A. Melnikov, P. Rogov, Li Wang, Andreas Gnirke, Tarjei S. Mikkelsen. 2014
185 citations recorded by the source corpus.
Deep mutational scanning has emerged as a promising tool for mapping sequence-activity relationships in proteins1–4, RNA5 and DNA6–8. In this approach, diverse variants of a sequence of interest are first ranked according to their activities in a relevant pooled assay, and this ranking is then used to infer the shape of the fitness landscape around the wild-type sequence. Little is currently know, 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 deep mutational scanning of APH(3’)II, a Tn5 transposon-derived kinase that confers resistance to aminoglycoside antibiotics9, in E. coli under selection with each of six structurally diverse antibiotics at a range of inhibitory concentrations. We found that the resulting fitness landscapes showed significant dependence on both antibiotic structure and concentration. This shows that the notion of essential amino acid residues is context-dependent, but also 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.
Subfields: Genetics, Molecular Biology
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Citations in the corpus
- A saturation mutagenesis approach to understanding PTEN lipid phosphatase activity and genotype-phenotypes relationships (2018, methodology): “We then introduced the yeast-preferred codon for each non-wild-type amino acid, stop codon, and single residue deletion at all PTEN codons en masse, utilizing a homologous recombination-based mutagenesis approach (Material and Methods, Figures 1B and S2A, Table S1).(32,33) To allow direct sequencing”
- Evolvability as a function of purifying selection in TEM-1 β-lactamase. (2015, unknown): “The tolerance of many proteins to mutations has been assessed in a number of important studies with high-throughput site-directed and random mutagenesis (Fowler et al., 2010; Guo et al., 2004; Huang et al., 1996; Jacquier et al., 2013; Loeb et al., 1989; McLaughlin et al., 2012; Melnikov et al., 201”