Adaptation proceeds through the selection of mutations. The distribution of mutant fitness effect and the forces shaping this distribution are therefore keys to predict the evolutionary fate of organisms and their constituents such as enzymes. Here, by producing and sequencing a comprehensive collection of 10,000 mutants, we explore the mutational landscape of one enzyme involved in the spread of antibiotic resistance, the beta-lactamase TEM-1. We measured mutation impact on the enzyme activity through the estimation of amoxicillin minimum inhibitory concentration on a subset of 990 mutants carrying a unique missense mutation, representing 64% of possible amino acid changes in that protein reachable by point mutation. We established that mutation type, solvent accessibility of residues, and the predicted effect of mutations on protein stability primarily determined alone or in combination changes in minimum inhibitory concentration of mutants. Moreover, we were able to capture the drastic modification of the mutational landscape induced by a single stabilizing point mutation (M182T) by a simple model of protein stability. This work thereby provides an integrated framework to study mutation effects and a tool to understand/define better the epistatic interactions.

Significance: DNA can adopt diverse structural conformations including duplexes, triplexes, and quadruplexes. Four-stranded structures known as G-quadruplexes have been implicated in cellular processes and found potential applications in therapeutics and nanotechnology. G-quadruplex structures are highly polymorphic, but so far, only right-handed helical forms have been observed. Here we present the NMR solution and X-ray crystal structures of an unprecedented left-handed DNA G-quadruplex, containing structural features that can be exploited as unique recognition elements.

Incorporation of noncanonical amino acids into cellular proteins often requires engineering new aminoacyl-tRNA synthetase activity into the cell. A screening strategy that relies on cell-surface display of reactive amino acid side-chains was used to identify a diverse set of methionyl-tRNA synthetase (MetRS) mutants that allow efficient incorporation of the methionine (Met) analog azidonorleucine (Anl). We demonstrate that the extent of cell-surface labeling in vivo is a good indicator of the rate of Anl activation by the MetRS variant harbored by the cell. By screening at low Anl concentrations in Met-supplemented media, MetRS variants with improved activities toward Anl and better discrimination against Met were identified.

The crystal structure of Escherichia coli methionyl‐tRNA_f^Met transformylase complexed with formyl‐methionyl‐tRNA_f^Met was solved at 2.8 Å resolution. The formylation reaction catalyzed by this enzyme irreversibly commits methionyl‐tRNA_f^Met to initiation of translation in eubacteria. In the three‐dimensional model, the methionyl‐tRNA_f^Met formyltransferase fills in the inside of the L‐shaped tRNA molecule on the D‐stem side. The anticodon stem and loop are away from the protein. An enzyme loop is wedged in the major groove of the acceptor helix. As a result, the C1‐A72 mismatch characteristic of the initiator tRNA is split and the 3′ arm bends inside the active centre. This recognition mechanism is markedly distinct from that of elongation factor Tu, which binds the acceptor arm of aminoacylated elongator tRNAs on the T‐stem side.