Site-directed spin labelling of proteins by Suzuki-Miyaura coupling via a genetically encoded aryliodide amino acid.

We report site-directed protein spin labelling via Suzuki-Miyaura coupling of a nitroxide boronic acid label with the genetically encoded amino acid 4-iodo-l-phenylalanine. The resulting spin label bears a rigid biphenyl linkage with lower flexibility than spin label R1. It is suitable to obtain defined electron paramagnetic resonance distance distributions and to report protein-membrane interactions and conformational transitions of α-synuclein.

Evolved sequence contexts for highly efficient amber suppression with noncanonical amino acids.

The expansion of the genetic code with noncanonical amino acids (ncAA) enables the function of proteins to be tailored with high molecular precision. In this approach, the ncAA is charged to an orthogonal nonsense suppressor tRNA by an aminoacyl-tRNA-synthetase (aaRS) and incorporated into the target protein in vivo by suppression of nonsense codons in the mRNA during ribosomal translation. Compared to sense codon translation, this process occurs with reduced efficiency. However, it is still poorly understood, how the local sequence context of the nonsense codon affects suppression efficiency. Here, we report sequence contexts for highly efficient suppression of the widely used amber codon in E. coli for the orthogonal Methanocaldococcus jannaschii tRNA(Tyr)/TyrRS and Methanosarcina mazei tRNA(Pyl)/PylRS pairs. In vivo selections of sequence context libraries consisting of each two random codons directly up- and downstream of an amber codon afforded contexts with strong preferences for particular mRNA nucleotides and/or amino acids that markedly differed from preferences of contexts obtained in control selections with sense codons. The contexts provided high amber suppression efficiencies with little ncAA-dependence that were transferrable between proteins and resulted in protein expression levels of 70-110% compared to levels of control proteins without amber codon. These sequence contexts represent stable tags for robust and highly efficient incorporation of ncAA into proteins in standard E. coli strains and provide general design rules for the engineering of amber codons into target genes.