a b s t r a c tPosttranslational modifications play a crucial role in modulating protein structure and function. Genetic incorporation of unnatural amino acids into a specific site of a protein facilitates the systematic study of protein modifications including acetylation. We here report the directed evolution of pyrrolysyl-tRNA synthetase (PylRS) from Methanosarcina mazei to create N-acetyl lysyl-tRNA synthetases (AcKRSs) using a new selection system based on the killing activity of the toxic ccdB gene product. The amino acid specificity of these and of published [1,2] AckRSs was tested in vitro and in vivo, and the enzyme-kinetic properties of the AckRSs were evaluated for the first time.

Analysis of protein dynamics using single-molecule fluorescence resonance energy transfer (smFRET) is widely used to understand the structure and function of proteins. Nonetheless, site-specific labeling of proteins with a pair of donor and acceptor dyes still remains a challenge. Here we present a general and facile method for site-specific dual labeling of proteins by incorporating two different, readily available, unnatural amino acids (p-acetylphenylalanine and alkynyllysine) for smFRET. We used newly evolved alkynyllysine-specific aminoacyl-tRNA synthetase/tRNA(UCA) and p-acetylphenylalanyl-tRNA synthetase/tRNA(CUA). The utility of our approach was demonstrated by analyzing the conformational change of dual-labeled calmodulin using smFRET measurements. The present labeling approach is devoid of major limitations in conventional cysteine-based labeling. Therefore, our method will significantly increase the repertoire of proteins available for FRET study and expand our ability to explore more complicated molecular dynamics.

Einbau von Phosphoserin: Ein allgemeiner Weg zu rekombinanten Histonen mit ortsspezifischer Serin‐Phosphorylierung nutzt das Engineering einer Phosphoseryl‐tRNA‐Synthetase (SepRS) und eines Elongationsfaktors Tu (EF‐Tu; siehe Bild). Serin‐phosphorylierte Nucleosome sind ein direkter Beweis für die gegenseitige Beeinflussung von Phosphorylierung und Acetylierung in Histonen.

No abstract

Einen selektiven Phosphoserin‐Einbau beschreiben H.‐S. Park et al. in ihrer Zuschrift Eine allgemeine Strategie für den Aufbau rekombinanter Histone mit ortsspezifischer Serinphosphorylierung wurde entwickelt, die auf der Modifizierung einer Phosphoseryl‐tRNA‐Synthetase (SepRS) und des Elongationsfaktors Tu (EF‐Tu) beruht. Die Methode dürfte die Erforschung der Histonphosphorylierung und kreuzregulatorischer Mechanismen vereinfachen.