期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2022
卷号：119
期号：29
DOI：10.1073/pnas.2201861119
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Significance
Systematic optimization of a pyrrolysyl–tRNA synthetase pair (tRNA: transfer ribonucleic acid) is shown to improve the incorporation of clickable amino acids into structured regions of proteins, in both bacterial and mammalian cells. The enhanced labeling of target proteins is demonstrated in fluorescence nanoscopy experiments of β-actin. MINFLUX measurements of filopodia at ∼2-nm three-dimensional precision contain segments with localization patterns that are consistent with a triangular filament bundling pattern, featuring interfilament separations close to 12 nm, that was previously only accessible via cryogenic imaging methods. This study highlights the potential of molecular-scale fluorescence nanoscopies when paired with minimally displacing labeling tags.
With few-nanometer resolution recently achieved by a new generation of fluorescence nanoscopes (MINFLUX and MINSTED), the size of the tags used to label proteins will increasingly limit the ability to dissect nanoscopic biological structures. Bioorthogonal (click) chemical groups are powerful tools for the specific detection of biomolecules. Through the introduction of an engineered aminoacyl–tRNA synthetase/tRNA pair (tRNA: transfer ribonucleic acid), genetic code expansion allows for the site-specific introduction of amino acids with “clickable” side chains into proteins of interest. Well-defined label positions and the subnanometer scale of the protein modification provide unique advantages over other labeling approaches for imaging at molecular-scale resolution. We report that, by pairing a new N-terminally optimized pyrrolysyl–tRNA synthetase (chPylRS
2020) with a previously engineered orthogonal tRNA, clickable amino acids are incorporated with improved efficiency into bacteria and into mammalian cells. The resulting enhanced genetic code expansion machinery was used to label β-actin in U2OS cell filopodia for MINFLUX imaging with minimal separation of fluorophores from the protein backbone. Selected data were found to be consistent with previously reported high-resolution information from cryoelectron tomography about the cross-sectional filament bundling architecture. Our study underscores the need for further improvements to the degree of labeling with minimal-offset methods in order to fully exploit molecular-scale optical three-dimensional resolution.
