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Multiplexed, bioorthogonal labeling of multicomponent, biomolecular complexes using genomically encoded, non-canonical amino acids

Authors :
Desai, Bijoy J.
Gonzalez Jr., Ruben L.
Publication Year :
2019
Publisher :
Cold Spring Harbor Laboratory, 2019.

Abstract

Stunning advances in the structural biology of multicomponent biomolecular complexes (MBCs) have ushered in an era of intense, structure-guided mechanistic and functional studies of these complexes. Nonetheless, existing methods to site-specifically conjugate MBCs with biochemical and biophysical labels are notoriously impracticable and/or significantly perturb MBC assembly and function. To overcome these limitations, we have developed a general, multiplexed method in which we genomically encode non-canonical amino acids (ncAAs) into multiple, structure-informed, individual sites within a target MBC; select for ncAA-containing MBC variants that assemble and function like the wildtype MBC; and site-specifically conjugate biochemical or biophysical labels to these ncAAs. As a proof-of-principle, we have used this method to generate unique single-molecule fluorescence resonance energy transfer (smFRET) signals reporting on ribosome structural dynamics that have thus far remained inaccessible to smFRET studies of translation.

Details

Database :
OpenAIRE
Accession number :
edsair.doi.dedup.....296ea06d236c365cc6d6dde7fbeb0f79
Full Text :
https://doi.org/10.1101/730465