Multiplexed genomic encoding of non-canonical amino acids for labeling large complexes.

Nature Chemical Biology
Bijoy J. Desai, Ruben L. Gonzalez

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.

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Citations

Jan 14, 2021·Nature Communications·Howard GamperYa-Ming Hou
Mar 24, 2021·Journal of the American Chemical Society·Miglena ManandharFloyd E Romesberg

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Methods Mentioned

BETA
X-ray
electron microscopy
FRET
in vitro transcription
size-exclusion chromatography

Software Mentioned

PyMOL
PyMOL Molecular Graphics System
μManager

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