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Multiplexed, bioorthogonal labeling of multicomponent, biomolecular complexes using genomically encoded, non-canonical amino acids
- 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.
- Subjects :
- chemistry.chemical_classification
0303 health sciences
Translation (biology)
Computational biology
010402 general chemistry
01 natural sciences
Ribosome
0104 chemical sciences
Amino acid
03 medical and health sciences
Förster resonance energy transfer
Structural biology
chemistry
polycyclic compounds
Bioorthogonal chemistry
Function (biology)
030304 developmental biology
Conjugate
Subjects
Details
- Database :
- OpenAIRE
- Accession number :
- edsair.doi.dedup.....296ea06d236c365cc6d6dde7fbeb0f79
- Full Text :
- https://doi.org/10.1101/730465