Multiplexed, bioorthogonal labeling of multicomponent, biomolecular complexes using genomically encoded, non-canonical amino acids
| Autor: | Desai, Bijoy J., Gonzalez Jr., Ruben L. |
|---|---|
| Rok vydání: | 2019 |
| Předmět: |
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 |
| DOI: | 10.1101/730465 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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