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.

多组分生物分子复合物 (MBC) 的结构生物学取得了惊人的进展，迎来了对这些复合物进行密集的、以结构为导向的机制和功能研究的时代。尽管如此，现有的将 MBC 与生化和生物物理标记进行位点特异性结合的方法是出了名的不切实际和/或显着扰乱 MBC 的组装和功能。为了克服这些限制，我们开发了一种通用的多路复用方法，在该方法中，我们将非规范氨基酸 (ncAAs) 基因组编码为目标 MBC 内的多个结构知情的单个位点；选择包含 ncAA 的 MBC 变体，这些变体的组装和功能类似于野生型 MBC；和特定站点将生化或生物物理标签与这些 ncAAs 结合。作为原理证明，