Novel in‐vitro tag‐and‐modify protein sample generation methods for multiplexed singlemolecule FRET screening

Abstract

Biomolecular systems exhibit many dynamic and biologically relevant properties, such as conformational fluctuations, multistep catalysis, transient interactions, folding, and allosteric structural transitions. These properties are challenging to detect and engineer using standard ensemble-based techniques. To address this drawback, single-molecule methods offer a way to access conformational distributions, transient states, and asynchronous dynamics inaccessible to these standard techniques. Fluorescence-based single molecule approaches are parallelizable and compatible with multiplexed detection; to date, however, they have remained limited to serial screens of small protein libraries. This stems from the current absence of methods for generating either individual dual-labeled protein samples at high throughputs or protein libraries compatible with multiplexed screening platforms. Here, we extend our recent work demonstrating that combinations of purified and reconstituted in-vitro translation, quantitative unnatural amino acid incorporation, and copper-catalyzed azide-alkyne cycloaddition can overcome these challenges. In particular, we demonstrate the ability to quantitatively label azide or alkyne-tagged proteins and ribosome-bound nascent polypeptide chains (RNCs) with pairs of dyes with greater flexibility and more specificity than was previously possible. This sample generation approach can provide protein samples suitable for highly-multiplexed single-molecule FRET-based conformational phenotyping. Importantly, dual-labeled RNC libraries enable single-molecule co-localization of genotypes with phenotypes, are well suited for multiplexed single-molecule screening of protein libraries, and can enable the in-vitro directed evolution of proteins with designer single-molecule conformational phenotypes of interest. Support or Funding Information California State University Program for Education and Research in Biotechnology New Investigator Award (Kambiz M. Hamadani) Ribosome-bound nascent polypeptide chains labeled with pairs of dyes for multiplexed single molecule FRET-based screening This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.