Kambiz M Hamadani
California State University, USA
Scientific Tracks Abstracts: Biochem Anal Biochem
�??Tag-and-modify�?� strategies to protein labeling often employ various combinations of unnatural amino acid incorporation and bio-orthogonal click chemistries to post-translationally attach fluorescent reporters, carbohydrates, or other functional groups onto purified target proteins. However, cell-based expression can limit the scope of such approaches. Purified and reconstituted in-vitro translation systems (prIVT) overcome many of these limitations at the expense of reduced product yield. Here we demonstrate that by combining prIVT technology, quantitative unnatural amino acid incorporation via sense codon reassignment, and either strain-promoted or copper-catalyzed azide-alkyne cycloaddition we can overcome the major hurdles which currently limit the scope and throughput of single molecule protein biophysics applications. Singlemolecule methods access biomolecular distributions, transient states, and asynchronous dynamics inaccessible to standard ensemble techniques. Although extremely powerful, the ability to screen large biomolecular libraries using fluorescence-based single-molecule detection platforms is still a challenge due to the lack of high-throughput methods for the generation and screening of large libraries of dye-labeled proteins. Here, we present a purificationfree and parallelizable in-vitro approach to generating dual-labeled proteins and ribosome-nascent-chain (RNC) libraries suitable for single-molecule FRET-based structural phenotyping. Importantly, dual-labeled RNC libraries enable single molecule co-localization of genotypes with phenotypes, and thus multiplexed single molecule screening of protein libraries. Such an approach to high-throughput posttranslational modification of large libraries of proteins may be useful for the in-vitro directed evolution of proteins with designer (single molecule) phenotypes.
Kambiz M Hamadani is primarily interested in studying protein folding on the ribosome using single molecule fluorescence methods. The methods he has developed to study this fundamental process have a wide range of applications in molecular evolution, structural biochemistry/biophysics, and glycobiology. He is very interested in collaborating with industry and academic partners that could possible make use of the labeling methods he’s developed for their own applications.