Facile tethering of stable and unstable proteins for optical tweezers experiments.

ABSTRACT: Single-molecule force spectroscopy with optical tweezers has emerged as a powerful tool for dissecting protein folding. The requirement to stably attach "molecular handles" to specific points in the protein of interest by preparative biochemical techniques is a limiting factor in applying this methodology, especially for large or unstable proteins that are difficult to produce and isolate. Here, we present a streamlined approach for creating stable and specific attachments using autocatalytic covalent tethering. The high specificity of coupling allowed us to tether ribosome-nascent chain complexes, demonstrating its suitability for investigating complex macromolecular assemblies. We combined this approach with cell-free protein synthesis, providing a facile means of preparing samples for s

SUBMITTER: Maciuba K 

PROVIDER: S-EPMC8390856 | biostudies-literature | 2021 Jul

REPOSITORIES: biostudies-literature