Integrated Method to Attach DNA Handles and Functionally Select Proteins to Study Folding and Protein-Ligand Interactions with Optical Tweezers.

ABSTRACT: Optical tweezers has emerged as a powerful tool to study folding, ligand binding, and motor enzymes. The manipulation of proteins with optical tweezers requires attaching molecular handles to the protein of interest. Here, we describe a novel method that integrates the covalent attachment of DNA handles to target proteins with a selection step for functional and properly folded molecules. In addition, this method enables obtaining protein molecules in different liganded states and can be used with handles of different lengths. We apply this method to study the cAMP binding domain A (CBD-A) of Protein kinase A. We find that the functional selection step drastically improves the reproducibility and homogeneity of the single molecule data. In contrast, without a functional selection step, pro

SUBMITTER: Hao Y 

PROVIDER: S-EPMC5589850 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature