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

5 years ago

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

Rodrigo A. Maillard, Clare Canavan, Yuxin Hao, Susan S. Taylor

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, proteins often display misfolded conformations. cAMP binding stabilizes the CBD-A against a denaturing force, and increases the folded state lifetime. Data obtained with handles of 370 and 70 base pairs are indistinguishable, but at low forces short handles provide a higher spatial resolution. Altogether, this method is flexible, selects for properly folded molecules in different liganded states, and can be readily applicable to study protein folding or protein-ligand interactions with force spectroscopy that require molecular handles.

Publisher URL: https://www.nature.com/articles/s41598-017-11214-z

DOI: 10.1038/s41598-017-11214-z