Validation of In Vivo Protein Surface Accessibility Method

Abstract

Measuring changes in surface accessibility is a way to probe conformational changes of proteins. Traditionally, in vitro methods of resolving protein structures have relied on the isolation and purification of proteins, but this artificial protein environment may not reflect its functional native conformation. Therefore, this project seeks to develop a novel approach to identify and quantify changes in protein surface accessibility caused by protein misfolding or altered biomolecule interactions in vivo on a proteome‐wide scale. We want to find out if this approach, chemically labeling the ɛ‐amino group of lysine residues, will yield the same results in vitro as it did in vivo, thus validating the accuracy of results generated with this technique. This dimethylation method is expected to yield biologically relevant information as it allows for the screening of proteomes for structural perturbations as well as identification of novel structural information for proteins not yet crystallized.Using GAPDH as a control, we showed that the GAPDH tetramer stays intact upon dimethylation using native gel electrophoresis. Using data from crystal structures of GAPDH, the GAPDH tetramer contains one predicted inaccessible lysine residue and we were able to detect this inaccessible lysine residue with our in vitro dimethylation experiment. In addition, under time‐limited conditions, the dimethylation reaction does not go to completion and appears to preferentially occur at certain lysine sites. The extent of dimethylation does not scale with relative surface accessibility and rather requires a minimal accessibility to the ɛ‐amino group of lysine residues.The dimethylation labeling method is reproducible in vitro as well as in vivo. The GAPDH experiments show that the method chemically labels proteins that are still in their native conformation. Limiting conditions of the dimethylation reaction provides additional structural information of the protein, which allow us to validate predicted structural models and/or data. This technique can be utilized to screen the proteome for structural alterations, which is especially important in diseases or drug interactions.Support or Funding InformationNIH NCI Enabling Technologies Grant (IMAT) R33CA212973‐01 to John R. Yates IIIThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.