Abstract
Gaining new structural information on proteins in their native cellular environments will shed light on many enzymatic reaction mechanisms and encourage the development of new therapeutic approaches. During the last decade, in cell electron paramagnetic resonance (EPR) spectroscopy experiments have provided high-resolution data on conformational changes of proteins within the cell. However, one of the major obstacles of EPR spectroscopy is the spin-labeling process, which until now was performed only outside the cellular environment (i.e., exogenously). The spin-labeled protein is then injected into the cell, which limits the protein size and the cellular system that can be used. Here, we describe a new spin-labeling approach that can be applied to over-expressed proteins in Escherichia coli (i.e., endogenously). This approach uses a Cu(II) ion bound to a ligand, which has high affinity to a dHis site in the protein of interest. The presence of a nearby 19F-phenylalanine residue can be exploited to verify that the Cu(II)-ligand indeed bound to the protein target. This new methodology allows for the study of any protein, regardless of size or the cellular system used.
Competing Interest Statement
The authors have declared no competing interest.
