Protein
production remains a major bottleneck in membrane protein
structural biology. In many cases, large-scale recombinant protein
expression is either unfeasible or impossible, driving structural
biologists to explore new production avenues. Several membrane proteins
have been successfully refolded from solubilized E. coli inclusion bodies. In recent years, a structure of the G-protein-coupled
receptor CXCR1 was obtained using refolded material from E.
coli inclusion bodies. However, aggregation during the refolding
process is a common difficulty, which is often addressed by immobilization
of the protein onto a solid support. Most spectroscopic methods are
incompatible with these light-scattering matrices, which renders automated
buffer exchange to screen refolding conditions impossible. This work
explores a potential approach to overcome this problem by utilizing
site-directed spin labeling and electron paramagnetic resonance (EPR)
spectroscopy of protein bound to standard, commercially available
Ni-NTA agarose resin. With this approach, the correct protein fold
is determined by activity, which is inferred from a protein conformational
response to a known stimulant. EPR spectra at each state of the refolding
workflow of spin-labeled Haloarcula marismortui bacteriorhodopsin-I
(HmbRI) are obtained, and refolded fractions of HmbRI with this platform
are quantitated using both protein from inclusion bodies and denatured
recombinant protein from E. coli membranes. The stimulant
used for HmbRI is visible light. The solid support allows for multiple
refolding trials through buffer exchanges, and the EPR spectra are
collected on the order of seconds under ambient conditions.