Stationary Phase EPR Spectroscopy for Monitoring Membrane Protein Refolding by Conformational Response
journal contributionposted on 26.11.2018, 00:00 by Aidin R. Balo, Jinyoung Lee, Oliver P. Ernst
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.
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screen refolding conditionsEPR spectraMonitoring Membrane Protein RefoldingStationary Phase EPR SpectroscopyG-protein-coupled receptor CXCR 1spin-labeled Haloarcula marismortui bacteriorhodopsin-IConformational Response Protein productionSeveral membrane proteinscoli inclusion bodiesNi-NTA agarose resin