Partially unfolded membrane protein has a compact conformation

Abstract

The 7-transmembrane helix protein bacteriorhodopsin is a good model for studies of membrane protein folding. The apoprotein, bacterio-opsin (BO), which lacks a retinal chromophore, folds into a nearly native conformation in lipid/bile salt mixed micelles from a partially unfolded state in dodecyl sulfate (SDS) micelles. Circular dichrosim studies showed around half of the native helix content for BO in SDS, but little else is known about this partially unfolded state. Using fluorescence resonance energy transfer (FRET), we now report that the distances from tryptophans 86 and 182 to a dansyl group at lysine 41 are about the same in SDS micelles as in lipid/bile salt mixed micelles. Thus, partially unfolded BO in SDS micelles has a compact structure. Because BO in SDS cannot form a native chromophore, we speculated that BO/SDS may have a more open structure, somewhat like the “molten globule” intermediate found in water-soluble proteins. We found that BO in lipid/bile salt mixed micelles shows slow deuterium/hydrogen (D/H) exchange, as measured by changes in tryptophan fluorescence (first-order rate constant = 0.0006 sec−1). By contrast, BO/SDS undergoes fast D/H exchange, indicating highly accessible tryptophans. The observed slow D/H exchange does not represent a kinetic isotope effect on folding: the rate of chromophore formation in D2O has a kinetic isotope effect of about 2, much less than would be observed if the slow D/H exchange corresponds to slow folding. (supported by NIH GM 08194)