Secondary and Tertiary Structure of Bacteriorhodopsin in the SDS Denatured State

Abstract

We characterized the structure of partially unfolded bacteriorhodopsin in sodium dodecyl sulfate (SDS) micelles and compared it with its in vitro refolded structure after reconstitution with dimyristoylphosphatidylcholine/3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (DMPC/CHAPS). Intrahelical and interhelical distances were mapped in the protein using strategically located spin-label pairs at helical ends, assayed by pulsed electron paramagnetic resonance spectroscopy (double electron-electron spin resonance, DEER). We find that in SDS the intrahelical end-to-end distances exhibit broad distributions, suggesting a heterogeneous ensemble of conformations with differing secondary structures. Nevertheless, a majority of the denatured population retains end-to-end distances similar to those in the native state. In contrast, the observed greatly increased interhelical distances, in addition to their very broad distributions, suggest that in the SDS micelles very little of the native tertiary structure remains.

Shifting hydrogen bonds may produce flexible transmembrane helices

“…In the case of Helix B specifically, where P50 resides, circular dichroism spectra of a B helix peptide indicates that roughly 19 of the residues remain helical in SDS, whereas a nuclear magnetic resonance structure of a fragment of bR from residues 1-71 finds that the region from 39-62 within helix B remains helical (23). Thus, the current evidence supports a model in which the B helix loses tertiary contacts and frays at the ends in SDS (22), but the hydrophobic center of the helix surrounding P50 maintains its helical character.…”

“…In the case of unfolding of bacteriorhodopsin in micelles, approximately 65% of the helix structure remains intact (11,21), and recent distance measurements by electron paramagnetic resonance throughout the protein are consistent with an unfolded state in sodium dodecyl sulfate (SDS) consisting of mostly helical structure with frayed ends (22). In the case of Helix B specifically, where P50 resides, circular dichroism spectra of a B helix peptide indicates that roughly 19 of the residues remain helical in SDS, whereas a nuclear magnetic resonance structure of a fragment of bR from residues 1-71 finds that the region from 39-62 within helix B remains helical (23).…”

Mapping protein conformational heterogeneity under pressure with site-directed spin labeling and double electron–electron resonance

“…The evidence reported to date indicates that the effects of flash-freezing in liquid nitrogen on R1 rotameric equilibria are minimal (32) and that conformational equilibria are effectively trapped (25,(34)(35)(36)(37)(38)(39)(40)(41). To compare the efficacy of rapid freezing in dry ice/ethanol with that in liquid nitrogen, we directly compared DEER distance distributions at atmospheric pressure for each doubly-labeled mutant of apoMb and holoMb using the two freezing methods (Fig.…”

“…However, the large temperature coefficient for the viscosity of glycerol-water mixtures (42) and, in some cases, large enthalpies of activation likely slow the exchange rates sufficiently during cooling to result in kinetic entrapment. This kinetic entrapment is a basic assumption in all published DEER experiments, and considerable empirical evidence is available to justify the assumption (25,(34)(35)(36)(37)(38)(39)(40)(41). In addition, distance distributions between spin pairs in T4 lysozyme were found to be only weakly dependent on cooling rates (32).…”

Conformational dynamics and distribution of nitroxide spin labels

“…Within the past decade, distance distribution measurements for pairs of spin labels with pulsed dipolar spectroscopy methods have been performed for a number of proteins with known structures [60,100,[128][129][130][131][132][133][134][135][136][137][138][139][140]. From these studies, a few were selected where mean distances were given and where the duration of the dipolar evolution was sufficient [21] for these mean distances to be reliable.…”