Slow α Helix Formation during Folding of a Membrane Protein

Abstract

Very little is known about the folding of proteins within biological membranes. A "two-stage" model has been proposed on thermodynamic grounds for the folding of alpha helical, integral membrane proteins, the first stage of which involves formation of transmembrane alpha helices that are proposed to behave as autonomous folding domains. Here, we investigate alpha helix formation in bacteriorhodopsin and present a time-resolved circular dichroism study of the slow in vitro folding of this protein. We show that, although some of the protein's alpha helices form early, a significant part of the protein's secondary structure appears to form late in the folding process. Over 30 amino acids, equivalent to at least one of bacteriorhodopsin's seven transmembrane segments, slowly fold from disordered structures to alpha helices with an apparent rate constant of about 0.012 s-1 at pH 6 or 0.0077 s-1 at pH 8. This is a rate-limiting step in protein folding, which is dependent on the pH and the composition of the lipid bilayer.

Site-directed mutagenesis combined with oxidative methionine labeling for probing structural transitions of a membrane protein by mass spectrometry

“…This core encompasses the regions surrounding the naturally occurring methionines of helices B, E, and G, as well as parts of C and F that were probed here through methionine substitutions (M93 and M179). An earlier circular dichroism study found a residual helicity of 42% for SDS-denatured BR, down from a native state value of 74% [72]. The results presented here are consistent with a scenario where this residual helicity is caused by partially intact helices B, C, E, F, and G. However, HDX measurements suggest that these helices are significantly more dynamic than in native BR.…”

Shifting hydrogen bonds may produce flexible transmembrane helices

“…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).…”

“…They create weak points for movement during catalytic cycles (1, 2); they enable the precise positioning of key side chains (3); they can help recruit water to functional sites (4); and they can prevent off-pathway folding (5). It is nevertheless surprising that distortions are much more common in transmembrane than soluble protein helices (6,7), because helices are more stable in the apolar membrane environment (8)(9)(10)(11) where backbone hydrogen bonds are stronger (12,13). It has therefore remained mysterious how distortions can possibly be generated by the evolutionary currency of random point mutations.…”

Stable folding core in the folding transition state of an α-helical integral membrane protein

“…The loss of the retinal chromophore, which has a characteristic absorbance band at 560 nm, was used as a folding probe. Circular dichroism measurements reveal that changes in retinal absorbance during SDS-unfolding are equivalent to a moderate reduction in secondary structure content from 74% to 42% associated with unfolding (8,32,33). The SDSunfolded state of bR thus retains a considerable amount of secondary structure, although some tertiary structure is lost (34,35); it might best be compared to a molten globule or late folding intermediate for an aqueous protein.…”