Slow α Helix Formation during Folding of a Membrane Protein

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 α helical, integral membrane proteins, the first stage of which involves formation of transmembrane α helices that are proposed to behave as autonomous folding domains. Here, we investigate α 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 α 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 α 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.