SPONTANEOUS, PH-DEPENDENT MEMBRANE INSERTION OF A TRANSBILAYER ALPHA-HELIX

A question of fundamental importance concerning the biosynthesis of in tegral membrane proteins is whether transmembrane secondary structure can insert spontaneously into a lipid bilayer. It has proven to be dif ficult to address this issue experimentally because of the poor solubi lity in aqueous solution of peptides and proteins containing these ext remely hydrophobic sequences. We have identified a system in which the kinetics and thermodynamics of alpha-helix insertion into lipid bilay ers can be studied systematically and quantitatively using simple spec troscopic assays. Specifically, we have discovered that a 36-residue p olypeptide containing the sequence of the C-helix of the integral memb rane protein bacteriorhodopsin exhibits significant solubility in aque ous buffers free of both detergents and denaturants. This helix contai ns two aspartic acid residues in the membrane-spanning region. At neut ral pH, the peptide associates with lipid bilayers in a nonhelical and presumably peripheral conformation. With a pK(a) of 6.0, the peptide inserts into the bilayer as a transbilayer alpha-helix. The insertion reaction proceeds rapidly at room temperature and is fully reversible.