Spontaneous, pH-Dependent Membrane Insertion of a Transbilayer α-Helix

• Published in: Biochemistry (Easton) Vol. 36; no. 49; pp. 15177 - 15192
• Main Authors: Hunt, John F., Rath, Parshuram, Rothschild, Kenneth J., Engelman, Donald M.
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 09.12.1997
• Subjects: Hydrogen-Ion Concentration; Kinetics; Lipid Bilayers - chemistry; Membrane Proteins - chemistry; Peptide Fragments - chemistry; Phospholipids - chemistry; Solubility; Spectrometry, Fluorescence; Spectroscopy, Fourier Transform Infrared; Thermodynamics
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi970147b

A question of fundamental importance concerning the biosynthesis of integral membrane proteins is whether transmembrane secondary structure can insert spontaneously into a lipid bilayer. It has proven to be difficult to address this issue experimentally because of the poor solubility in aqueous solution of peptides and proteins containing these extremely hydrophobic sequences. We have identified a system in which the kinetics and thermodynamics of α-helix insertion into lipid bilayers can be studied systematically and quantitatively using simple spectroscopic assays. Specifically, we have discovered that a 36-residue polypeptide containing the sequence of the C-helix of the integral membrane protein bacteriorhodopsin exhibits significant solubility in aqueous buffers free of both detergents and denaturants. This helix contains two aspartic acid residues in the membrane-spanning region. At neutral 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 α-helix. The insertion reaction proceeds rapidly at room temperature and is fully reversible.