Transmembrane orientation of hydrophobic alpha-helices is regulated both by the relationship of helix length to bilayer thickness and by the cholesterol concentration.
A fluorescence-based approach to evaluate the regulation of transmembrane orientation of alpha-helices has been developed to examine the behavior of a membrane-inserted alpha-helical peptide with a 19 residue hydrophobic sequence. The emission lambdamax of a Trp residue in the helix was used to determine its location in the bilayer. To calibrate this method, Trp lambdamax and depth (determined by parallax analysis of fluorescence quenching) were measured for transmembrane peptides with Trp at different positions. Transmembrane orientation of the alpha-helix was found to be destabilized by differences between the width of the bilayer and the length of the hydrophobic sequence (i.e., hydrophobic mismatch). When bilayer width exceeded the length of the hydrophobic segment, mismatch induced formation of a nontransmembraneous orientation close to the polar/hydrocarbon interface. By manipulation of bilayer width in situ, it was found that the transmembrane and nontransmembrane orientations could interconvert. Cholesterol altered the transmembrane/nontransmembrane equilibrium to a degree consistent with its tendency to increase bilayer thickness. Evaluation of the energetics of transmembrane vs nontransmembrane insertion showed increased mismatch of a helix with bilayer width by the equivalent of just one hydrophobic residue can destabilize transmembrane orientation by roughly 0.5 kcal/mol. Inclusion of 30 mol % cholesterol in a bilayer can alter transmembrane insertion stability by 3-5 kcal/mol. Thus, physiologically relevant variations in both the hydrophobic helix length/membrane thickness ratio and the cholesterol levels influence transmembrane insertion significantly.
Ren
J
J
Department of Biochemistry and Cell Biology, State University of New York at Stony Brook, Stony Brook, New York 11794-5215, USA.
Lew
S
S
Wang
Z
Z
London
E
E
D000595:Amino Acid Sequence; D002784:Cholesterol / Q000737:chemistry*; D002942:Circular Dichroism; D008051:Lipid Bilayers / Q000737:chemistry*; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D008051:Lipid Bilayers; D002784:Cholesterol