PEPTIDES MODELED ON THE TRANSMEMBRANE REGION OF THE SLOW VOLTAGE-GATED ISK POTASSIUM CHANNEL - STRUCTURAL CHARACTERIZATION OF PEPTIDE ASSEMBLIES IN THE BETA-STRAND CONFORMATION

A 27-residue peptide, having a sequence corresponding to the transmemb rane domain of the IsK protein with slow voltage-gated potassium chann el activity, has been incorporated into synthetic saturated-chain phos pholipid membranes. The peptide-lipid complexes have been characterize d by attenuated-total-reflection Fourier-transform-infrared spectrosco py (ATR-FTIR), spin-label electron spin resonance (ESR) spectroscopy, P-31 and H-2 nuclear magnetic resonance (NMR) spectroscopy, differenti al scanning calorimetry, and low-angle X-ray diffraction. From FTIR sp ectroscopy, it is found that, when reconstituted into membranes by dia lysis from 2-chloroethanol, the peptide has a predominantly beta-stran d secondary structure in which the peptide backbone is oriented at an angle of approximately 56 degrees relative to the membrane normal in d ry films of phosphatidylcholines. Hydration of the dry film in the gel phase does not appear to affect the orientation of the peptide backbo ne, and a relatively small change in orientation occurs when the bilay er undergoes the transition to the fluid phase. The ESR and NMR spectr a from spin-labeled and H-2-labeled phospholipids, respectively, indic ate that the incorporated peptide restricts the rotational motion of t he lipids, without appreciably affecting the chain order, in a way sim ilar to that found for integral membrane proteins. The characteristic two-component ESR spectra from spin-labeled lipids further indicate a selectivity in the interaction of anionic phospholipids with the pepti de. The motional restriction of the chains of the spin-labeled phospha tidylcholine and the reduction in the enthalpy of the lipid chain-melt ing transition indicate that, on average, approximately two to three p hospholipid molecules interact directly with each peptide monomer. whi ch is consistent with a limited degree of aggregation of the beta-shee t structures. Both P-31 NMR spectroscopy and X-ray diffraction indicat e that the lipid-peptide complexes have a lamellar structure up to the highest peptide concentration studied (R(p) = 0.2). The surface area occupied by lipid molecules (ca. 30 Angstrom(2) per chain) in the pept ide complexes, deduced from the lamellar repeat spacings at defined wa ter content, is very similar to that in pure fluid lipid bilayers, con sistent with the H-2 NMR results. The additional membrane surface area contributed by the peptide is approximately 112 Angstrom(2) per monom er. This large value for the peptide area in the fluid bilayer is cons istent with the ATR studies of dry peptide/lipid films which suggest t hat the long axis of the beta-strand is strongly tilted with respect t o the bilayer normal (56 degrees in the dry film).