Peptides Modeled on the Transmembrane Region of the Slow Voltage-Gated IsK Potassium Channel: Structural Characterization of Peptide Assemblies in the β-Strand Conformation

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Abstract
A 27-residue peptide, having a sequence corresponding to the transmembrane domain of the IsK protein with slow voltage-gated potassium channel activity, has been incorporated into synthetic saturated-chain phospholipid membranes. The peptide−lipid complexes have been characterized by attenuated-total-reflection Fourier-transform-infrared spectroscopy (ATR-FTIR), spin-label electron spin resonance (ESR) spectroscopy, 31P and 2H nuclear magnetic resonance (NMR) spectroscopy, differential scanning calorimetry, and low-angle X-ray diffraction. From FTIR spectroscopy, it is found that, when reconstituted into membranes by dialysis from 2-chloroethanol, the peptide has a predominantly β-strand secondary structure in which the peptide backbone is oriented at an angle of approximately 56° relative to the membrane normal in dry films of phosphatidylcholines. Hydration of the dry film in the gel phase does not appear to affect the orientation of the peptide backbone, and a relatively small change in orientation occurs when the bilayer undergoes the transition to the fluid phase. The ESR and NMR spectra from spin-labeled and 2H-labeled phospholipids, respectively, indicate that the incorporated peptide restricts the rotational motion of the lipids, without appreciably affecting the chain order, in a way similar 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 peptide. The motional restriction of the chains of the spin-labeled phosphatidylcholine and the reduction in the enthalpy of the lipid chain-melting transition indicate that, on average, approximately two to three phospholipid molecules interact directly with each peptide monomer, which is consistent with a limited degree of aggregation of the β-sheet structures. Both 31P NMR spectroscopy and X-ray diffraction indicate that the lipid−peptide complexes have a lamellar structure up to the highest peptide concentration studied (RP = 0.2). The surface area occupied by lipid molecules (ca. 30 Å2 per chain) in the peptide complexes, deduced from the lamellar repeat spacings at defined water content, is very similar to that in pure fluid lipid bilayers, consistent with the 2H NMR results. The additional membrane surface area contributed by the peptide is approximately 112 Å2 per monomer. This large value for the peptide area in the fluid bilayer is consistent with the ATR studies of dry peptide/lipid films which suggest that the long axis of the β-strand is strongly tilted with respect to the bilayer normal (56° in the dry film).