A technique of neutron in-plane scattering for studying the structures of peptide pores in membranes is described. Alamethicin in the inserted state was prepared and undeuterated and deuterated dilauroyl phosphatidylcholine (DLPC) hydrated with D2O or H2O. Neutron in-plane scattering showed a strong dependence on deuteration, clearly indicating that water is a part of the high-order structure of inserted alamethicin. The data are consistent with the simple barrel-stave model originally proposed by Baumann and Mueller. The theoretical curves computed with this model at four different deuteration conditions agree with the data in all cases. Both the diameter of the water pore and the effective outside diameter of the channel are determined accurately. Alamethicin forms pores in a narrow range of size. In a given sample condition, > 70% of the peptide forms pores of n and n +/- 1 monomers. The pore size varies with hydration and with lipid. In DLPC, the pores are made of n = 8-9 monomers, with a water pore approximately 18 A in diameter and with an effective outside diameter of approximately 40 A. In diphytanoyl phosphatidylcholine, the pores are made of n approximately 11 monomers, with a water pore approximately 26 A in diameter, with an effective outside diameter of approximately 50 A.

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Despite the growing number of atomic-resolution membrane protein structures, direct structural information about proteins in their native membrane environment is scarce. This problem is particularly relevant in the case of the highly-charged S1–S4 voltage-sensing domains responsible for nerve impulses, where interactions with the lipid bilayer are critical for the function of voltage-activated potassium channels. Here we use neutron diffraction, solid-state nuclear magnetic resonance spectroscopy, and molecular dynamics simulations to investigate the structure and hydration of bilayer membranes containing S1–S4 voltage-sensing domains. Our results show that voltage sensors adopt transmembrane orientations, cause a modest reshaping of the surrounding lipid bilayer, and that water molecules intimately interact with the protein within the membrane. These structural findings reveal that voltage sensors have evolved to interact with the lipid membrane while keeping the energetic and structural perturbations to a minimum, and that water penetrates into the membrane to hydrate charged residues and shape the transmembrane electric field.