title: Interaction between soluble and membrane-embedded potassium channel peptides monitored by Fourier transform infrared spectroscopy.
abstract: Recent studies have explored the utility of Fourier transform infrared spectroscopy (FTIR) in dynamic monitoring of soluble protein-protein interactions. Here, we investigated the applicability of FTIR to detect interaction between synthetic soluble and phospholipid-embedded peptides corresponding to, respectively, a voltage-gated potassium (Kv) channel inactivation domain (ID) and S4-S6 of the Shaker Kv channel (KV1; including the S4-S5 linker "pre-inactivation" ID binding site). KV1 was predominantly α-helical at 30°C when incorporated into dimyristoyl-l-α-phosphatidylcholine (DMPC) bilayers. Cooling to induce a shift in DMPC from liquid crystalline to gel phase reversibly decreased KV1 helicity, and was previously shown to partially extrude a synthetic S4 peptide. While no interaction was detected in liquid crystalline DMPC, upon cooling to induce the DMPC gel phase a reversible amide I peak (1633 cm(-1)) consistent with novel hydrogen bond formation was detected. This spectral shift was not observed for KV1 in the absence of ID (or vice versa), nor when the non-inactivating mutant V7E ID was applied to KV1 under similar conditions. Alteration of salt or redox conditions affected KV1-ID hydrogen bonding in a manner suggesting electrostatic KV1-ID interaction favored by a hairpin conformation for the ID and requiring extrusion of one or more KV1 domains from DMPC, consistent with ID binding to S4-S5. These findings support the utility of FTIR in detecting reversible interactions between soluble and membrane-embedded proteins, with lipid state-sensitivity of the conformation of the latter facilitating control of the interaction.
authors.affiliation: Department of Pharmacology, School of Medicine, University of California Irvine, Irvine, California, United States of America. abbottg@uci.edu
authors.forename: Geoffrey W
authors.initials: GW
authors.lastname: Abbott
authors.forename: Bala
authors.initials: B
authors.lastname: Ramesh
authors.forename: Surjit K
authors.initials: SK
authors.lastname: Srai
mesh_terms: D004134:Dimyristoylphosphatidylcholine / Q000737:chemistry; D006860:Hydrogen Bonding; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D060066:Protein Interaction Maps; D017434:Protein Structure, Tertiary; D051637:Shaker Superfamily of Potassium Channels / Q000737:chemistry / Q000378:metabolism; D051670:Shaw Potassium Channels / Q000737:chemistry / Q000378:metabolism; D012995:Solubility; D017550:Spectroscopy, Fourier Transform Infrared
chemical_list: C492015:KCNC4 protein, human; D008051:Lipid Bilayers; D008565:Membrane Proteins; D010455:Peptides; D051637:Shaker Superfamily of Potassium Channels; D051670:Shaw Potassium Channels; D004134:Dimyristoylphosphatidylcholine