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.
Highlights
Despite recent dramatic advances in membrane protein crystallography and other structural techniques, development of systems in which dynamic protein-protein interactions can be detected and studied is still warranted
Using 25 mM sodium dodecyl sulphate (SDS) in DBS as a solvent, but solubilizing by the thin film method, again resulted in a predominantly aggregated peptide, with a major infrared amide I absorbance at 1632 cm21, and a secondary component arising from a-helix at 1654 cm21 (Fig. 1E)
Reconstitution by thin film method in 50 mg/ml LPC gave a predominant infrared amide I absorbance at 1654 cm21, attributable to a-helical structures, and a secondary sharp peak at 1630 cm21 arising from aggregated structures (Fig. 1F)
Summary
Despite recent dramatic advances in membrane protein crystallography and other structural techniques, development of systems in which dynamic protein-protein interactions can be detected and studied is still warranted. While Fourier transform infrared spectroscopy (FTIR) is a relatively low resolution technique in terms of structural information compared to e.g., X-ray crystallography, FTIR can detect changes in protein conformation, or interaction via novel hydrogen-bonding, via spectral shifts in the amide I region [1,2]. Some Kv channels include a cytoplasmic inactivation domain (ID) that facilitates rapid ‘‘N-type’’ channel inactivation following voltage-dependent activation of the channel. The ID is a cytoplasmic tethered ‘‘ball’’ that can bind to the intracellular S4–S5 linker region following depolarization-initiated activation of
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