A single-residue deletion alters the lipid selectivity of a K+ channel-associated peptide in the beta-conformation: spin label electron spin resonance studies

Abstract

Lipid-peptide interactions with the 27-residue peptide of sequence KLEALYILMVLGFFGFFTLGIMLSYIR reconstituted as beta-sheet assemblies in dimyristoylphosphatidylcholine bilayers have been studied by electron spin resonance (ESR) spectroscopy with spin-labeled lipids. The peptide corresponds to residues 42-68 of the IsK voltage-gated K+ channel protein and contains the single putative transmembrane span of this protein. Lipid-peptide interactions give rise to a second component in the ESR spectra of lipids spin-labeled on the 14C atom of the chain that corresponds to restriction of the lipid mobility by direct interaction with the peptide assemblies. From the dependence on the lipid/peptide ratio, the stoichiometry of lipid interaction is found to be about two phospholipids/peptide monomer. The sequence of selectivity for lipid association with the peptide assemblies is in the order phosphatidic acid > stearic acid = phosphatidylserine > phosphatidylglycerol = phosphatidylcholine. Comparison with previous data for a corresponding 26-residue mutant peptide with a single deletion of the apolar residue Leu2 (Horvath et al., 1995. Biochemistry 34:3893-3898), indicates a very similar mode of membrane incorporation for native and mutant peptides, but a strongly modified pattern and degree of specificity for the interaction with negatively charged lipids. The latter is interpreted in terms of the relative orientations of the charged amino acid side chains in the beta-sheet assemblies of the native and deletion-mutant peptides.

Reconstitution of KCNE1 into Lipid Bilayers: Comparing the Structural, Dynamic, and Activity Differences in Micelle and Vesicle Environments

“…Given that we have confirmed that KCNE1 is largely helical in the vesicles it is very unlikely that its secondary structure would be dramatically altered during the process of protein transfer from the vesicles to the oocyte membranes. This suggests that KCNQ1 associates with the mostly-helical form of KCNE1, not a beta sheet-dominated structure as was previously seen for a peptide corresponding to the TMD of KCNE1 following reconstitution of this peptide into vesicles by co-solubilizing the peptide with lipid in an organic solvent and then dialyzing out the solvent in an aqueous reservoir ( 35 ).…”

Analytical Approaches for Studying Transporters, Channels and Porins

“…SDSL experiments immediately provided links between open and closed states of the protein, by observing dynamic results of the transmembrane helices of the channel − as well as monitoring the limited reactivity of cystine centers buried within the channel region. , At the most ambitious, SDSL experiments can be used to refine crystal structures by observing small-molecule (i.e., oxygen and nickel ethylenediaminediacetic acid) collision rates at labeled sites within the protein . Beyond simply confirming or refining the crystallography results, and perhaps adding some detail on open and closed states, SDSL was used to observe interactions between the channel and solvent and lipid. − These results are significant in the study of ion channel diseases in part because of the indication that single-site mutations (a common genetic link to channelopathies) can alter the way that membrane-spanning segments interact with lipid bilayers . EPR has also provided a link between the voltage-sensing and pore domains of KvAP .…”

Association of Spin-Labeled Lipids with β-Barrel Proteins from the Outer Membrane of Escherichia coli

“…Lipid−protein interactions with a large range of integral proteins have been studied in this way, but so far these studies have been confined to α-helical proteins ( , ). The sole exceptions are the β-sheet configurations of certain transmembrane peptides ( − ). So far, no such studies have been undertaken with the β-barrel proteins that are characteristic for the outer membranes of Gram-negative bacteria and of mitochondria.…”