Premium
Secondary structure, membrane localization, and coassembly within phospholipid membranes of synthetic segments derived from the N‐ and C‐termini regions of the ROMK1 K + channel
Author(s) -
BenEfraim Iris,
Shai Yechiel
Publication year - 1996
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560051115
Subject(s) - membrane , chemistry , phospholipid , förster resonance energy transfer , popc , transmembrane protein , biophysics , peptide , crystallography , quenching (fluorescence) , stereochemistry , fluorescence , biochemistry , biology , physics , receptor , quantum mechanics
The hydropathy plot of the inwardly rectifying ROMK1 K + channel, which reveals two transmembrane and a pore region domains, also reveals areas of intermediate hydrophobicity in the N terminus (M0) and in the C terminus (post‐M2). Peptides that correspond to M0, post‐M2, and a control peptide, pre‐M0, were synthesized and characterized for their structure, affinity to phospholipid membranes, organizational state in membranes, and ability to self‐assemble and coassemble in the membrane‐bound state. CD spectroscopy revealed that both M0 and post‐M2 adopt highly α‐helical structures in 1% SDS and 40% TFE/water, whereas pre‐M0 is not α‐helical in either 1% SDS or 40% TFE/water. Binding experiments with NBD‐labeled peptides demonstrated that both M0 and post‐M2, but not pre‐M0, bind to zwitterionic phospholipid membranes with partition coefficients of 10 3 –10 5 M −1 . A surface localization for both post‐M2 and M0 was indicated by NBD shift, tryptophan quenching experiments with brominated phospholipids, and enzymatic cleavage. Resonance energy transfer measurements between fluorescently labeled pairs of donor (NBD)/acceptor (rhodamine) peptides revealed that M0 and post‐M2 can coassemble in their membrane‐bound state, but cannot self‐associate when membrane‐bound. The results are in agreement with recent data indicating that amino acids in the carboxy terminus of inwardly rectifying K + channels have a major role in specifying the pore properties of the channels (Taglialatela M, Wible BA, Caporaso R, Brown AM, 1994, Science 264 :844–847; Pessia M, Bond CT, Kavanaugh MP, Adelman JP, 1995, Neuron 14 :1039–1045). The relevance of the results presented herein to the suggested model for the structure of the ROMK1 channel and to general aspects of molecular recognition between membrane‐bound polypeptides are also discussed.