Secondary structure analysis of the putative membrane-associated domains of the inward rectifier K+ channel ROMK1.
The inward rectifier K+ channels contain two putative membrane-spanning domains per subunit (M1, M2) and a 'pore' (P) region, which is similar to the H5 domain of voltage-gated K+ channels. Here we have used Fourier transform infrared (FTIR) and CD spectroscopy to analyse the secondary structures of synthetic peptides corresponding to the M1, M2 and P regions of ROMK1 in aqueous solution, in organic solvents and in phospholipid membranes. A previous CD study was unable to provide any structural data on a similar P peptide [Ben-Efraim and Shai (1997) Biophys. J. 72, 85-96]. However, our FTIR and CD spectroscopic analyses indicate that this peptide adopts an alpha-helical structure when reconstituted into dimyristoyl phosphatidylcholine vesicles and lysophosphatidyl choline (LPC) micelles as well as in trifluoroethanol (TFE) solvent. This result is in good agreement with a previous study on a peptide corresponding to the pore domain of a voltage-gated K+ channel [Haris, Ramesh, Sansom, Kerr, Srai and Chapman (1994) Protein Eng. 7, 255-262]. FTIR spectra of the M1 peptide in LPC micelles displayed a strong absorbance characteristic of an intermolecular beta-sheet structure, suggesting aggregation of the M1 peptide. Sucrose gradient centrifugation was used to separate aggregated peptide from peptide incorporated into micelles in an unaggregated manner; subsequent analysis by FTIR suggested that the M1 peptide adopted an alpha-helical structure when incorporated into phospholipid membranes. FTIR and CD spectra of the M2 peptide in phospholipids and high concentrations of TFE suggest that this peptide adopts an alpha-helical structure. The structural data obtained in these experiments have been used to propose a model for the structure of the membrane-associated core (M1-P-M2) of the inward rectifier K+ channel protein.
Brazier
S P
SP
Department of Biochemistry and Molecular Biology, Royal Free Hospital School of Medicine, Rowland Hill Street, Hampstead, London NW3 2PF, UK.
Ramesh
B
B
Haris
P I
PI
Lee
D C
DC
Srai
S K
SK
D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000378:metabolism; D002942:Circular Dichroism; D004134:Dimyristoylphosphatidylcholine / Q000737:chemistry; D008051:Lipid Bilayers; D008244:Lysophosphatidylcholines / Q000737:chemistry; D008969:Molecular Sequence Data; D015221:Potassium Channels / Q000737:chemistry* / Q000378:metabolism*; D024661:Potassium Channels, Inwardly Rectifying; D017433:Protein Structure, Secondary; D017550:Spectroscopy, Fourier Transform Infrared; D014270:Trifluoroethanol / Q000737:chemistry; D014867:Water
D008051:Lipid Bilayers; D008244:Lysophosphatidylcholines; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D014867:Water; D014270:Trifluoroethanol; D004134:Dimyristoylphosphatidylcholine