Expression and characterization of the FXYD ion transport regulators for NMR structural studies in lipid micelles and lipid bilayers

• Published in: Biochimica et biophysica acta 2003-01, Vol.1645 (1), p.15-21
• Main Authors: Crowell, Kevin J, Franzin, Carla M, Koltay, Anita, Lee, Sangmin, Lucchese, Anna Maria, Snyder, Bradley C, Marassi, Francesca M
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; CHIF; Circular Dichroism; Cloning, Molecular; DNA, Complementary - biosynthesis; DNA, Complementary - chemistry; Electrophoresis, Polyacrylamide Gel; Escherichia coli - metabolism; FXYD; Intracellular Signaling Peptides and Proteins; Lipid Bilayers - chemistry; Lipids - chemistry; Magnetic Resonance Spectroscopy; Mat8; Membrane protein; Membrane Proteins - biosynthesis; Membrane Proteins - chemistry; Membrane Proteins - genetics; Micelles; Molecular Sequence Data; Neoplasm Proteins - biosynthesis; Neoplasm Proteins - chemistry; Neoplasm Proteins - genetics; NMR; PLM; Potassium Channels - biosynthesis; Potassium Channels - chemistry; Potassium Channels - genetics; Protein Conformation; Sequence Alignment; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
• ISSN: 1570-9639; 0006-3002; 1878-1454
• DOI: 10.1016/S1570-9639(02)00473-9

The proteins PLM (phospholemman), CHIF (channel inducing factor), and Mat8 (mammary tumor protein 8 kDa) are members of the FXYD family of ion transport regulatory membrane proteins. Here we describe their cloning and expression in Escherichia coli, and their purification for NMR structural studies in lipid micelles and lipid bilayers. The molecular masses of the purified recombinant FXYD proteins, determined from SDS-PAGE and from MALDI TOF mass spectrometry, reflect monomeric species. The solution NMR and CD spectra in SDS micelles show that they adopt helical conformations. The solid-state NMR spectra in lipid bilayers give the first view of their transmembrane architecture.