Downscaling Fourier transform infrared spectroscopy to the micrometer and nanogram scale: secondary structure of serotonin and acetylcholine receptors.
High signal-to-noise Fourier transform infrared (FTIR) spectra of the 5-hydroxytryptamine (serotonin) receptor (5-HT(3)R) and the nicotinic acetylcholine receptor (nAChR) were obtained by microscope FTIR spectroscopy using micrometer-sized, fully hydrated protein films. Because this novel procedure requires only nanogram quantities of membrane proteins, which is 4-5 orders of magnitude less than the amount of protein typically used for conventional FTIR spectroscopy, it opens the possibility to access the structure and dynamics of many important mammalian receptor proteins. The secondary structure of detergent-solubilized 5-HT(3)R determined by curve fitting of the amide I band yielded 36% alpha-helix, 33% beta-strand, 15% beta-turn, and 16% nonregular structures, which remained unchanged upon reconstitution in lipid membranes. From hydrogen-deuterium exchange, the secondary structure of the water-accessible part of 5-HT(3)R was determined as 14% alpha-helix, 16% beta-strand, 26% beta-turn, and 14% nonregular structures. Interestingly, we found that both the overall and the water-accessible nAChR secondary structures were nearly identical to those of 5-HT(3)R, in agreement with predicted structures of this class of receptors. This is the first time that structural investigations were obtained for two closely related ligand-gated ion channels under strictly identical experimental conditions.
Rigler
Per
P
Laboratoire de Chimie Physique des Polymères et Membranes, Ecole Polytechnique Fédérale de Lausanne, CH-1015 Ecublens, Switzerland.
Ulrich
Wolf-Peter
WP
Hovius
Ruud
R
Ilegems
Erwin
E
Pick
Horst
H
Vogel
Horst
H
D000577:Amides / Q000737:chemistry; D000818:Animals; D003902:Detergents; D003903:Deuterium / Q000737:chemistry; D041961:Deuterium Exchange Measurement; D007700:Kinetics; D051379:Mice; D036103:Nanotechnology / Q000379:methods*; D017433:Protein Structure, Secondary; D011522:Protons; D011978:Receptors, Nicotinic / Q000737:chemistry*; D044406:Receptors, Serotonin, 5-HT3 / Q000737:chemistry*; D012995:Solubility; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*; D014101:Torpedo; D014867:Water / Q000737:chemistry
D000577:Amides; D003902:Detergents; D011522:Protons; D011978:Receptors, Nicotinic; D044406:Receptors, Serotonin, 5-HT3; D014867:Water; D003903:Deuterium