Intrahelical arrangement in the integral membrane protein rhodopsin investigated by site-specific chemical cleavage and mass spectrometry.
Site-specific cleavage on the interhelical loop I on the cytoplasmic face of rhodopsin has been observed after activation of a Cu-phenanthroline tethered cleavage reagent attached on the cytoplasmic loop IV. The characterization of the reaction products by mass spectrometry, both of the membrane-bound protein and of the CNBr-cleaved peptides, allows the site of cleavage to be determined precisely. The specific cleavage of the peptide bond between Q64 and H65 on loop I leaves the N-terminal peptide (M1-Q64) intact, confirmed by MALDI-MS detection of the two N-linked glycosyl groups near the N-terminus of rhodopsin. The limited extension of the tether side chain requires a interresidue distance between the cleavage site, Q64, and the site of ligand attachment, C316, of less than 12 A. Upon photoactivation of the receptor, no change in the cleavage pattern is observed; however, a simulated Meta II intermediate activation state indicates a much more complex cleavage pattern. The development of this cleavage method, previously used primarily as a "chemical nuclease", in combination with mass spectrometry, may provide a powerful method on membrane protein conformation studies that can be used to complement other biophysical characterizations.
Gelasco
A
A
Department of Cell Pharmacology, Medical University of South Carolina, Charleston, South Carolina 29425, USA.
Crouch
R K
RK
Knapp
D R
DR
D000595:Amino Acid Sequence; D001205:Ascorbic Acid / Q000378:metabolism; D003300:Copper / Q000378:metabolism; D003488:Cyanogen Bromide / Q000378:metabolism; D003545:Cysteine / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009942:Organometallic Compounds / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D010618:Phenanthrolines / Q000378:metabolism; D017433:Protein Structure, Secondary; D012243:Rhodopsin / Q000737:chemistry* / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
C095824:5-(bromoacetamido)-1,10-phenanthroline copper complex; D008565:Membrane Proteins; D009942:Organometallic Compounds; D010446:Peptide Fragments; D010618:Phenanthrolines; D003300:Copper; D012243:Rhodopsin; D003545:Cysteine; D003488:Cyanogen Bromide; D001205:Ascorbic Acid; D010100:Oxygen