Solution NMR of signal peptidase, a membrane protein.
Useful solution nuclear magnetic resonance (NMR) data can be obtained from full-length, enzymatically active type I signal peptidase (SPase I), an integral membrane protein, in detergent micelles. Signal peptidase has two transmembrane segments, a short cytoplasmic loop, and a 27-kD C-terminal catalytic domain. It is a critical component of protein transport systems, recognizing and cleaving amino-terminal signal peptides from preproteins during the final stage of their export. Its structure and interactions with the substrate are of considerable interest, but no three-dimensional structure of the whole protein has been reported. The structural analysis of intact membrane proteins has been challenging and only recently has significant progress been achieved using NMR to determine membrane protein structure. Here we employ NMR spectroscopy to study the structure of the full-length SPase I in dodecylphosphocholine detergent micelles. HSQC-TROSY spectra showed resonances corresponding to approximately 3/4 of the 324 residues in the protein. Some sequential assignments were obtained from the 3D HNCACB, 3D HNCA, and 3D HN(CO) TROSY spectra of uniformly 2H, 13C, 15N-labeled full-length SPase I. The assigned residues suggest that the observed spectrum is dominated by resonances arising from extramembraneous portions of the protein and that the transmembrane domain is largely absent from the spectra. Our work elucidates some of the challenges of solution NMR of large membrane proteins in detergent micelles as well as the future promise of these kinds of studies.
Musial-Siwek
Monika
M
Department of Molecular and Cell Biology, The University of Connecticut, Storrs, CT 06269, USA.
Kendall
Debra A
DA
Yeagle
Philip L
PL
D000595:Amino Acid Sequence; D003902:Detergents / Q000494:pharmacology; D004795:Enzyme Stability / Q000187:drug effects; D007553:Isotope Labeling; D009682:Magnetic Resonance Spectroscopy; D008565:Membrane Proteins / Q000737:chemistry* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000302:isolation & purification; D012996:Solutions; D013696:Temperature
D003902:Detergents; D008565:Membrane Proteins; D012996:Solutions; D012697:Serine Endopeptidases; C026515:type I signal peptidase