Glutamic acid residues of bacteriorhodopsin at the extracellular surface as determinants for conformation and dynamics as revealed by site-directed solid-state 13C NMR.
We recorded (13)C NMR spectra of [3-(13)C]Ala- and [1-(13)C]Val-labeled bacteriorhodopsin (bR) and a variety of its mutants, E9Q, E74Q, E194Q/E204Q (2Glu), E9Q/E194Q/E204Q (3Glu), and E9Q/E74Q/E194Q/E204Q (4Glu), to clarify contributions of the extracellular (EC) Glu residues to the conformation and dynamics of bR. Replacement of Glu-9 or Glu-74 and Glu-194/204 at the EC surface by glutamine(s) induced significant conformational changes in the cytoplasmic (CP) surface structure. These changes occurred in the C-terminal alpha-helix and loops, and also those of the EC surface, as viewed from (13)C NMR spectra of [3-(13)C]Ala- and [1-(13)C]Val-labeled proteins. Additional conformational changes in the transmembrane alpha-helices were induced as modified retinal-protein interactions for multiple mutants involving the E194Q/E204Q pair. Significant dynamic changes were induced for the triple or quadruple mutants, as shown by broadened (13)C NMR peaks of [1-(13)C]Val-labeled proteins. These changes were due to acquired global fluctuation motions of the order of 10(-4)-10(-5) s as a result of disorganized trimeric form. In such mutants (13)C NMR signals from Val residues of [1-(13)C]Val-labeled triple and quadruple mutants near the CP and EC surfaces (including 8.7-A depth from the surface) were substantially suppressed, as shown by comparative (13)C NMR studies with and without 40 micro M Mn(2+) ion. We conclude that these Glu residues at the EC surface play an important role in maintaining the native secondary structure of bR in the purple membrane.
Saitô
Hazime
H
Department of Life Science, Graduate School of Science, Himeji Institute of Technology, Harima Science Garden City, Kamgiori, Hyogo, Japan 678-1297. saito@sci.himeji-tech.ac.jp
Yamaguchi
Satoru
S
Ogawa
Keiji
K
Tuzi
Satoru
S
Márquez
Mercedes
M
Sanz
Carolina
C
Padrós
Esteve
E
D019943:Amino Acid Substitution; D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000528:radiation effects*; D002247:Carbon Isotopes; D002462:Cell Membrane / Q000737:chemistry; D005110:Extracellular Space / Q000737:chemistry*; D018698:Glutamic Acid / Q000737:chemistry* / Q000528:radiation effects*; D008027:Light; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D009038:Motion; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation / Q000528:radiation effects; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship
D002247:Carbon Isotopes; D018698:Glutamic Acid; D001436:Bacteriorhodopsins