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.
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
Hazime
H
Saitô
Satoru
S
Yamaguchi
Keiji
K
Ogawa
Satoru
S
Tuzi
Mercedes
M
Márquez
Carolina
C
Sanz
Esteve
E
Padrós
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