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Glutamic Acid Residues of Bacteriorhodopsin at the Extracellular Surface as Determinants for Conformation and Dynamics as Revealed by Site-Directed Solid-State ¹³C NMR

Hazime Saitô ^*, Satoru Yamaguchi ^*, Keiji Ogawa ^*, Satoru Tuzi ^*, Mercedes Márquez , Carolina Sanz  and Esteve Padrós 

^* Department of Life Science, Graduate School of Science, Himeji Institute of Technology, Harima Science Garden City, Kamgiori, Hyogo, Japan 678-1297; and Unitat de Biofísica, Departament de Bioquímica i de Biologia Molecular, Facultat de Medicina, Universitat Autònoma de Barcelona, Bellaterra, Barcelona 08193, Spain

Correspondence: Address reprint request to Dr. Hazime Saitô, Dept. of Life Science, Himeji Institute of Technology, Harima Science Garden City, Kouto 3-chome, Kamigori, Hyogo 678-1297, Japan. Tel./Fax: +81-78-856-2876; E-mail: saito{at}sci.himeji-tech.ac.jp.

We recorded ¹³C NMR spectra of [3-¹³C]Ala- and [1-¹³C]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 -helix and loops, and also those of the EC surface, as viewed from ¹³C NMR spectra of [3-¹³C]Ala- and [1-¹³C]Val-labeled proteins. Additional conformational changes in the transmembrane -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 ¹³C NMR peaks of [1-¹³C]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 ¹³C NMR signals from Val residues of [1-¹³C]Val-labeled triple and quadruple mutants near the CP and EC surfaces (including 8.7-Å depth from the surface) were substantially suppressed, as shown by comparative ¹³C NMR studies with and without 40 µM Mn²⁺ 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.