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Originally published In Press as doi:10.1074/jbc.M104836200 on August 27, 2001
J. Biol. Chem., Vol. 276, Issue 44, 40788-40794, November 2, 2001
Contribution of Extracellular Glu Residues to the Structure and
Function of Bacteriorhodopsin
PRESENCE OF SPECIFIC CATION-BINDING SITES*
Carolina
Sanz §¶,
Mercedes
Márquez¶,
Alex
Perálvarez,
Samir
Elouatik ,
Francesc
Sepulcre**,
Enric
Querol,
Tzvetana
Lazarova, and
Esteve
Padrós§§
From the Unitat de Biofísica, Departament de
Bioquímica i de Biologia Molecular, Facultat de Medicina,
Universitat Autònoma de Barcelona, Bellaterra (Cerdanyola del
Vallès), Barcelona 08193, Spain, the ** Departament
d'Enginyeria Química, Escola Universitària d'Enginyeria
Tècnica Industrial, Universitat Politècnica de Catalunya,
Barcelona 08036, Spain, and the Institut de
Biotecnologia i Biomedicina, Universitat Autònoma de Barcelona,
Bellaterra (Cerdanyola del Vallès),
Barcelona 08193, Spain
Single and multiple mutants of extracellular Glu
side chains of bacteriorhodopsin were analyzed by acid and calcium
titration, differential scanning calorimetry, and thermal difference
spectrophotometry. Acid titration spectra show that the second group
protonating with Asp85 is revealed in E204Q in the
absence of Cl but is not observed in the triple mutant
E9Q/E194Q/E204Q or in the quadruple mutant E9Q/E74Q/E194Q/E204Q.
The results point to Glu9 as the second group protonating
cooperatively with Asp85. Comparison of the apparent
pKa of Asp85 protonation in water and
in the deionized forms and results of calcium titration suggest that
cation-binding sites are of low affinity in the multiple Glu mutants.
Like for deionized wild type bacteriorhodopsin, differential scanning
calorimetry reveals a lack of the pretransition in the multiple
mutants, whereas in E9Q it appears at lower temperature and with lower
cooperativity. Additionally, at neutral pH the band at 630 nm arising
from cation release upon temperature increase is absent for the
multiple mutants. Based on these results, we propose the presence of
two cation-binding sites in the extracellular region of
bacteriorhodopsin having as ligands Glu9,
Glu194, Glu204, and water molecules.
*
This work was supported by Dirección General de
Investigación Grant BMC2000-0121, Secretaría de Estado de
Educación y Universidades Grant SAB1999-0102, and
Direcció General de Recerca Grant 1999SGR-102 and fellowships (to
S. E. and to M. M.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Present address: Dept. of Biochemistry, University of Cambridge, 80 Tennis Court Rd., Cambridge CB2 1GA, UK.
¶
These authors contributed equally to this work.
Present address: Dept. de Chimie, Université de
Montréal, C.P. 6128, Montréal, PQ H3C 3J7, Canada.
§§
To whom correspondence should be addressed. E-mail:
esteve.padros@uab.es.
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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Copyright © 2001 by the American Society for Biochemistry and Molecular Biology.
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