title: Identification of Specific Effect of Chloride on the Spectral Properties and Structural Stability of Multiple Extracellular Glutamic Acid Mutants of Bacteriorhodopsin.
abstract: In the present work we combine spectroscopic, DSC and computational approaches to examine the multiple extracellular Glu mutants E204Q/E194Q, E204Q/E194Q/E9Q and E204Q/E194Q/E9Q/E74Q of bacteriorhodopsin by varying solvent ionic strength and composition. Absorption spectroscopy data reveal that the absorption maxima of multiple EC Glu mutants can be tuned by the chloride concentration in the solution. Visible Circular dichroism spectra imply that the specific binding of Cl- can modulate weakened exciton chromophore coupling and reestablish wild type-like bilobe spectral features of the mutants. The DSC data display reappearance of the reversible thermal transition, higher Tm of denaturation and an increase in the enthalpy of unfolding of the mutants in 1 M KCl solutions. Molecular dynamics simulations indicate high affinity binding of Cl- to Arg82 and to Gln204 and Gln194 residues in the mutants. Analysis of the experimental data suggests that simultaneous elimination of the negatively charged side chain of Glu194 and Glu204 is the major cause for mutants' alterations. Specific Cl- binding efficiently coordinates distorted hydrogen bonding interactions of the EC region and reconstitutes the conformation and structure stability of mutated bR in WT-like fashion.
authors.affiliation: Unitat de Biofísica, Departament de Bioquímica i de Biologia Molecular, Facultat de Medicina, and Centre d'Estudis en Biofísica, Universitat Autònoma de Barcelona, Barcelona, Spain.
authors.forename: Tzvetana
authors.initials: T
authors.lastname: Lazarova
authors.affiliation: Faculty of Chemistry, Biological and Chemical Research Centre, University of Warsaw, Warsaw, Poland.
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Mlynarczyk
authors.affiliation: Institut de Biomedicina i Biotecnologia, Universitat Autònoma de Barcelona, Barcelona, Spain.
authors.forename: Enric
authors.initials: E
authors.lastname: Querol
authors.affiliation: Department of Medical Physics and Biophysics, Faculty of Medicine, Medical University - Sofia, Sofia, Bulgaria.
authors.forename: Boris
authors.initials: B
authors.lastname: Tenchov
authors.affiliation: Faculty of Chemistry, Biological and Chemical Research Centre, University of Warsaw, Warsaw, Poland.
authors.forename: Slawomir
authors.initials: S
authors.lastname: Filipek
authors.affiliation: Unitat de Biofísica, Departament de Bioquímica i de Biologia Molecular, Facultat de Medicina, and Centre d'Estudis en Biofísica, Universitat Autònoma de Barcelona, Barcelona, Spain.
authors.forename: Esteve
authors.initials: E
authors.lastname: Padrós