The effect of triple glutamic mutations E9Q/E194Q/E204Q on the structural stability of bacteriorhodopsin

Authors

  • Tzvetana Lazarova,

    Corresponding author
    1. Unitat de Biofísica, Departament de Bioquímica i de Biologia Molecular, Facultat de Medicina, Universitat Autònoma de Barcelona, Spain
    2. Centre d'Estudis en Biofísica, Universitat Autònoma de Barcelona, Spain
    • Correspondence

      T. Lazarova and E. Padrós, Unitat de Biofísica, Facultat de Medicina, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain

      Fax: +34 93 5811907

      Tel: +34 93 5811870

      E-mails: tzvetana.lazarova@uab.cat; esteve.padros@uab.cat

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    • These authors contributed equally to this work.
  • Krzysztof Mlynarczyk,

    1. Faculty of Chemistry, University of Warsaw, Poland
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    • These authors contributed equally to this work.
  • Slawomir Filipek,

    1. Faculty of Chemistry, University of Warsaw, Poland
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  • Michal Kolinski,

    1. Bioinformatics Laboratory, Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland
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  • Tsjerk A. Wassenaar,

    1. Groningen Institute for Biomolecular Research and Biotechnology, Zernike Institute for Advanced Materials, University of Groningen, The Netherlands
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  • Enric Querol,

    1. Institut de Biomedicina i Biotecnologia, Universitat Autònoma de Barcelona, Spain
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  • Venkatesan Renugopalakrishnan,

    1. Children's Hospital, Harvard Medical School, Boston, MA, USA
    2. Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA, USA
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  • Sowmya Viswanathan,

    1. Newton Wellesley Hospital/Partners Healthcare System, MA, USA
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  • Esteve Padrós

    Corresponding author
    1. Unitat de Biofísica, Departament de Bioquímica i de Biologia Molecular, Facultat de Medicina, Universitat Autònoma de Barcelona, Spain
    2. Centre d'Estudis en Biofísica, Universitat Autònoma de Barcelona, Spain
    • Correspondence

      T. Lazarova and E. Padrós, Unitat de Biofísica, Facultat de Medicina, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain

      Fax: +34 93 5811907

      Tel: +34 93 5811870

      E-mails: tzvetana.lazarova@uab.cat; esteve.padros@uab.cat

    Search for more papers by this author

Abstract

In the present study, we report on the structural features of the bacteriorhodopsin triple mutant E9Q/E194Q/E204Q (3Glu) of bacteriorhodopsin by combining experimental and molecular dynamics (MD) approaches. In 3Glu mutant, Glu9, Glu194 and Glu204 residues located at the extracellular side of the protein were mutated altogether to glutamines. UV-visible and differential scanning calorimetry experiments served as diagnostic tools for monitoring the resistance against thermal stress of the active site and the tertiary structures of the 3Glu. The analyses of the UV-visible thermal difference spectra demonstrate that the spectral forms at room temperature and the thermal unfolding path differ in the wild-type bacteriorhodopsin and the 3Glu. Even with these spectral differences, the thermal unfolding of the active site occurs at rather similar melting temperatures in both proteins. A noteworthy consequence of the mutations is the altered two-dimensional packing revealed by the lack of the pre-transition peak in differential scanning calorimetry traces of 3Glu mutant, as previously detected in wild-type and the corresponding single mutants. The infrared spectroscopy data agree with the loss of paracrystalinity, illustrating a substantial conversion of αII to αI helical conformation in the 3Glu mutant. Molecular dynamics simulations show higher dynamics flexibility of most of the extracellular regions of 3Glu, which may account for the somewhat lower tertiary structural stability of the mutated protein. Finally, hydrogen bond analysis reveals that the mutated Glu194 and Glu204 residues create ~ 50% less hydrogen bonds with water molecules compared to wild-type bacteriorhodopsin. These results exemplify the role of the water hydrogen-bonding network for structural integrity and conformational flexibility of bacteriorhodopsin.

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