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Studying Substrate Binding to Reconstituted Secondary Transporters by Attenuated Total Reflection Infrared Difference Spectroscopy

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Book cover Membrane Protein Structure and Dynamics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 914))

Abstract

The determination of protein conformational changes induced by the interaction of substrates with secondary transporters is an important step toward the elucidation of their transport mechanism. Since conformational changes in a protein alter its vibrational patterns, they can be detected with high sensitivity by infrared difference (IRdiff) spectroscopy without the need for external probes. We describe a general procedure to obtain substrate-induced IRdiff spectra by alternating perfusion of buffers over an attenuated total reflection (ATR) crystal containing an adhered film of a membrane protein reconstituted in lipids. As an example, we provide specific protocols to obtain melibiose and Na+-induced ATR-IRdiff spectra of reconstituted melibiose permease, a sodium/melibiose co-transporter from E. coli. The presented methodology is applicable in principle to any membrane protein, provided that it can be purified and reconstituted in functional form, and appropriate substrates are available.

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Acknowledgments

This work was supported by a Marie Curie Reintegration Grant PIRG03-6A-2008-231063 (to V.L.-F.), and by Ministerio de Ciencia e Innovación grants BMC2003-04941, BFU2006-04656/BMC, and BFU2009-08758/BMC, the Direcció General de Recerca (DURSI, Generalitat de Catalunya), 1999SGR-0102 grant, and the European Commission Bio4-CT97-2119 grant.

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Authors and Affiliations

  1. 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

    Víctor A. Lórenz-Fonfría & Esteve Padrós

  2. Centre de Biotecnologia Animal i Teràpia Gènica, Universitat Autònoma de Barcelona, Barcelona, Spain

    Xavier León

Authors
  1. Víctor A. Lórenz-Fonfría
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  2. Xavier León
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  3. Esteve Padrós
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Corresponding authors

Correspondence to Víctor A. Lórenz-Fonfría or Esteve Padrós .

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Editors and Affiliations

  1. , Division of Immunology, Beckman Research Institute of the City o, E. Duarte Road 1500, Duarte, 91010, USA

    Nagarajan Vaidehi

  2. , Department of Structural Biology, University of Pittsburgh School of Medic, Fifth Avenue 3501, Pittsburgh, 15261, USA

    Judith Klein-Seetharaman

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Lórenz-Fonfría, V.A., León, X., Padrós, E. (2012). Studying Substrate Binding to Reconstituted Secondary Transporters by Attenuated Total Reflection Infrared Difference Spectroscopy. In: Vaidehi, N., Klein-Seetharaman, J. (eds) Membrane Protein Structure and Dynamics. Methods in Molecular Biology, vol 914. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-023-6_7

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  • DOI: https://doi.org/10.1007/978-1-62703-023-6_7

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-022-9

  • Online ISBN: 978-1-62703-023-6

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