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NMR Relaxation Dispersion Experiments to Study Phosphopeptide Recognition by SH2 Domains: The Grb2-SH2–Phosphopeptide Encounter Complex

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SH2 Domains

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2705))

Abstract

Protein interactions are at the essence of life. Proteins evolved not to have stable structures, but rather to be specialized in participating in a network of interactions. Every interaction involving proteins comprises the formation of an encounter complex, which may have two outcomes: (i) the dissociation or (ii) the formation of the final specific complex. Here, we present a methodology to characterize the encounter complex of the Grb2-SH2 domain with a phosphopeptide. This method can be generalized to other protein partners. It consists of the measurement of 15N CPMG relaxation dispersion (RD) profiles of the protein in the free state, which describes the residues that are in conformational exchange. We then acquire the dispersion profiles of the protein at a semisaturated concentration of the ligand. At this condition, the chemical exchange between the free and bound state leads to the observation of dispersion profiles in residues that are not in conformational exchange in the free state. This is due to fuzzy interactions that are typical of the encounter complexes. The transient “touching” of the ligand in the protein partner generates these new relaxation dispersion profiles. For the Grb2-SH2 domain, we observed a wider surface at SH2 for the encounter complex than the phosphopeptide (pY) binding site, which might explain the molecular recognition of remote phosphotyrosine. The Grb2-SH2–pY encounter complex is dominated by electrostatic interactions, which contribute to the fuzziness of the complex, but also have contribution of hydrophobic interactions.

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Author information

Authors and Affiliations

  1. National Center for Structural Biology and Bioimaging (CENABIO)/National Center for Nuclear Magnetic Resonance (CNRMN), Federal University of Rio de Janeiro, Rio de Janeiro, Brazil

    Fabio C. L. Almeida & Icaro P. Caruso

  2. Institute of Medical Biochemistry – IBqM, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil

    Fabio C. L. Almeida

  3. Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, Australia

    Karoline Sanches

  4. ARC Centre for Fragment-Based Design, Monash University, Parkville, VIC, Australia

    Karoline Sanches

  5. Multiuser Center for Biomolecular Innovation (CMIB), Department of Physics, São Paulo State University (UNESP), Sao Jose do Rio Preto, Sao Paulo, Brazil

    Icaro P. Caruso & Fernando A. Melo

Authors
  1. Fabio C. L. Almeida
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  2. Karoline Sanches
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  3. Icaro P. Caruso
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  4. Fernando A. Melo
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Corresponding author

Correspondence to Fabio C. L. Almeida .

Editor information

Editors and Affiliations

  1. School of Biosciences, College of Life and Environmental Sciences, University of Birmingham, Birmingham, UK

    Teresa Carlomagno

  2. Signalling Research Centres BIOSS &CIBSS, University of Freiburg, Freiburg, Germany

    Maja Köhn

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Almeida, F.C.L., Sanches, K., Caruso, I.P., Melo, F.A. (2023). NMR Relaxation Dispersion Experiments to Study Phosphopeptide Recognition by SH2 Domains: The Grb2-SH2–Phosphopeptide Encounter Complex. In: Carlomagno, T., Köhn, M. (eds) SH2 Domains. Methods in Molecular Biology, vol 2705. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3393-9_8

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  • DOI: https://doi.org/10.1007/978-1-0716-3393-9_8

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3392-2

  • Online ISBN: 978-1-0716-3393-9

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