Single-shot NMR measurement of protein unfolding landscapes

Enrico Rennella, Alessandra Corazza, Luca Codutti, Araldo Causero, Vittorio Bellotti, Monica Stoppini, Paolo Viglino, Federico Fogolari, Gennaro Esposito

Research output: Contribution to journalArticle

Abstract

The transient unfolding events from the native state of a protein towards higher energy states can be closely investigated by studying the process of hydrogen exchange. Here, we present BLUU-Tramp (Biophysics Laboratory University of Udine - Temperature ramp), a new method to measure the rates for the exchange process and the underlying equilibrium thermodynamic parameters, using just a single sample preparation, in a single experiment that lasts some 20 to 60 h depending on the protein thermal stability, to record hundreds of points over a virtually continuous temperature window. The method is suitable also in presence of other proteins in the sample, if only the target protein is 15N-labelled. This allows the complete thermodynamic description of the unfolding landscape at an atomic level in the presence of small or macromolecular ligands or cosolutes, or in physiological environments. The method was successfully tested with human ubiquitin. Then the unfolding thermodynamic parameters were satisfactorily determined for the amyloidogenic protein β2-microglobulin, in aqueous buffer and in synovial liquid, that is the natural medium of amyloid deposition in joints.

Original languageEnglish (US)
Pages (from-to)842-849
Number of pages8
JournalBiochimica et Biophysica Acta - Proteins and Proteomics
Volume1824
Issue number6
DOIs
StatePublished - Jun 1 2012

Keywords

  • Energy landscape
  • H-D exchange
  • Protein NMR
  • Protein thermal denaturation
  • Protein unfolding
  • Unfolding thermodynamics

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biophysics
  • Biochemistry
  • Molecular Biology

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  • Cite this

    Rennella, E., Corazza, A., Codutti, L., Causero, A., Bellotti, V., Stoppini, M., Viglino, P., Fogolari, F., & Esposito, G. (2012). Single-shot NMR measurement of protein unfolding landscapes. Biochimica et Biophysica Acta - Proteins and Proteomics, 1824(6), 842-849. https://doi.org/10.1016/j.bbapap.2012.04.002