title: Single-shot NMR measurement of protein unfolding landscapes.
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 60h 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 (15)N-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 β(2)-microglobulin, in aqueous buffer and in synovial liquid, that is the natural medium of amyloid deposition in joints.
authors.affiliation: Dipartimento di Scienze Mediche e Biologiche, Università di Udine, Udine, Italy.
authors.forename: Enrico
authors.initials: E
authors.lastname: Rennella
authors.forename: Alessandra
authors.initials: A
authors.lastname: Corazza
authors.forename: Luca
authors.initials: L
authors.lastname: Codutti
authors.forename: Araldo
authors.initials: A
authors.lastname: Causero
authors.forename: Vittorio
authors.initials: V
authors.lastname: Bellotti
authors.forename: Monica
authors.initials: M
authors.lastname: Stoppini
authors.forename: Paolo
authors.initials: P
authors.lastname: Viglino
authors.forename: Federico
authors.initials: F
authors.lastname: Fogolari
authors.forename: Gennaro
authors.initials: G
authors.lastname: Esposito
mesh_terms: D000595:Amino Acid Sequence; D001499:Bayes Theorem; D002021:Buffers; D041961:Deuterium Exchange Measurement; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D055550:Protein Stability; D017433:Protein Structure, Secondary; D058767:Protein Unfolding; D013582:Synovial Fluid / Q000737:chemistry; D013816:Thermodynamics; D025801:Ubiquitin / Q000737:chemistry*; D001613:beta 2-Microglobulin / Q000737:chemistry*
chemical_list: D002021:Buffers; D025801:Ubiquitin; D001613:beta 2-Microglobulin