title: Determining the energy landscape of proteins by a fast isotope exchange NMR approach.
abstract: We present a new and efficient NMR method, BLUU-Tramp (Biophysics Laboratory University of Udine temperature ramp), for the collection of hydrogen-deuterium exchange experiments as a function of time and temperature for small and medium-sized proteins. Exchange rates can be determined to extract the underlying thermodynamic equilibrium or kinetic parameters by sampling hundreds of points over a virtually continuous temperature ramp. Data are acquired in a single experimental session that lasts some 20-60 h, depending on the thermal stability of the protein. Subsequent analysis provides a complete thermodynamic description of the protein energy landscape. The global thermal unfolding process and the partial or local structure opening events can be fully determined at the single-residue resolution level. The proposed approach is shown to work successfully with the amyloidogenic protein β(2)-microglobulin. With (15)N-labeling, the unfolding landscape of a protein can also be studied in the presence of other unlabeled proteins and, in general, with ligands or cosolutes or in physiological environments.
authors.affiliation: Dipartimento di Scienze Mediche e Biologiche, Università di Udine, I-33100 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: 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: D007554:Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011506:Proteins / Q000737:chemistry*; D013816:Thermodynamics
chemical_list: D007554:Isotopes; D011506:Proteins