Determining the Energy Landscape of Proteins by a Fast Isotope Exchange NMR Approach

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 β2-microglobulin. With 15N-labeling, the unfolding landscape of a protein can also be studied in the presence of other unla...