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 15N-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.
Original language | English (US) |
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Pages (from-to) | 4457-4460 |
Number of pages | 4 |
Journal | Journal of the American Chemical Society |
Volume | 134 |
Issue number | 10 |
DOIs | |
State | Published - Mar 14 2012 |
ASJC Scopus subject areas
- Catalysis
- General Chemistry
- Biochemistry
- Colloid and Surface Chemistry