Single-shot NMR measurement of protein unfolding landscapes
June 2012
Publication year: 2012
Source:Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, Volume 1824, Issue 6
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 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 ?2-microglobulin, in aqueous buffer and in synovial liquid, that is the natural medium of amyloid deposition in joints.
Graphical abstract
Highlights
? NMR hydrogen–deuterium exchange measurements to study protein unfolding landscape. ? BLUU-Tramp, new NMR method to measure isotope exchange thermal kinetics. ? BLUU-Tramp: time gain, single sample and experimental session, routine instruments. ? Identification of stability determinants in proteins and protein complexes. ? Application to amyloidogenic systems, physiological milieu, basic folding studies.
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