22] Structural characterization of protein folding intermediates by proton magnetic resonance and hydrogen exchange

Abstract

The ability to resolve resonances from a large number of individual nuclei, especially by two-dimensional nuclear magnetic resonance (NMR) methods, permits a detailed characterization of the conformational changes associated with folding-unfolding transitions. The nuclear overhauser effect (NOE) can in principle provide quantitative structural information on stable analogs of folding intermediates. The primary advantage of NMR for protein folding applications lies, however, in its temporal information content. The dynamics of structural fluctuations and transitions can be probed over a wide time scale by using relaxation methods, line-shape analysis, magnetization transfer, and hydrogen exchange. Much of the initial effort in such protein folding projects will have to be devoted to exploring conditions for unfolding and refolding, and testing of the reversibility and quench efficiency. It is important to choose unfolding conditions where the protein is as much as possible devoid of residual structure or aggregation. The presence of intra- or intermolecular H bonds would retard hydrogen exchange and lead to erroneous interpretation of the trapping results. The most pertinent test is to perform hydrogen exchange measurements under the conditions chosen for unfolding, and to compare the results with free peptide rates. Under most circumstances rapid mixing is required for these measurements.