Protein vivisection reveals elusive intermediates in folding.
Although most folding intermediates escape detection, their characterization is crucial to the elucidation of folding mechanisms. Here, we outline a powerful strategy to populate partially unfolded intermediates: A buried aliphatic residue is substituted with a charged residue (e.g., Leu-->Glu(-)) to destabilize and unfold a specific region of the protein. We applied this strategy to ubiquitin, reversibly trapping a folding intermediate in which the beta5-strand is unfolded. The intermediate refolds to a native-like structure upon charge neutralization under mildly acidic conditions. Characterization of the trapped intermediate using NMR and hydrogen exchange methods identifies a second folding intermediate and reveals the order and free energies of the two major folding events on the native side of the rate-limiting step. This general strategy may be combined with other methods and have broad applications in the study of protein folding and other reactions that require trapping of high-energy states.
Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637, USA.
Zhongzhou
Z
Zheng
Tobin R
TR
Sosnick
D041961:Deuterium Exchange Measurement; D008956:Models, Chemical; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D017510:Protein Folding; D012542:Scattering, Radiation; D013816:Thermodynamics; D025801:Ubiquitin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D025801:Ubiquitin