Solution NMR as an Investigational Tool for Disordered and Partly Folded Proteins

The chaperone Hsp90 interacts with a relatively large set of proteins, termed client proteins, but their state, whether folded, partly folded or alternatively folded is not clear. NMR experiments on client proteins in the presence and absence of Hsp90 provide unprecedented insights into the conformational states of client proteins. As well, the NMR spectra of the chaperone itself in the presence of the client protein and of co-chaperones give information on interaction sites. NMR experiments on such large proteins and protein complexes are difficult both of execution and interpretation, but allow the formulation of hypotheses that can be tested by other spectroscopic means. We find that several client proteins form molten globule-like states when in the presence of Hsp90. The interactions between one client protein, the p53 DNA-binding domain, and fragments of Hsp90 of various sizes, comprising 1 and 2 domains of the protein, up to the full-length dimeric protein showed loss of signal intensity of the p53 resonances in the complex, from which we infer the prtesence of a state with secondary structure indistinguishable from that of the free protein, but containing a manifold of states that are in intermediate exchange on the NMR time scale. Further evidence for the loose and flexible nature of the bound p53 client is provided by the fluorescence behavior of the dye 1-anilinonaphthalene-8-sulfonic acid (ANS) and by comparison of H/D exchange rates of the amide protons of the p53 DNA binding domain. Hsp90 itself appears to make highly dynamic interactions with the client protein, which are modified in the presence of co-chaperones such as p23. We conclude that the interaction between Hsp90 and p53 is complex, and involves a structural change in the client protein to a loosened state.