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Protein Folding

Real-time NMR of Protein Folding
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Interactions that govern protein folding, one of the most fundamental biochemical processes, are often determined by NMR under equilibrium conditions. Because of the chemical specificity provided by chemical shift, NMR spectroscopy is also ideally positioned for observing structural changes that occur in protein folding. Traditionally, NMR spectroscopy requires a long signal averaging time, which is incompatible with the observation of transient processes on the sub-second to second time scale. We are applying real-time, hyperpolarization enhanced NMR spectroscopy for accessing the protein folding process in this time window. Using a rapid sample injection technique developed in-house, we are able to preserve substantial spin polarization on polypeptides despite their large molecular weight, which would otherwise cause prohibitive spin relaxation. Proteins are hyperpolarized by dynamic nuclear polarization, and dissolved under denaturing conditions. The protein solution is then mixed with a different buffer to carry out a pH jump and trigger folding. Folding is monitored using a series of NMR experiments at different time points. Decomposing each transient into signal contributions from unfolded and folded protein fractions yields the folding rate constant. Additionally, resolved signals from individual sites carry specific information tied to the structure of the protein.


Case Study: Folding Upon Binding of p27 Kinase Inhibitor
picture 2In a real-time, hyperpolarization enhanced experiment, spin-relaxation plays an important role in addition to reaction kinetics. This effect can be exploited for the characterization of intermediates that may arise in protein folding. Here, hyperpolarized p27 kinase inhibitor is mixed with its target protein complex Cdk2/Cyclin A. Signal evolution in the entire 13C NMR spectrum is then monitored on the second time scale. This time scale of the DNP-NMR experiment is seemingly placed in-between a fast and slow phase of association of these proteins. The signal decay rates, calculated for each position in the spectrum, are then dominated by spin relaxation. A comparison with relaxation theory indicates decay rates consistent with folded portions of the polypeptide in the spectrum of p27 mixed with the binding partner, but not in the spectrum of p27 alone. These differences can be seen, for example, in decay rates (red) near 178 ppm in the figure shown. Protein structural features on this time scale are not readily accessible by other methods, illustrating the unique information content in real-time D-DNP NMR spectra.