Molecular Motions and Protein Folding:  Characterization of the Backbone Dynamics and Folding Equilibrium of α₂D Using¹³C NMR Spin Relaxation

De novo protein design enables systematic exploration of the relationship between the amino acid sequences, conformations, and thermodynamics of proteins. The polypeptide α 2 D is a de novo designed dimeric four-helix bundle with a native-like three-dimensional structure [Hill, R. B.; DeGrado, W. F. J. Am. Chem. Soc. 1998, 120, 1138−1145]. The roles of intramolecular conformational dynamics and folding kinetics in determining the equilibrium properties of α 2 D have been investigated using novel NMR spin relaxation methods. To facilitate these experiments, the four leucine residues in the α 2 D monomer were labeled specifically with 13 C at the C α position. Reduced spectral densities [Farrow, N. A.; Zhang, O.; Szabo, A.; Torchia, D. A.; Kay, L. E. J. Biomol. NMR 1995, 6, 153−162] were obtained from spin relaxation data recorded at four static magnetic fields and were interpreted using the model-free formalism [Lipari, G.; Szabo, A. J. Am. Chem. Soc. 1982, 104, 4546−4559]. Generally, the backbone mobility of α 2 D is typical of natural proteins. High C α order parameters indicate that motions are restricted on the picosecond to nanosecond time scale. Slightly lower order parameters and longer internal correlation times are observed for the most N-terminal and C-terminal sites. Chemical exchange linebroadening is manifest for all leucine 13 C α spins and results from the folding equilibrium of α 2 D. The chemical exchange process was characterized using the relaxation-compensated Carr−Purcell−Meiboom−Gill experiment [Loria, J. P.; Rance, M.; Palmer, A. G., III. J. Am. Chem. Soc. 1999, 121, 2331−2332]. The folding and unfolding rate constants were measured to be (4.7 ± 0.9) × 10 6 Μ - 1 s - 1 and 15 ± 3 s - 1, respectively, and agree with the equilibrium constant for folding of α 2 D. The 13 C α chemical shifts for unfolded and folded forms of α 2 D, obtained from this analysis, indicate that the ensemble of unfolded states includes transiently structured helical conformations. The results both confirm the success of the de novo design strategy and suggest avenues for further improvement of the native-like properties of α 2 D.