De nova protein design enables systematic exploration of the relationship between the amino acid sequences, conformations, and thermodynamics of proteins. The polypeptide alpha D-2 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. Sec. 1998, 120, 1138-1145]. The roles of intramolecular conformational dynamics and folding kinetics in determining the equilibrium properties of alpha D-2 have been investigated using novel NMR spin relaxation methods To facilitate these experiments, the four leucine residues in the alpha D-2 monomer were labeled specifically with C-13 at the Ca position. Reduced spectral densities [Farrow,N. A.;Zhang, O.; Szabo, A.; Torchia, D. A.; Kayi 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. Sec. 1982, 104, 4546-4559]. Generally,: the backbone mobility of alpha D-2 is typical of natural proteins. High Ca 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 C-13(alpha) spins and results from the folding equilibrium of alpha D-2. The chemical exchange process was characterized using the relaxation-compensated Carr-Purcell-Meiboom-Gill experiment [Loria, J: P:; Rance, M.; Palmer, A. G., ill. J. Am. Chem..Soc. 1999, fil 2331-2332].,The folding and unfolding rate constants were measured to be (4.7 +/-0.9):x 10(6) M-1 s(-1) and 15 +/- 3 s(-1), respectively, and agree with the equilibrium constant for folding of alpha D-2. The C-13(alpha) chemical shifts for unfolded and folded forms of alpha D-2, 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 alpha D-2.