Molecular motions and protein folding: Characterization of the backbone dynamics and folding equilibrium of alpha D-2 using C-13 NMR spin relaxation

De nova protein design enables systematic exploration of the relationship b etween the amino acid sequences, conformations, and thermodynamics of prote ins. The polypeptide alpha D-2 is a de novo designed dimeric four-helix bun dle 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 relaxa tion methods To facilitate these experiments, the four leucine residues in the alpha D-2 monomer were labeled specifically with C-13 at the Ca positio n. 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 interprete d using the model-free formalism [Lipari, G.; Szabo, A. J. Am. Chem. Sec. 1 982, 104, 4546-4559]. Generally,: the backbone mobility of alpha D-2 is typ ical of natural proteins. High Ca order parameters indicate that motions ar e restricted on the picosecond to nanosecond time scale. Slightly lower ord er parameters and longer internal correlation;times are observed for the mo st N-terminal and C-terminal sites. Chemical exchange linebroadening is man ifest for all leucine C-13(alpha) spins and results from the folding equili brium of alpha D-2. The chemical exchange process was characterized using t he relaxation-compensated Carr-Purcell-Meiboom-Gill experiment [Loria, J: P :; Rance, M.; Palmer, A. G., ill. J. Am. Chem..Soc. 1999, fil 2331-2332].,T he 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 equil ibrium constant for folding of alpha D-2. The C-13(alpha) chemical shifts f or unfolded and folded forms of alpha D-2, obtained from this analysis, ind icate 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-l ike properties of alpha D-2.