Backbone dynamics of apocytochrome b5 in its native, partially folded state

The backbone dynamics in the native state of apocytochrome bs were studied using N-15 nuclear magnetic spin relaxation measurements. The field (11.7 and 14.1 T) and temperature (10-25 degrees C) dependence of the relaxation parameters (R-1, R-2, and R-1 rho) and the H-1-N-15 NOE established that the protein undergoes multiple time scale internal motions related to the secondary structure. The relaxation data were analyzed with the reduced spectral density mapping approach and within the extended model-free framework. The apoprotein was confirmed to contain a disordered heme-binding loop of approximately 30 residues with dynamics on the sub-nanosecond time scale (0.6 < S-2 < 0.7, 100 ps < tau(e) < 500 Ps). This loop is attached to a structured hydrophobic core, rigid on the picosecond time scale (S-2 > 0.75, tau(e) < 50 ps). The inability to fit the data for several residues with the model-free protocol revealed the presence of correlated motion. An exchange contribution was detected in the transverse relaxation rate (R-2) Of all residues. The differential temperature response of R-2 along the backbone supported slower exchange rates for residues in the loop (tau(ex) > 300 mu s) than for the folded polypeptide chain (tau(ex) < 150 mu s). The distribution of the reduced spectral densities at the H-1 and N-15 frequencies followed the dynamic trend and predicted the slowing of the internal motions at 10 degrees C. Comparison of the dynamics with those of the holoprotein [Dangi, B., Sarma, S., Yan, C., Banville, D. L., and Guiles, R. D. (1998) Biochemistry 37, 8289-8302] demonstrated that binding of the heme alters the time scale of motions both in the heme-binding loop and in the structured hydrophobic core.