Internal motion time scales of a small, highly stable and disulfide-rich protein: A N-15, C-13 NMR and molecular dynamics study

Motions of the backbone CalphaHalpha and threonine CbetaHbeta bonds of toxi n alpha were investigated using natural abundance C-13 NMR and molecular dy namics. Measurement of the C-13 longitudinal and transverse relaxation rate s employed ACCORDION techniques together with coherence selection by pulsed field gradients and sensitivity enhancement through the use of preservatio n of equivalent pathway, thus allowing a considerable reduction of the requ ired spectrometer time. C-13 R-1, R-2, H-1 -->C-13 NOE were obtained, as we ll as the variations of R-1 rho(90 degrees ) as a function of the rf field strength. These data were compared to those recorded by H-1 and N-15 NMR on a labelled sample of the toxin [Guenneugues et al. (1997) Biochemistry, 36 , 16097-16108]. Both sets of data showed that picosecond to nanosecond time scale motions are well correlated to the secondary structure of the protei n. This was further reinforced by the analysis of a 1 ns molecular dynamics simulation in water. Several CalphaHalpha and threonine CbetaHbeta experim entally exhibit fast motions with a correlation time longer than 500 ps, th at cannot be sampled along the simulation. In addition, the backbone exhibi ts motions on the microsecond to millisecond time scale on more than half o f its length. Thus, toxin alpha, a highly stable protein (T-m = 75 degrees C at acidic pH) containing 61 amino acids and 4 disulfides, shows important internal motions on time scales ranging from 0.1-0.5 ps, to 10-100 ps, 1 n s, and about 30 mu s to 10 ms.