STRUCTURAL AND DYNAMIC CHARACTERIZATION OF THE UREA DENATURED STATE OF THE IMMUNOGLOBULIN BINDING DOMAIN OF STREPTOCOCCAL PROTEIN-G BY MULTIDIMENSIONAL HETERONUCLEAR NMR-SPECTROSCOPY

The structure and dynamics of the urea-denatured B1 immunoglobulin bin ding domain of streptococcal protein G (GB1) has been investigated by multidimensional heteronuclear NMR spectroscopy. Complete H-1, N-15, a nd C-13 assignments are obtained by means of sequential through-bond c orrelations. The nuclear Overhauser enhancement, chemical shift, and ( 3)J(HN alpha) coupling constant data provide no evidence for the exist ence of any significant population of residual native or nonnative ord ered structure. N-15 relaxation measurements at 500 and 600 MHz, howev er, provide evidence for conformationally restricted motions in three regions of the polypeptide that correspond to the second beta-hairpin, the N-terminus of the alpha-helix, and the middle of the alpha-helix in the native protein. The time scale of these motions is longer than the apparent overall correlation time (similar to 3 ns) and could rang e from about 6 ns in the case of one model to between 4 mu s and 2 ms in another; it is not possible to distinguish between these two cases with certainty because the dynamics are highly complex and hence the a nalysis of the time scale of this slower motion is highly model depend ent. It is suggested that these three regions may correspond to nuclea tion sites for the folding of the GB1 domain. With the exception of th e N- and C-termini, where end effects predominate, the amplitude of th e subnanosecond motions, on the other hand, are fairly uniform and mod el independent, with an overall order parameter S-2 ranging from 0.4 t o 0.5.