Establishing a quantitative relationship between backbone-backbone hydrogen-bond (H-bond) length observed in protein crystal structures and recently observed 3h J NC′ couplings across such bonds is limited by the coordinate precision of the X-ray structure. For an immunoglobulin binding domain of streptococcal protein G, very high-resolution X-ray structures are available. It is demonstrated that over the small range of N-O H-bond lengths (2.8-3.3 Å) for which 3h J NC′ couplings are observable, the 32 measured 3h J NC′ values can be fit to: 3h J NC′ ) -59000 exp(-4R NO ) ( 0.09 Hz, or R NO ) 2.75 -0.25 ln(-3h J NC′ ) ( 0.06 Å. Backbone amide to side-chain carboxyl hydrogen bonds were also investigated, and the measured 3h J NC′ values tend to be smaller than expected from their crystallographically determined H-bond lengths. The sign of 3h J NC′ , determined from a zero-quantum/double-quantum experiment, is found to be the same as that of the 1 J NH coupling, i.e., negative.

The structure and dynamics of the urea-denatured B1 immunoglobulin binding domain of streptococcal protein G (GBl) has been investigated by multidimensional heteronuclear NMR spectroscopy. Complete 'H, "N, and I3C assignments are obtained by means of sequential through-bond correlations. The nuclear Overhauser enhancement, chemical shift, and 3JHN, coupling constant data provide no evidence for the existence of any significant population of residual native or nonnative ordered structure. "N relaxation measurements at 500 and 600 MHz, however, provide evidence for conformationally restricted motions in three regions of the polypeptide that correspond to the second P-hairpin, the N-terminus of the a-helix, and the middle of the a-helix in the native protein. The time scale of these motions is longer than the apparent overall correlation time (-3 ns) and could range from about 6 ns in the case of one model to between 4 ps 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 analysis of the time scale of this slower motion is highly model dependent. It is suggested that these three regions may correspond to nucleation sites for the folding of the GB1 domain. With the exception of the N-and C-termini, where end effects predominate, the amplitude of the subnanosecond motions, on the other hand, are fairly uniform and model independent, with an overall order parameter S2 ranging from 0.4 to 0.5. Keywords: B1 domain; backbone dynamics; I5N relaxation; protein G ; structure; unfolded stateThe two-state model of protein unfolding in chemical denaturants is commonly used to analyze the stability of proteins. In this model, there is an unfolded state and a native state and the relative populations of these states are changed by the addition of the chemical denaturant. The energetics of this transition can be related to structural features of the native state, although, strictly speaking, the energetics depend on the difference in structure between the two states (Dobson, 1992;Shortle, 1993). It is possible that chemically denatured proteins have residual structure, thereby affecting the kinetics and thermodynamics of

A library of core mutants of the GB1 domain of streptococcal protein G was created, and the structure and stability of selected members was assessed by 1H-lSN heteronuclear correlation NMR spectroscopy and fluorescence. All mutants comprised changes in ~-sheet residues, with sidechains at positions 5 (Leu), 7 (Leu), 52 (Phe) and 54 (Val) forming the ~-sheet side of the sheet-helix core interface. A solvent exposed position Ile-6 was chosen as a control. Randomization of bases at codon positions 1 and 3 with thymine at position 2 introduces five possible hydrophobic amino acids, namely Leu, Val, Ile, Phe, and Met. The distribution of encoded amino acids at all five positions is approximately as expected theoretically and indicates that no major bias was introduced towards particular residues. The overall structural integrity of several mutants, as assessed by NMR, ranges from very close to wild type to fully unfolded. Interestingly, the stability of the mutants is not strictly correlated with the number of changes or residue volume.