Structural Investigations of Solid Proteins at Natural Abundance Using 2D Multiple-Pulse NMR

Results from multiple-pulse 2D 1H−13C correlation experiments are described for natural proteins in the solid state. Detailed HETCOR experiments on the complex silk fibroin proteins Samia cynthia ricini (S. c. ricini) and Bombyx mori (B. mori) demonstrate that while the expected dipolar-mediated 13C and 1H correlations are observed, additional chemical shift and coupling information involving amide linkages and dilute peptides are also detected indirectly. This is an important result since we show that the 1H chemical shifts for −NH groups in S. c. ricini are not resolved in the CRAMPS experiments, thereby preventing their direct measurement. Chemical shifts via dipolar couplings (two-spin and multiple-spin interactions) are also detected for dilute peptides in the silk fibroins, again providing key structure information for these functional residues that is completely absent in the CRAMPS data. Relative dynamics of bulky side groups from dilute amino acid residues are apparent from the 2D multiple-pulse data. Differential spin-diffusion behavior is observed in a modified HETCOR experiment for S. c. ricini relative to the B. mori protein, suggesting morphological differences in the arrangement of crystalline segments in the proteins. Information regarding hydrogen bonding and constrained interchain spin pairs is provided based on comparison of cross-peak intensities for several nearest-neighbor C−H spin pairs within the chain and based upon the presence of CO correlations with specific α-hydrogens.