We recorded X-ray diffraction, 13C CP-MAS NMR, and infrared spectra of silk fibroin samples from Bombyx mori in the solid state, prepared under different conditions, in order to compare conformational features obtained by the respective methods. These three methods provided consistent data as to the presence of the most stable silk II form. Silk I and random-coil forms were distinguishable only by the extent of line broadening in 13C NMR peaks, although the chemical shifts are the same. The most obvious advantage of the 13C CP-MAS NMR approach is that this method is intrinsically nondestructive and very convenient for evaluation of the relative proportion of silk I and II forms in the same sample. We found that powder formation even at liquid-nitrogen temperature caused a change from silk I to random-coil form, although the amount of silk II form remains unchanged during this procedure. In view of the sample preparation conditions as well as the similarity of the 13C chemical shifts and infrared spectra between the silk I and random-coil forms, it is plausible that the random-coil form is a mixture of silk I type forms whose long-range crystalline packing is distorted to the extent to give halo X-ray diffraction.

High-resolution 13C NMR (75.46 MHz) spectra of silk fibroins in the solid state were recorded by the cross-polarization-magic angle spinning method with emphasis on revealing conformational features of the dimorphic structures, silk I and II, of Bombyx mori fibroin prepared under different conditions. It was found that the 13C chemical shifts of Gly, Ala, and Ser residues of silk II samples from B. mori fibroin by different preparations and of cocoon samples from several silkworms gave identical values with those of corresponding model polypeptides having the /3-sheet conformation. Thus, identification of the silk II type form is easily performed by examining their 13C chemical shift values. The 13C chemical shifts of samples having the silk I form are significantly displaced from those of the silk II form, and can be used for diagnostic purposes. As expected, the 13C chemical shifts of Ala and Gly residues of the silk I samples were identical with those of (Ala-Gly)"II. However, we found that none of the 13C chemical shifts predicted from the crankshaft model [Lotz, B.; Keith, H. D. J. Mol. Biol. 1971, 61, 201-215] in which Ala and Gly residues are close to the /S-sheet and -helix conformations, respectively, was in agreement with the 13C chemical shifts of (Ala-Gly)"II and silk I type form. Instead, we found that predicted 13C chemical shifts from the loose helix proposed by Konishi and Kurokawa [Konishi, T.; Kurokawa, M. Sen'i Gakkaishi 1968, 24, 550-554] on the basis of the calculated 13C contour map of chemical shifts for Ala residue are in good agreement with the displacement of the l3C chemical shifts.

SynopsisWe have recorded high-resolution l3C-nmr spectra of collagen fibrils in the solid state by the cross-polarization-magic-angle-spinning (CP-MAS) method and analyzed the spectra with reference to those of collagenlike polypetides. We used two kinds of model polypeptides to obtain reference 13C chemical shifts of major amino acid residues of collagen (Gly, Pro, Ala, and Hyp): the 3,-helical polypeptides [(GlyIJI, (Pro)JI, (Hyp),, and (Ala-Gly-Gly).II], and the triple-helical polypeptides [(Pro--Gly-Pro), and (Pr+Ala-Gly)n]. Examination of the I3C chemical shifts of these polypeptides, together with our previous data, showed that the 13C chemical shifts of individual amino acid residues are the same, within experimental error (k0.5 ppm), among different polypeptides with different primary sequences, if the conformations are the same. We found that the I3C chemical shifts of Ala residues of the 3,-helical (Ala-Gly--Gly), and triplehelical (Pro-Ala--Gly), are significantly displaced, compared with those of the a-helix, &sheet, and silk I form, and can be utilized as excellent probes to examine conformational features of collagen-like polypeptides. Further, the I3C chemical shifts of Gly and Pro residues in the triple-helical polypeptides are substantially displaced from those found in (Gly),II and fPro),II of the 3,-helix, reflecting further conformational change from the 3,-helix to the supercoiled triple helix. In particular, the I3C chemical shifts of Gly C =O carbons of the triple-helical polypeptides are substantially displaced upfield (4.1-5.1 ppm), with respect to those of the 3,-helical polypeptides. These displacements are interpreted by that Gly C=O of the former is not involved in NH . . . O=C hydrogen bonds, while this carbon of the latter is linked by these kinds of hydrogen bonds.* To whom all correspondence should be addressed. Biopolymers, Vol. 23, 2279-2297 On the basis of these 13C chemical shifts, as reference data for the collagenlike structure, we were able to assign the l3C-nmr peaks of Gly, Ala, Pro, and Hyp residues of collagen fibrils, which are in good agreement with the values expected from the model polypeptides mentioned above. We also discuss a plausible conformational change of collagen fibrils during denaturation.

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Solid state high resolution 13C NMR spectra of the silk fibroins from Bombyx mori (B. mori) and Philosamia cynthia ricini (P. c. ricini) together with those of appropriate model peptides were recorded by cross polarization-magic angle spinning method. We found by examining 13C chemical shifts that anti-parallel β-sheet and α-helix forms were straightforwardly ascribed to the fibroins from B. mori and P. c. ricini, respectively.