A high‐resolution ¹³C‐nmr study of collagenlike polypeptides and collagen fibrils in solid state studied by the cross‐polarization–magic angle‐spinning method. Manifestation of conformation‐dependent ¹³C chemical shifts and application to conformational characterization

Abstract

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.

Solid-state NMR Study Reveals Collagen I Structural Modifications of Amino Acid Side Chains upon Fibrillogenesis

“…1 and supplemental Fig. S1), and previous work on a collagen-like peptide (8) and native collagen I (9,10,17). No ambiguity was found for carbon resonances except for some C␣ and C␤ for which we observed overlaps in the two-dimensional { 1 H}- 13 C INEPT MAS spectra obtained in native conditions.…”

“…1 H NMR has only shown moderate potential to solve collagen-like peptide structure because of many overlaps of the resonances (8). Moreover, reported 13 C NMR experiments did not display a sufficient spectral resolution to allow a clear distinction among the different amino acid carbon resonances (9,10). NMR studies using specific residue labeling have given valuable information on dynamics (11)(12)(13).…”

“…NMR studies using specific residue labeling have given valuable information on dynamics (11)(12)(13). To deduce the backbone conformation, Saitô et al (9) have compared 13 C chemical shifts of fibrillar collagen with those obtained on small triple helical peptides. Torchia et al (14) have compared native and denatured resonances of collagen fragment CB2.…”

Some aspects of the NMR chemical shift/structure correlation in the structural characterization of polymers and biopolymers

“…However, the C ¼ O chemical shifts do not seem to be affected by the residue structure and thus can be used to determine the main-chain conformation. This method has been applied for the structural characterization of collagen proteins, 42 wool keratin, 43,44 silk protein, 45 polypeptide liquid crystals, 46,47 polypeptide gels, 48,49 and polypeptide blends. 50,51 Other peptide systems analyzed by this method are reviewed in Saito et al 40,52 It is known that changes in the helix sense of polypeptides in the solid state often occur because of changes in external conditions, such as temperature and so on, and changes in the side-chain conformation also often induce changes in the helix sense of the main-chain or the other conformations.…”

“…By comparing the experimental data and the predicted values given by this chemical shift map, the 13 C chemical shifts of Ala residues in polypeptides and proteins can be successfully predicted. This has been used to understand the 13 C chemical shift behavior of collagen protein 42 and silk proteins 45 in the solid state. Furthermore, ab initio calculations for the NMR chemical shifts have become available for medium-size molecules as a consequence of remarkable advances in the performance of workstations, personal computers and supercomputers.…”