Structural Analysis of Alanine Tripeptide with Antiparallel and Parallel β-Sheet Structures in Relation to the Analysis of Mixed β-Sheet Structures in Samia cynthia ricini Silk Protein Fiber Using Solid-State NMR Spectroscopy

Abstract

The structural analysis of natural protein fibers with mixed parallel and antiparallel beta-sheet structures by solid-state NMR is reported. To obtain NMR parameters that can characterize these beta-sheet structures, (13)C solid-state NMR experiments were performed on two alanine tripeptide samples: one with 100% parallel beta-sheet structure and the other with 100% antiparallel beta-sheet structure. All (13)C resonances of the tripeptides could be assigned by a comparison of the methyl (13)C resonances of Ala(3) with different [3-(13)C]Ala labeling schemes and also by a series of RFDR (radio frequency driven recoupling) spectra observed by changing mixing times. Two (13)C resonances observed for each Ala residue could be assigned to two nonequivalent molecules per unit cell. Differences in the (13)C chemical shifts and (13)C spin-lattice relaxation times (T(1)) were observed between the two beta-sheet structures. Especially, about 3 times longer T(1) values were obtained for parallel beta-sheet structure as compared to those of antiparallel beta-sheet structure, which could be explicable by the difference in the hydrogen-bond networks of both structures. This very large difference in T(1) becomes a good measure to differentiate between parallel or antiparallel beta-sheet structures. These differences in the NMR parameters found for the tripeptides may be applied to assign the parallel and antiparallel beta-sheet (13)C resonances in the asymmetric and broad methyl spectra of [3-(13)C]Ala silk protein fiber of a wild silkworm, Samia cynthia ricini.

Studying natural structural protein fibers by solid‐state nuclear magnetic resonance

“…A complementary approach exists which consists in the synthesis and investigation of bio-inspired simplified peptides that provide invaluable information in the understanding of their natural counterparts (69,71,78,139). The study of specific properties found in natural fibers isolated in synthetic polypeptides should lead us to a better understanding of their functioning and the production of improved synthetic materials.…”

“…Alternatively, the 13 C-13 C dipolar coupling can be reintroduced by coherent schemes such as radio-frequency driven recoupling (RFDR) (68). Performed on fully 13 C-labeled alanine tripeptides, Asakura et al (69) used 2D 13 C- 13 C RFDR MAS spectra to assign 13 C resonances in parallel and antiparallel b-sheet structures. This method, applicable to natural fibers, revealed subtle differences in chemical shifts and interpeptides contacts at long mixing times.…”

Structural studies of amphiphilic oligopeptides composed of alternating alanine and ionizable amino-acid residues using CD and 13C CP/MAS NMR spectroscopy

“…15 The shape of the Ala Cb peak is known to be related not only to the conformation of the peptide but also to the intermolecular arrangement of the peptides. [16][17][18] Regarding the Ala Cb signal in the spectrum for AE12, the broad peak at 18.2 p.p.m. was assigned to the random coil peptides, and the other peaks at lower field were all derived from the b-sheet structures.…”

Application of Electron Paramagnetic Resonance and Solid-state 13C Nuclear Magnetic Resonance of Cross-polarization/Magic Angle Spinning to Study Enzymatic Degradation of Silk Fabrics

“…NMR signals arising from the major amino acid residues were well resolved and easily assigned to the peaks of Gly, Ala, Ser, Tyr and the amorphous region (including random coil and α-helix), 18 as shown in Fig. 3.…”

“…The chemical shifts centered at 172 ppm was assigned to the peak of Gly on the crystalline region. 18,19 That peak intensity of the 5-day aged sample increased, while with the increasing degrading times, it gradually decreased. As shown in Fig.…”