Parallel β-Sheet Structure of Alanine Tetrapeptide in the Solid State As Studied by Solid-State NMR Spectroscopy

The structural analysis of alanine oligopeptides is important for understanding the crystalline region in silks from spiders and wild silkworms and also the mechanism of cellular toxicity of human diseases arising from expansion in polyalanine sequences. The atomic-level structures of alanine tripeptide and tetrapeptide with antiparallel beta-sheet structures (AP-Ala(3), and AP-Ala(4), respectively) together with alanine tripeptide with parallel beta-sheet structures (P-Ala(3)) have been determined, but alanine tetrapeptide with a parallel beta-sheet structure (P-Ala(4)) has not been reported yet. In this article, first, we established the preparation protocol of P-Ala4 from more stable AP-Ala4. Second, complete assignments of the C-13, N-15, and H-1 solid-state NMR spectra were performed with C-13- and N-15-labeled Ala(4) samples using several solid-state NMR techniques. Then, the structural constraints were obtained, for example, the amide proton peaks of P-Ala4 in the H-1 double-quantum magic-angle spinning NMR spectrum were heavily overlapped and observed at about 7.4 ppm, which was a much higher field than that of 8.7-9.1 ppm observed for AP-Ala4, indicating that the intermolecular hydrogen-bond lengths across strands (N-H center dot center dot center dot O=C) were considerably longer for P-Ala(4), that is, 2.21-2.34 A, than those reported for AP-Ala(4), that is, 1.8-1.9 angstrom. The structural model was proposed for P-Ala4 by NMR results and MD calculations.