Toward Understanding the Silk Fiber Structure: 13C Solid-State NMR Studies of the Packing Structures of Alanine Oligomers before and after Trifluoroacetic Acid Treatment.

Polyalanine (poly-A) sequences with tightly packed antiparallel ß sheet (AP-ß) structures are frequently observed in silk fibers and serve as a key contributor to the exceptionally high-fiber tensile strength. In general, the poly-A sequence embedded in the amorphous glycine-rich regions has different lengths depending on the fiber type from spiders or wild silkworms. In this paper, the packing structures of AP-ß alanine oligomers with different lengths were studied using 13C solid-state NMR as a model of the poly-A sequences. These included alanine oligomers with and without the protection groups (i.e., 9-fluorenylmethoxycarbonyl and polyethylene glycol groups at the N- and C-terminals, respectively). The fractions of the packing structures as well as the conformations were determined by deconvolution analyses of the methyl NMR peaks. Trifluoroacetic acid was used to promote the staggered packing structures, and the line shapes changed significantly for oligomers without the protected groups but only slightly for oligomers with the protected groups. Through NMR analysis of the 3-13C singly labeled alanine heptamer and refined crystal structure of the staggered packing units, a possible mechanism of the staggered packing formation is proposed for the AP-ß alanine heptamer.

High field 17O solid-state NMR study of alanine tripeptides.

(17)O chemical shifts of Ala-Ala-Ala, with parallel and anti-parallel beta-sheet structures, are observed using a 930-MHz high-resolution solid-state NMR spectrometer. Ala-Ala-Ala serves as a model sheet-forming peptide because it can be easily prepared as either a parallel or an anti-parallel beta-sheet structure. Spectral differences between the two samples are observed which arise from molecular packing differences between the two sheet structures. DFT chemical shift calculations are performed for the two samples, and the calculated spectra are in good agreement with the experimental spectra. The DFT calculations provide insight into the nature of the chemical shift differences observed between the two sheet structures.

High-field 1H MAS and 15N CP-MAS NMR studies of alanine tripeptides and oligomers: distinction of antiparallel and parallel beta-sheet structures and two crystallographically independent molecules.

beta-Strand peptides are known to assemble into either antiparallel (AP) or parallel (P) beta-sheet forms which are very important motifs for protein folding and fibril formations occurring in silk fibroin or amyloid proteins. Well-resolved 1H NMR signals including NH protons were observed for alanine tripeptides (Ala)3 with the AP and P structures as well as (Ala)n (n = 4-6) by high-field/fast magic-angle spinning NMR. Amide NH and amino NH3+ 1H signals of (Ala)3 with the P structure were well resonated at 7.5 and 8.9 ppm, respectively, whereas they were not resolved for the AP structure. Notably, NH 1H signals of (Ala)3 and (Ala)4 taking the P structure are resonated at higher field than those of the AP structure by 1.0 and 1.1 ppm, respectively. Further, NH 15N signals of (Ala)3 with the AP structure were resonated at lower field by 2 to 5 ppm than those of (Ala)3 with the P structure. These relative 1H and 15N hydrogen bond shifts of the P structure with respect to those of the AP structure are consistent with the relative hydrogen bond lengths of the interstrand N-H...O=C bonds. Distinction between the two crystallographically independent chains present in the AP and P structures was feasible by 15N chemical shifts but not by 1H chemical shifts because of insufficient spectral resolution in the latter. Calculated 1H and 15N shielding constants by density functional theory are generally consistent with the experimental data, although some discrepancies remain depending upon the models used.

Heterogeneity in the conformation of valine in the elastin mimetic (LGGVG)6 as shown by solid-state 13C NMR SPEctroscopy.

Elastin is an abundant protein found in vertebrates and is the source of elasticity in connective tissues and blood vessels. The repeating polypeptide sequences found in the hydrophobic domains of elastin have been the focus of many studies that attempt to understand the function of the native protein on a molecular scale. In this communication, the (LGGVG)6 elastin mimetic is characterized by solid-state 13C NMR spectroscopy. Through the use of a combination of a statistical analysis based on the Protein Data Bank, one-dimensional cross-polarization magic-angle-spinning NMR spectroscopy, and two-dimensional off-magic-angle-spinning spin-diffusion experiments, it is determined that this tandem repeat does not form a regular, highly ordered structure. Instead, like the poly(VPGVG) elastin mimetics, the valine has a twofold heterogeneity, although the conformations of these two populations differ from one peptide to the other.

Structural analysis of alanine tripeptide with antiparallel and parallel beta-sheet structures in relation to the analysis of mixed beta-sheet structures in Samia cynthia ricini silk protein fiber using solid-state NMR spectroscopy.

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.