Fibroin, orientation

Normal transmission IRLD can also be used to characterize polymeric fibers, although scattering can induce sloping baselines. Raman spectroscopy then becomes a convenient alternative. Rutledge et al. have recently probed the orientation in electrospun nanofibers composed of a core of Bombyx mori fibroin and an outer shell of poly (ethylene oxide) [24], The orientation values were low, less than 0.1, as is often the case in electrospun fibers. 

Rossle, M., Panine, P., Urban, V. S., and Riekel, C. (2004). Structural evolution of regenerated silk fibroin under shear Combined wide- and small-angle x-ray scattering experiments using synchrotron radiation. Biopolymers 74, 316-327. Rousseau, M. E., Lefevre, T., Beaulieu, L., Asakura, T., and Pezolet, M. (2004). Study of protein conformation and orientation in silkworm and spider silk fibres using Raman microspectroscopy. Biomacromolecules 5, 2247-2257. 

Fiber diffraction is a technique used to determine the structures of molecules that are oriented to form fibers by virtue of a parallel assembly of molecules. Certain materials, such as cellulose, keratin and fibroin (the silk protein) occur naturally in this form. Some polymers can be drawn out to form fibers in which the same type of orientation occurs. This was the technique used by Rosalind Franklin to study DNA. More recently. 

Polypeptides of low complexity, the constitution of which can be ascertained, have been obtained from fibroin by the controlled action of enzymes. Such evidence leads to the conclusion that the orientated regions consist of amino acid residues arranged in the following order. 

As mentioned above, the determination of atomic level structure, i.e., the backbone torsion angles for an oriented protein fiber, is possible by using both solid-state NMR method described here and specifically isotope labeling. This is basically to obtain the angle information. Another structural parameter is distance between the nuclei for atomic coordinate determination. The observation of Nuclear Overhauser Enhancements (NOEs) between hydrogen atoms is a well known technique to determine the atomic coordinates of proteins in solution [14]. In the field of solid-state NMR, REDOR (rotational echo double resonance) for detection of weak heteronuclear dipole interactions, such as those due to C and N nuclei [15, 16] or R (rotational resonance) for detection of the distance between homonuclei, are typical methods for internuclear distance determination [17,18]. The REDOR technique has been applied to structure determination of a silk fibroin model compound [19]. In general, this does not require orientation of the samples in the analysis, but selective isotope labeling between specified nuclear pairs in the samples is required which frequently becomes a problem. A review of these approaches has appeared elsewhere [16]. 

To extract information on molecular orientation distribution from experimental data, the most widely known technique, the Legendre moment expansion approach can be taken. In this section, this approach will be discussed first, followed by methods to elucidate atomic resolution details of the structures of ordered polymers with orientation-dependent NMR interactions, such as those from chemical shielding, dipole-dipole and quadrupolar coupling. Then, solid-state NMR studies of the torsion angles of the peptide backbone of highly ordered silk fibroin fiber, a protein that has been studied extensively as a model for fibrous proteins, will be described. 

Fig. 8.5. Solid-state N NMR spectra obtained from the oriented [ N]Gly silk fibroin fiber sample. Spectra were observed with the fiber axis both (A) parallel and (B) perpendicular to the magnetic field. The best-fit simulated line shapes are superimposed on the experimental spectra. Parameters for spectral simulation were found from error analysis as a function of ap and Pf(C).

Figure 8.8 shows the solid-state NMR spectra (solid line) obtained from blocks of oriented natural abundance silk fibroin fiber, Poly(p-pheny-... 

When the values, udnh and /Sdnh determined for appropriate model compounds are used [31, 17], the angle, can be obtained as a function of ttp and jSp as described in Section 8.3.1. The o-paraiiei o- so values are calculated by searching all of the and /3p space (0°-180°) for ap and jSp pairs that yield simultaneous solutions to both Equations (8.10) and (8.11) in Section 8.3.1. The orientational restriction for silk fibroins and PBLG are shown by the region shown by the horizontal lines in Fig. 8.8. Similarly, the region shown by the vertical lines is allowed region for PPTA. The latter... 

Fig. 8.8. Solid-state NMR spectra of the oriented block samples of natural abundance B. mori silk fibroin, poly(p-phenylene terephthal amide) (PPTA), and poly(7-benzyl L-glutamate) (PBLG).

The less stable form, silk I, has remained poorly understood [37]. In general, attempts to induce orientation of its polymer chain tend to cause the silk I to convert to the more stable silk II. If an oriented silk fibroin sample with silk I form can be obtained, the solid-state NMR analysis for an oriented molecular system described here may be used to determine the atomic coordinates of silk fibroin with the silk I form. 

Fig. 23.8. Solid-state C NMR spectra of an oriented block of [T CJGly silk fibroin fibers as a function of the angles between the oriented fiber axis and Bo-...

Table 23.2. The Euler angles, f and Pp and bond orientation data, and 0nc for [ N]Gly sites of silk fibroins with silk II form ...

Fujiwara et al. [69] applied solid-state NMR to a structural study on oriented [l- C]Ala silk fibroin fiber from B. mori. They found that the Euler angles obtained from the simulated lineshapes of the Ala carbonyl group are slightly different from that of a typical anti parallel j8-sheet, [30] raising questions concerning the accuracy of the current models for silk II structure. However, our data are in agreement with the X-ray diffraction model within experimental error. 

Fig. 23.9. (A) quadrupole echo spectrum of (A) an oriented block and (B) unaligned silk fibroin fiber of [2,2- H2]Gly-labeled samples.

Figure 23.9(A) shows the quadrupole echo spectrum of an oriented block of [2,2- H2]Gly labeled silk fibroin fibers when the fiber axis was set parallel to the magnetic field direction. The quadrupole echo spectrum, Fig. 23.9(B), of unaligned [2,2- H2]Gly labeled silk fibroin fiber is also observed as a powder pattern. Both spectra are split into doublets, which give the value of the quadrupole splitting, Avq as 117.8 kHz for the [2,2- H2]Gly site the values are the same in each case. The full rigid-lattice width of about 126 kHz should be observed when the motion is frozen [72]. Thus, it is concluded that the motion of the methylene groups of the Gly residue is almost frozen at room temperature, which is in agreement with the prediction from the intermolecular hydrogen bonding network in the silk fibroin backbone chain with an antiparallel /3-sheet conformation.

The quadrupole splitting, Ai q of 117.8 kHz observed for the oriented block of [2,2- H2]Gly labeled silk fibroin fiber is the same as the value in the spectrum of an unaligned sample as shown in Fig. 23.9(B). Therefore, if the latter quadrupole splitting is used to provide a value of the proportionality constant, (3/4)e qQ/h, which describes the relationship between the quadrupole splitting and the angle CD (the angle of the C H bond vector of Gly relative to the fiber axis) > CD is calculated as 90°. By taking into account an... 

The solid-state NMR spectrum of an oriented block of the [3,3,3- HsjAla labeled silk fibroin fiber was also observed. The quadrupole splitting, Avq, is 39.8 kHz and is the same as the value in the spectrum of the unaligned sample. This is the same as the case of the [2,2- H2]Gly labeled silk fibroin fiber. The smaller value of the quadrupole splitting for the Ala site indicates the presence of three-fold fast rotation about the Ca-C/3 axis [50, 72]. This... 

A restricted (<, ifi) region for Gly in the Ramachandran map (-180° > 0°, 0° > i/r> 180°) was obtained as the overlap of each region obtained experimentally from the NH, NC, CN, and CO bond orientations as described above. The quadrupole echo spectrum of the oriented [2,2- HaJGly labeled silk fibroin fiber yields an angle, 0 = 90 2°, for the C H2 bond vector of Gly residue relative to the fiber axis. A further narrow... 

From the ultraviolet dichroism at 2500 A. of oriented silk fibroin films it was inferred that the axes of the C=N peptenol chromophors were inclined at angles of ca. 45° to the stretching axis of the films, which was also taken to be the direction of the extended polypeptide chains. 

Recently, Kameda etal. characterized and compared the Ser side-chain dynamics of B. mori and S.c. ricini silk fibroin using solid-state -H NMR.- - A detailed lineshape analysis has provided quantitative data on the rate of motion and on the fractions of two distinct dynamic populations. In addition, the torsion angle of the Ser C -C/j bond and its orientational distribution was measured in uniformly aligned silk fibers. Figures 16(a) and (b) show the -H NMR spectra of [3,3--H2]Ser-labeled silk fibroin from B. mori and S.c. ricini, respectively. 

Because of their two-fold symmetry, the phenolic side-chains of Tyr can execute a 7r-flip motion about the Cp-Cy bond between two orientations of locally equal energy. Generally, the effect of molecular motion reduces the quadrupole coupling to a time-averaged value which is smaller than the rigid lattice constant. Thus, the small inner doublet with a splitting of 30 kHz, which is observed both for B. mori and S.c. ricini silk fibroin, is attributed to a fast... 

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