We recorded X-ray diffraction, 13C CP-MAS NMR, and infrared spectra of silk fibroin samples from Bombyx mori in the solid state, prepared under different conditions, in order to compare conformational features obtained by the respective methods. These three methods provided consistent data as to the presence of the most stable silk II form. Silk I and random-coil forms were distinguishable only by the extent of line broadening in 13C NMR peaks, although the chemical shifts are the same. The most obvious advantage of the 13C CP-MAS NMR approach is that this method is intrinsically nondestructive and very convenient for evaluation of the relative proportion of silk I and II forms in the same sample. We found that powder formation even at liquid-nitrogen temperature caused a change from silk I to random-coil form, although the amount of silk II form remains unchanged during this procedure. In view of the sample preparation conditions as well as the similarity of the 13C chemical shifts and infrared spectra between the silk I and random-coil forms, it is plausible that the random-coil form is a mixture of silk I type forms whose long-range crystalline packing is distorted to the extent to give halo X-ray diffraction.

A survey of recent high-resolution solid-state OC NMR studies is given to illustrate how and to what extent OC chemical shifts of a variety of molecular systems are displaced upon conformational changes. Examples are taken from studies of alkanes, polysaccharides, polypeptides, and naturally occurring and synthetic ionophores, in which more than single conformations are available owing to either crystalline polymorphs or a variety of metal-iigand interactions. The magnitude of the conformation-dependent "C chemical shifts are in many instances as large as 8 ppm and are well related to a set of torsion angles of nearby single bonds. The application of this concept to conformational characterization is also discussed.

Conformational transitions of human calcitonin~hCT! during fibril formation in the acidic and neutral conditions were investigated by high-resolution solid-state 13 C NMR spectroscopy. In aqueous acetic acid solution~pH 3.3!, a local a-helical form is present around Gly10, whereas a random coil form is dominant as viewed from Phe22, Ala26, and Ala31 in the monomer form on the basis of the 13 C chemical shifts. On the other hand, a local b-sheet form as viewed from Gly10 and Phe22, and both b-sheet and random coil as viewed from Ala26 and Ala31 were detected in the fibril at pH 3.3. The results indicate that conformational transitions from a-helix to b-sheet, and from random coil to b-sheet forms occurred in the central and C-terminus regions, respectively, during the fibril formation. The increased 13 C resonance intensities of fibrils after a certain delay time suggests that the fibrillation can be explained by a two-step reaction mechanism in which the first step is a homogeneous association to form a nucleus, and the second step is an autocatalytic heterogeneous fibrillation. In contrast to the fibril at pH 3.3, the fibril at pH 7.5 formed a local b-sheet conformation at the central region and exhibited a random coil at the C-terminus region. Not only a hydrophobic interaction among the amphiphilic a-helices, but also an electrostatic interaction between charged side chains can play an important role for the fibril formation at pH 7.5 and 3.3 acting as electrostatically favorable and unfavorable interactions, respectively. These results suggest that hCT fibrils are formed by stacking antiparallel b-sheets at pH 7.5 and a mixture of antiparallel and parallel b-sheets at pH 3.3.

High-resolution 13C NMR (75.46 MHz) spectra of silk fibroins in the solid state were recorded by the cross-polarization-magic angle spinning method with emphasis on revealing conformational features of the dimorphic structures, silk I and II, of Bombyx mori fibroin prepared under different conditions. It was found that the 13C chemical shifts of Gly, Ala, and Ser residues of silk II samples from B. mori fibroin by different preparations and of cocoon samples from several silkworms gave identical values with those of corresponding model polypeptides having the /3-sheet conformation. Thus, identification of the silk II type form is easily performed by examining their 13C chemical shift values. The 13C chemical shifts of samples having the silk I form are significantly displaced from those of the silk II form, and can be used for diagnostic purposes. As expected, the 13C chemical shifts of Ala and Gly residues of the silk I samples were identical with those of (Ala-Gly)"II. However, we found that none of the 13C chemical shifts predicted from the crankshaft model [Lotz, B.; Keith, H. D. J. Mol. Biol. 1971, 61, 201-215] in which Ala and Gly residues are close to the /S-sheet and -helix conformations, respectively, was in agreement with the 13C chemical shifts of (Ala-Gly)"II and silk I type form. Instead, we found that predicted 13C chemical shifts from the loose helix proposed by Konishi and Kurokawa [Konishi, T.; Kurokawa, M. Sen'i Gakkaishi 1968, 24, 550-554] on the basis of the calculated 13C contour map of chemical shifts for Ala residue are in good agreement with the displacement of the l3C chemical shifts.

No abstract

Glu-194 near the extracellular surface of bacteriorhodopsin is indispensable for proton release to the medium upon protonation of Asp-85 during light-driven transport. As for Glu-204, its replacement with glutamine (but not aspartate) abolishes both proton release and the anomalous titration of Asp-85 that originates from coupling between the pKa of this buried aspartate and those of the other acidic groups. Unlike the case of Glu-204, however, replacement of Glu-194 with aspartate raises the pKa for proton release. In Fourier transform infrared spectra of the E194D mutant a prominent positive band is observed at 1720 cm-1. It can be assigned from [4-13C]aspartate and D2O isotope shifts to the C&dbd;O stretch of protonated Asp-194. Its rise correlates with proton transfer from the retinal Schiff base to Asp-85. Its decay coincides with the appearance of a proton at the surface, detected under similar conditions with fluorescein covalently bound to Lys-129 and with pyranine. Its amplitude decreases with increasing pH, with a pKa of about 9. We show that this pKa is likely to be that of the internal proton donor to Asp-194, the Glu-204 site, before photoexcitation, while 13C NMR titration indicates that Asp-194 has an initial pKa of about 3. We propose that there is a chain of interacting residues between the retinal Schiff base and the extracellular surface. After photoisomerization of the retinal the pKa's change so as to allow (i) Asp-85 to become protonated by the Schiff base, (ii) the Glu-204 site to transfer its proton to Asp-194 in E194D, and therefore to Glu-194 in the wild type, and (iii) residue 194 to release the proton to the medium.

No abstract

No abstract