Biomedical subjects

Marie-Eve Rousseau

Publications and source records attributed to Marie-Eve Rousseau.

4 recordsLinked to original sources

PubMed2006

Quantitative mapping of the orientation of fibroin beta-sheets in B. mori cocoon fibers by scanning transmission X-ray microscopy.

The spatial distribution of the linear dichroic signal associated with aligned beta-sheets in a microtomed section of a Bombyx mori cocoon silk fiber was derived from scanning transmission X-ray microscopy (STXM). The intense C 1s --> pi(amide) peak at 288.25 eV was found to have negligible dichroic signal in transverse sections but a large dichroic signal in longitudinal sections. This is consistent with other measurements of the orientation of the aligned beta-sheets in silk fibers, in particular with those obtained by polarized Raman microspectroscopy to which our results are compared. When the dichroic signal strength is mapped at better than 100 nm spatial resolution, microscopic variations are found. Although the magnitude of the dichroic signal changes over a fine spatial scale, the direction of the maximum signal at any position does not change. We interpret the spatial variation of the intensity of the dichroic signal as a map of the quality of local orientation of the beta-sheets in the fiber. At sufficiently high magnification and resolution, this technique should image individual beta-sheet crystallites, although the present implementation does not achieve that. A map of the orientation parameter P(2) is derived. The average value of P(2) (-0.20 +/- 0.04) from STXM is smaller than that derived from the analysis of the amide I band in polarized Raman spectra (-0.41 +/- 0.03). This deviation is attributed to the fact that the STXM results also include the signal from unaligned regions of the protein.

Animals↗

PubMed2006

Characterization by Raman microspectroscopy of the strain-induced conformational transition in fibroin fibers from the silkworm Samia cynthia ricini.

Raman microspectroscopy has been used to quantitatively study the effect of a mechanical deformation on the conformation and orientation of Samia cynthia ricini (S. c. ricini) silk fibroin. Samples were obtained from the aqueous solution stored in the silk gland and stretched at draw ratios (lambda) ranging from 0 to 11. Using an appropriate band decomposition procedure, polarized and orientation-insensitive spectra have been analyzed to determine order parameters and the content of secondary structures, respectively. The data unambiguously show that, in response to mechanical deformation, S. c. ricini fibroin undergoes a cooperative alpha-helix to beta-sheet conformational transition above a critical draw ratio of 4. The alpha-helix content decreases from 33 to 13% when lambda increases from 0 to 11, while the amount of beta-sheets increases from 15 to 37%. In comparison, cocoon silk is devoid of alpha-helical structure and always contains a larger amount of beta-sheets. Although the presence of isosbestic points in different spectral regions reveals that the conformational change induced by mechanical deformation is a two-state process, our results suggest that part of the glycine residues might be incorporated into beta-poly(alanine) structures. The beta-sheets are initially isotropically distributed and orient along the fiber axis as lambda increases, but do not reach the high level of orientation found in the cocoon fiber. The increase in the orientation level of the beta-sheets is found to be concomitant with the alpha --> beta conformational conversion, whereas alpha-helices do not orient under the applied strain but are rather readily converted into beta-sheets. The components assigned to turns exhibit a small orientation perpendicular to the fiber axis in stretched samples, showing that, overall, the polypeptide chains are aligned along the stretching direction. Our results suggest that, in nature, factors other than stretching contribute to the optimization of the amount of beta-sheets and the high degree of orientation found in natural cocoon silk.

Algorithms↗

PubMed2006

Orientation-insensitive spectra for Raman microspectroscopy.

Separating effects due to molecular conformation from those due to orientation in the spectra of oriented samples obtained by Raman microspectroscopy is a complex issue. To solve this problem, we propose a procedure to calculate an orientation-insensitive spectrum (so-called isotropic spectrum) from polarized spectra obtained by Raman microspectroscopy that is valid for systems that exhibit a uniaxial symmetry. The method has first been tested on highly oriented samples of high-density polyethylene (HDPE). Polarized and isotropic spectra of a highly oriented HDPE cylindrical rod and an isotropic HDPE sample have been compared. The differences in the relative intensities, which occur in the polarized spectra and are due to orientation of the polyethylene chains, are nearly cancelled in the isotropic spectra, showing that the orientation-insensitive spectrum adequately represents the molecular conformation without contributions of orientation. Second, spectra of silk fibroins have been compared in the amide I region for Bombyx mori cocoon silk fibers and methanol-treated regenerated fibroin films. The similarity of the shape of the amide I band of the isotropic spectra indicates that the secondary structure of the fibroins is very close in both samples. These experimental results support the conclusion that the molecular conformation can be efficiently characterized from the intensity and the shape of Raman bands in the orientation-insensitive spectrum.

Animals↗

PubMedDate not supplied

Study of protein conformation and orientation in silkworm and spider silk fibers using Raman microspectroscopy.

Raman microspectroscopy has been used for the first time to determine quantitatively the orientation of the beta-sheets in silk monofilaments from Bombyx mori and Samia cynthia ricini silkworms, and from the spider Nephila edulis. It is shown that, for systems with uniaxial symmetry such as silk, it is possible to determine the order parameters P2 and P4 of the orientation distribution function from intensity ratios of polarized Raman spectra. The equations allowing the calculation of P2 and P4 using polarized Raman microspectroscopy for a vibration with a cylindrical Raman tensor were first derived and then applied to the amide I band that is mostly due to the C=O stretching vibration of the peptide groups. The shape of the Raman tensor for the amide I vibration of the beta-sheets was determined from an isotropic film of Bombyx mori silk treated with methanol. For both the Bombyx mori and Samia cynthia ricini fibroin fibers, the values of P2 and P4 obtained are equal to -0.36 +/- 0.03 and 0.19 +/- 0.02, respectively, even though the two types of silkworm fibroins strongly differ in their primary sequences. For the Nephila edulis dragline silk, values of P2 and P4 of -0.32 +/- 0.02 and 0.13 +/- 0.02 were obtained, respectively. These results clearly indicate that the carbonyl groups are highly oriented perpendicular to the fiber axis and that the beta-sheets are oriented parallel to the fiber axis, in agreement with previous X-ray and NMR results. The most probable distribution of orientation was also calculated from the values of P2 and P4 using the information entropy theory. For the three types of silk, the beta-sheets are highly oriented parallel to the fiber axis. The orientation distributions of the beta-sheets are nearly Gaussian functions with a width of 32 degrees and 40 degrees for the silkworm fibroins and the spider dragline silk, respectively. In addition to these results, the comparison of the Raman spectra recorded for the different silk samples and the polarization dependence of several bands has allowed to clarify some important band assignments.

Animals↗