Protein Secondary Structure and Orientation in Silk as Revealed by Raman Spectromicroscopy

Abstract

Taking advantage of recent advances in polarized Raman microspectroscopy, and based on a rational decomposition of the amide I band, the conformation and orientation of proteins have been determined for cocoon silks of the silkworms Bombyx mori and Samia cynthia ricini and dragline silks of the spiders Nephila clavipes and Nephila edulis. This study distinguished between band components due to beta-sheets, beta-turns, 3(1)-helices, and unordered structure for the four fibers. For B. mori, the beta-sheet content is 50%, which matches the proportion of residues that form the GAGAGS fibroin motifs. For the Nephila dragline and S. c. ricini cocoon, the beta-sheet content (36-37% and 45%, respectively) is higher than the proportion of residues that belong to polyalanine blocks (18% and 42%, respectively), showing that adjacent GGA motifs are incorporated into the beta-sheets. Nephila spidroins contain fewer beta-sheets and more flexible secondary structures than silkworm fibroins. The amorphous polypeptide chains are preferentially aligned parallel to the fiber direction, although their level of orientation is much lower than that of beta-sheets. Overall, the results show that the four silks exhibit a common molecular organization, with mixtures of different amounts of beta-sheets and flexible structures, which are organized with specific orientation levels.

Structure of silk by raman spectromicroscopy: From the spinning glands to the fibers

“…Typically, the b-sheet content that is found by this method is 37 6 3% for the dragline, 45 6 3% for the cocoon silk of S. c. ricini and 50 6 3% for that of B. mori, 33 which is basically consistent with crystallinity measurements. 11,[44][45][46][47] Comparison between the b-sheet content determined from the spectroscopic data and the percentage of amino acids that may potentially be included into the b-sheets lead to the conclusion that for the dragline fiber the AG and GGA motifs adjacent to the (A) n are incorporated into the b-sheets, 33 which is consistent with the literature. [48][49][50] Other results have shown that the minor ampullate (Mi) and cylindriform (Cyl) silks belong to the same structural family than the Ma silk as they are composed of highly oriented b-sheets (35-37%) dispersed in an amorphous matrix made of other slightly oriented secondary structures.…”

“…33 On the basis of these assumptions, the orientation distribution function, N(h), which represents the probability of finding a given structural unit oriented at an angle h with respect to the fiber axis, can be expanded into a series of Legendre polynomials P ' (cos h) such as 31 :…”

“…26 This methodology has been applied to various types of silk. 26,33,37 For instance, the values obtained from the height of the amide I band of silkworm silk and of the dragline of N. edulis are P 2 5 20.36 and P 2 5 20.32, respectively. 26 The limiting values of P 2 are 20.5 P 2 1, 20.5 corresponding to a perfect orientation of all the structural units (Dirac distribution) perpendicular to the fiber axis, 1 corresponding to a perfect orientation of all structural units parallel to the fiber axis.…”

“…14 Another approach consists in peak-fitting the amide I band using ''synthetic'' bands and a least square-based method to perform an iterative adjustment of the height, width and position of the components. 33,38 However, to be valuable, the procedure has to be based on a rigorous model, else, mathematically correct, but physically meaningless results may be obtained. Therefore, to be robust, the model has to be based on reliable spectroscopic information, on available structural insights derived from other techniques as well as on a control of the band parameters.…”

Molecular and Nanostructural Mechanisms of Deformation, Strength and Toughness of Spider Silk Fibrils

“…1a) [6][7][8]. Thereby, the anti-parallel beta-sheet nanocrystals [9][10][11][12] play a key role in defining the mechanical properties as they provide stiff orderly cross-linking domains embedded in a semiamorphous matrix that consists of less orderly beta-structures, 3 1 helices and beta-turns [6,13,14].…”