Orientation of silk III at the air-water interface

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Abstract

A threefold helical crystal structure of Bombyx mori silk fibroin has been observed in films prepared from aqueous silk fibroin solutions using the Langmuir–Blodgett (LB) technique. The films were studied using a combination of transmission electron microscopy and electron diffraction techniques. Films prepared at a surface pressure of 16.7 mN/m have a uniaxially oriented crystalline texture, with the helical axis oriented perpendicular to the plane of the LB film. Films obtained from the air-water interface without compression have a different orientation, with the helical axes lying roughly in the plane of the film. In both cases the d-spacings observed in electron diffraction are the same and match a threefold helical model crystal structure, silk III, described in previous publications. Differences in the relative intensities of the observed reflections in both types of oriented samples, as compared to unoriented samples, allows estimations of orientation distributions and the calculations of orientation parameters. The orientation of the fibroin chain axis in the plane of the interfacial film for uncompressed samples is consistent with the amphiphilic behavior previously postulated to drive the formation of the threefold helical silk III conformation.

Introduction

A new crystal structure of Bombyx mori silk fibroin was observed in films taken from the air-water interface of aqueous fibroin solutions [1], [2], [3]. The crystal structure, silk III, involves an approximately hexagonal packing of silk molecules in a left handed threefold helical chain conformation. Because this conformation separates the serine and alanine residues, creating a hydrophilic column of residues parallel to the helical axis, surfactant behavior of fibroin at the air-water interface is believed to play a role in selecting the conformation and subsequent crystal structure at the interface. However, much of the data used to characterize the structure was for LB films compressed to 16.7 mN/m. These films possess a uniaxial crystallite orientation similar to a sedimented mat, with the crystallite c (or chain) axes predominantly oriented perpendicular to the plane of the film. If the fibroin is behaving as a surfactant, assuming a threefold helical conformation that separates its hydrophobic and hydrophilic residues, the chain axis of the fibroin molecules and of any resulting crystallites should lie in the plane of the film. While it is possible that compression on the Langmuir trough could reorient the fibroin molecules at the interface, data on the orientation of uncompressed films are needed for comparison.

Solid film formation has been observed previously on uncompressed surfaces of aqueous fibroin solution, but only a cursory examination of the morphology with no supporting diffraction data was reported [4]. In the current study, electron diffraction and TEM morphology data have been obtained for uncompressed surface films. The electron diffraction data clearly indicates a silk III crystal structure with the helical axes lying in the plane of the uncompressed film. The relative intensities in the diffraction patterns are altered in a systematic fashion, consistent with this orientation, i.e. the intensities of 00l reflections are enhanced and the intensity of hk0 reflections are attenuated. The oriented crystalline textures observed are uniform, and can be controlled by varying the surface compression treatments.

Section snippets

Experimental

Bombyx mori cocoons were degummed in order to remove sericin, yielding pure fibroin. This was achieved by boiling cocoon silk for about 1.5 h in distilled water with 1.1 wt.% CaCO3 and 6.6 weight percent sodium dodecyl sulfate (SDS). After this initial treatment, the fibroin was rinsed with distilled water and then boiled a second time in distilled water with 0.4 weight percent CaCO3 for about 1 h. Amino acid analysis has been used to assess the protein composition of fibroin prepared in this

Results and discussion

The silk protein films which form at interfaces are dominated by surface phenomena. The regenerated silk solutions used to prepare our thin films exhibit a marked tendency to foam, indicating surfactancy, and the residue sequence in the crystallizable regions of B. mori fibroin suggests a mechanism for the surface activity of this molecule. The six residue repeating sequence of the crystallizable portion of silk fibroin, [Gly-Ala-Gly-Ala-Gly-Ser]n, in a sterically reasonable, left handed

Conclusions

A plausible explanation of the orientation behavior in the compressed silk LB films is possible which is consistent with our observations. The silk excess layer that forms at the air-water interface results in aggregates of the threefold helical structure with an asymmetric shape. These aggregates possess the orientation one would intuitively expect, with the chain axis lying in the plane of the interface. When the trough is compressed these aggregates realign so that they each occupy a minimum

Acknowledgements

Funding from the National Science Foundation in the form of a CAREER grant (SPG), DMR 9624306, is gratefully acknowledged. Use of central facilities in the NSF funded Materials Research Science and Engineering Center (MRSEC) at the University of Massachusetts Amherst is also acknowledged as is the use of the facilities of the W.M. Keck Electron Microscopy Laboratory.

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