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Belton, D.J., Plowright, R., Kaplan, D.L. and Perry, C.C. 
ORCID: 0000-0003-1517-468X,
2018.
A robust spectroscopic method for the determination of protein conformational composition - application to the annealing of silk.
Acta Biomaterialia, 73, pp. 355-364.
ISSN 1742-7061
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Abstract
The physical and mechanical properties of structural proteins such as silk fibroin can be modified by controlled conformational change, which is regularly monitored by Fourier transform infrared spectroscopy by peak fitting of the amide I band envelope. Although many variables affecting peak shape are well established, there is no fixed methodology to compare and follow secondary structural differences without significant operator input especially where low frequency spectral noise is a problem.
The aim of this contribution is to establish a method for such analyses to be carried at high levels of autonomy to prevent subjective or erroneous fitting. A range of approaches was trialled with optimal peak parameters selected based on overall goodness of fit and reproducibility of fit of replicate sample spectra. The method was successfully tested against reference proteins having contrasting β content and the rationale for parameter selection is presented.
Further, we applied this method to measure the effect of conformational change on the energy of the amide I band of silk fibroin during annealing. Energy changes were ca. 400 kJ mol−1 of fibroin. To confirm that this energy change was a consequence of increased hydrogen bonding we used a Thioflavin T staining method typically used to identify β aggregate type structures in amyloid plaques.
We propose that the approach described herein can aid in the development of silk based materials for biomedical applications where tuning of the physical and mechanical properties of the silk are needed to guarantee optimum activity.
| Item Type: | Journal article | ||||||
|---|---|---|---|---|---|---|---|
| Publication Title: | Acta Biomaterialia | ||||||
| Creators: | Belton, D.J., Plowright, R., Kaplan, D.L. and Perry, C.C. | ||||||
| Publisher: | Elsevier | ||||||
| Date: | June 2018 | ||||||
| Volume: | 73 | ||||||
| ISSN: | 1742-7061 | ||||||
| Identifiers: |
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| Divisions: | Schools > School of Science and Technology | ||||||
| Record created by: | Jonathan Gallacher | ||||||
| Date Added: | 11 May 2018 14:30 | ||||||
| Last Modified: | 06 Jun 2018 10:08 | ||||||
| URI: | https://irep.ntu.ac.uk/id/eprint/33545 |
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