A robust spectroscopic method for the determination of protein conformational composition - application to the annealing of silk

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:
NumberType
10.1016/j.actbio.2018.03.058DOI
S1742706118301909Publisher Item Identifier
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: http://irep.ntu.ac.uk/id/eprint/33545

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