Naturally Stable Free Radical in the Silk Fibroin and Its Structure Environment Studied by EPR and DFT

ABSTRACT

EPR spectra of Bombyx mori silk fibroin show a singlet peak at g = 2.0030 ± 0.0005, which is thought to be a signal of the naturally stable free radical. We found that the intensity of EPR signal increases with the increase in pH of the prepared samples and that the signal recorded at ambient temperature is more symmetric and sharper than that recorded at 100 K. We calculated the ¹H hyperfine coupling constants by the density functional theory (DFT) and simulated the EPR spectrum by the EasySpin software for the tyrosyl radical models. We found that the EPR spectrum of the tyrosyl radical models is dependent sensitively on the local structure of the protein. Furthermore, the calculation demonstrated that the EPR signal of the tyrosyl radical model is isotropic in the model silk fibroin in Silk I conformation, while the signal is anisotropic in the model silk fibroin in Silk II conformation. Therefore, we conclude that the naturally stable free-radical signals observed in the silk fibroin are most likely from the tyrosyl radicals and that the radicals reside in a highly hydrophobic silk fibroin domain in Silk I conformation. It is important to understand the formation mechanism and the lifetime of the free radicals caused by the external factors including molecular structure, pH, heat, light, mechanic force, and the like in the silk fibroin for development of silk applications in the biomaterial field.

KEYWORDS:

• conformation
• DFT
• protein
• Tyrosyl radical

ACKNOWLEDGMENTS

This work was supported by Natural Science Foundations of China (No. 10475017, 20673022, and 21074025) and postdoctoral foundation from Shanghai. The authors give many thanks to High-End Computing Centre in Fudan University for the Gaussian calculation with 16 CPUs of Inter Xeon 3.06 GHz and also thank Prof. Pan, Prof. Zhou, and Prof. Shi of Research Center of Analysis and Test in East China University of Science and Technology for their kind help in the EPR measurements.

Notes

^a The anisotropic ¹H hyperfine couplings A (A _xx , A _yy , A _zz ) include Fermi interaction (A _iso ) and dipolar components T (T _xx , T _yy , T _zz ).

^a The anisotropic ¹H hyperfine couplings A (A _xx , A _yy , A _zz ) include Fermi interaction (A _iso ) and dipolar components T (T _xx , T _yy , T _zz ).

^a The anisotropic ¹H hyperfine couplings A (A _xx , A _yy , A _zz ) include Fermi interaction (A _iso ) and dipolar components T (T _xx , T _yy , T _zz ).