Application of circular dichroism and fluorescence spectroscopies to assess photostability of water-soluble porcine lens proteins

DOI: 10.1021/acsomega.9b04234

Authors: Claudia Honisch (Institute of Biomolecular Chemistry of CNR; University of Padova) , Viola Donadello (Institute of Biomolecular Chemistry of CNR) , Rohanah Hussain (Diamond Light Source) , Daniele Peterle (University of Padova) , Vincenzo De Filippis (University of Padova) , Giorgio Arrigoni (University of Padova) , Claudio Gatto (Alchilife Srl) , Laura Giurgola (Alchilife Srl) , Giuliano Siligardi (Diamond Light Source) , Paolo Ruzza (Institute of Biomolecular Chemistry of CNR)
Co-authored by industrial partner: Yes

Type: Journal Paper
Journal: Acs Omega

State: Published (Approved)
Published: February 2020
Diamond Proposal Number(s): 8034 , 20210

Abstract: The eye lens is mainly composed of the highly ordered water-soluble (WS) proteins named crystallins. The aggregation and insolubilization of these proteins lead to progressive lens opacification until cataract onset. Although this is a well-known disease, the mechanism of eye lens protein aggregation is not well understood; however, one of the recognized causes of proteins modification is related to the exposure to UV light. For this reason, the spectroscopic properties of WS lens proteins and their stability to UV irradiation have been evaluated by different biophysical methods including synchrotron radiation circular dichroism, fluorescence, and circular dichroism spectroscopies. Moreover, dynamic light scattering, gel electrophoresis, transmission electron microscopy, and protein digestion followed by tandem LC–MS/MS analysis were used to study the morphological and structural changes in protein aggregates induced by exposure to UV light. Our results clearly indicated that the exposure to UV radiation modified the protein conformation, inducing a loss of ordered structure and aggregation. Furthermore, we confirmed that these changes were attributable to the generation of reactive oxygen species due to the irradiation of the protein sample. This approach, involving the photodenaturation of proteins, provides a benchmark in high-throughput screening of small molecules suitable to prevent protein denaturation and aggregation.

Journal Keywords: Peptides and proteins; Electromagnetic radiation; Protein structure; Optical properties; Irradiation

Subject Areas: Biology and Bio-materials, Chemistry


Instruments: B23-Circular Dichroism

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