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Diselenide crosslinks for enhanced and simplified oxidative protein folding

DOI: 10.1038/s42004-021-00463-9 DOI Help

Authors: Reem Mousa (The Hebrew University of Jerusalem) , Taghreed Hidmi (The Hebrew University of Jerusalem) , Sergei Pomyalov (The Hebrew University of Jerusalem) , Shifra Lansky (The Hebrew University of Jerusalem) , Lareen Khouri (The Hebrew University of Jerusalem) , Deborah E. Shalev (The Hebrew University of Jerusalem) , Gil Shoham (The Hebrew University of Jerusalem) , Norman Metanis (The Hebrew University of Jerusalem)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Communications Chemistry , VOL 4

State: Published (Approved)
Published: March 2021

Open Access Open Access

Abstract: The in vitro oxidative folding of proteins has been studied for over sixty years, providing critical insight into protein folding mechanisms. Hirudin, the most potent natural inhibitor of thrombin, is a 65-residue protein with three disulfide bonds, and is viewed as a folding model for a wide range of disulfide-rich proteins. Hirudin’s folding pathway is notorious for its highly heterogeneous intermediates and scrambled isomers, limiting its folding rate and yield in vitro. Aiming to overcome these limitations, we undertake systematic investigation of diselenide bridges at native and non-native positions and investigate their effect on hirudin’s folding, structure and activity. Our studies demonstrate that, regardless of the specific positions of these substitutions, the diselenide crosslinks enhanced the folding rate and yield of the corresponding hirudin analogues, while reducing the complexity and heterogeneity of the process. Moreover, crystal structure analysis confirms that the diselenide substitutions maintained the overall three-dimensional structure of the protein and left its function virtually unchanged. The choice of hirudin as a study model has implications beyond its specific folding mechanism, demonstrating the high potential of diselenide substitutions in the design, preparation and characterization of disulfide-rich proteins.

Journal Keywords: Protein design; Protein folding

Subject Areas: Chemistry, Biology and Bio-materials


Instruments: I04-Macromolecular Crystallography

Added On: 17/03/2021 09:26

Documents:
s42004-021-00463-9.pdf

Discipline Tags:

Life Sciences & Biotech Structural biology Chemistry Biochemistry

Technical Tags:

Diffraction Macromolecular Crystallography (MX)