Diselenide crosslinks for enhanced and simplified oxidative protein folding

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.

One-Pot Chemical Protein Synthesis Utilizing Fmoc-Masked Selenazolidine to Address the Redox Functionality of Human Selenoprotein F

“…We also checked the folding of hirudin as an alternative candidate, where it folds to the native state through heterogenous pathway that involve the formation of non-native intermediates. 42 However, under anaerobic conditions and in the presence of catalytic amounts of SELENOFTrx, hirudin folded through the established pathway 39,42 showing no difference when compared to the control experiment (Fig. S12c, d).…”

Integrated Top-Down and Bottom-Up Mass Spectrometry for Characterization of Diselenide Bridging Patterns of Synthetic Selenoproteins

“…Assessing the best way to decipher diselenide-bridged peptides utilizing the predicted digestion products also allows more facile analysis of peptides containing unanticipated diselenide bridges. During the proteolytic digestion process used for bottom-up analysis, the possibility of diselenide scrambling must be considered because disulfide scrambling has been reported to occur during digestion. , The replacement of sulfur with diselenide for cysteine bridges, however, has repeatedly been shown to lead to improved stability. ,, To mitigate diselenide scrambling, the digestion was performed at pH 6, which has previously been shown to limit disulfide scrambling. , After establishing the conditions to limit scrambling, the identification of unanticipated diselenide bonds is possible.…”

A shelf stable Fmoc hydrazine resin for the synthesis of peptide hydrazides

“…[17] This method avoids the need for strong oxidizing conditions, enabling broader compatibility with common synthetic peptide functionalities, and has been widely adopted in a broad range of applications. [20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37] The increased use of C-terminal α-hydrazides in peptide chemistry creates a demand for robust synthetic tools for accessing these moieties. A number of methods have been published to allow access to C-terminal hydrazides on both synthetic [15,16,38] and expressed [15,39,40] peptides and proteins.…”