Diselenide Crosslinks for Enhanced and Simplified Oxidative Protein Folding

Abstract

The 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, which plague its folding rate and yield in vitro. Aiming to overcome these limitations, we undertook a systematic investigation of diselenide bridges at native and non-native positions and investigated their effect on hirudin’s folding, structure and activity. Our studies demonstrated that, regardless of the specific positions of these substitutions, the diselenide crosslinks enhanced the folding rate and yield of the corresponding hirudin analogs, while reducing the complexity and heterogeneity of the process. Moreover, crystal structure analysis confirmed that the diselenide substitutions maintained the overall structure of the protein and left the 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 SELENOF Trx , hirudin folded through the established pathway [39,42] showing no difference when compared to the control experiment (Figure S12c and d).…”

“…Weissman and Kim studied BPTI folding in the presence of PDI, [37] which increased dramatically both the yield and the rate of the native state formation. In BPTI folding, PDI functions as a catalyst for the rate‐determining intramolecular rearrangement from the trapped intermediates N’ and N* to form , the direct precursor to N. Furthermore, previously the Sec‐substituted BPTI [38] and hirudin [39] analogs, as well as other proteins [40] have been investigated, and in all cases, Sec was found to enhance the folding kinetics and yields of these proteins. We have initiated our studies on BPTI by following previously reported folding conditions [40] .…”

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. [ 20–37 ]…”

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.…”