Enhancement of protein stability by an additional disulfide bond designed in human neuroglobin

Abstract

A disulfide bond of Cys120 and Cys15 was rationally designed in human neuroglobin (Ngb) by A15C mutation, which caused minimal structural alterations, whereas enhanced both chemical and pH stability, with a thermal stability higher than 100 °C.

The X‐ray crystal structure of human A15C neuroglobin reveals both native/de novo disulfide bonds and unexpected ligand‐binding sites

“…The A15C Ngb mutant was expressed and purified as reported previously. 19 To obtain crystals for the mutant with good quality, we screened different crystallization conditions using the crystal screen reagent kits from Hampton research. We found that the mutant formed crystals suitable for X-ray diffraction with high quality (Figure 1, top-left) at 16 C within 2 weeks by using the number 23 reagent (1.6 mol/L ammonium sulfate, 0.1 mol/L 2-MES monohydrate pH 6.5, 10% v/v 1,4-dioxane).…”

“…The A15C Ngb mutant was expressed and purified as reported previously 19 . To obtain crystals for the mutant with good quality, we screened different crystallization conditions using the crystal screen reagent kits from Hampton research.…”

“…Motivated by these studies, we envisaged that the design of a de novo disulfide bond with Cys120 might be able to not only protect the cysteine from modification by ROS/RNS but also enhance the protein stability. For this purpose, in a previous study, we introduced an additional Cys at position 15 close to Cys120 based on the structural information 19 . Experimental studies showed that the A15C Ngb mutant exhibited both enhanced chemical and pH stabilities compared to those of WT Ngb, with thermal stability >100°C.…”

Impact of zinc oxide nanoparticles on thioredoxin‐interacting protein and asymmetric dimethylarginine as biochemical indicators of cardiovascular disorders in gamma‐irradiated rats

“…Further, cysteine residues consider as an antioxidant defense system either directly by reacting with reactive ROS and RNS, or can be represented as cofactors for several enzymes. Additionally, cysteine residues are important modifying the structure and function of many proteins . In turn, oxidized Trx‐1 is reactivated by NADPH‐dependent flavoprotein TrxR, a homodimeric, selenium comprising flavoprotein oxidoreductase to reduce the disulfide bonds of proteins through thiol‐disulfide exchange reactions .…”

Evidence for a synergistic effect of post‐translational modifications and genomic composition of eEF‐1α on the adaptation of Phytophthora infestans

“…A disulfide bridge is the covalent bond formed by linking sulfur atoms of two spatially close cysteine residues. This type of PTM can serve either as a structural or a biochemical role by affecting protein folding, stability, and function (Creighton et al., 1995; Xiao Liu et al., 2019; Zhang et al., 2018). There are three types of disulfide bonds in cells (Cook & Hogg, 2013).…”