The Modification of Cystine — Cleavage of Disulfide Bonds
Roger L. Lundblad in Chemical Reagents for Protein Modification, 2020
There are several approaches to the cleavage of disulfide bonds in proteins. The majority of studies involve the cleavage of the disulfide bond of cystine to the free thiol group of cysteine by reduction. Reduction has been generally accomplished with a mild reducing agent such as β-mercaptoethanol or cysteine. Gorin and co-workers1 have examined the rate of reaction of lysozyme with various thiols. At pH 10.0 (0.025 M borate), the relative rates of reaction were β-mercaptoethanol (2-mercaptoethanol), 0.2; dithiothreitol, 1.0; 3-mercaptopropionate, 0.4; and 2-aminoethanol, 0.01. The results with aminoethanethiol were somewhat surprising since the reaction (disulfide exchange) involves the thiolate anion and 2-aminoethanethiol would be more extensively ionized than the other mercaptans. Dithiothreitol has been a useful reagent in the reduction of disulfide bonds in proteins2 as introduced by Cleland. Dithiothreitol and the isomeric form, dithioerythritol, are each capable of the quantitative reduction of disulfide bonds in proteins. Furthermore, the oxidized form of dithiothreitol has an absorbance maximum at 283 nm (Δϵ = 273) which can be used to determine the extent of disulfide bond cleavage.2 The UV spectra of dithiothreitol and oxidized dithiothreitol are shown in Figure 1. Insolubilized dihydrolipoic acid has also been proposed for use in the quantitative reduction of disulfide bonds.4
The Glutathione Redox State and Zinc Mobilization from Metallothionein and Other Proteins with Zinc–Sulfur Coordination Sites
Christopher A. Shaw in Glutathione in the Nervous System, 2018
As GSSG is biosynthesized from GSH, whose concentration varies in the range 0.1–10 mM (Meister 1988), its concentration is regulated by that of GSH. GSSG is also the major end product of the decomposition of S–nitrosoglutathione (Hogg, Singh, and Kalyanaraman 1996; Singh et al. 1996), a compound that is now considered a biologically significant donor of nitric oxide. Alternatively, the concentration of GSSG can change independently if the redox state changes. In the cytosol, where the GSH/GSSG ratio typically is between 30:1 and 100:1, reducing conditions prevail. However, this does not apply to all cellular compartments. Examination of the GSH/GSSG ratio in the endoplasmic reticulum reveals that it is more oxidizing by a factor between 10 and 100 than that of the cytosol, resulting in GSH/GSSG ratios between 3:1 and 1:1 (Hwang, Sinskey, and Lodish 1992). These are thought to be necessary for disulfide formation in protein folding. Even small changes of the GSH/GSSG redox ratio modify physiological responses (Ziegler 1985; Gilbert 1990), but how such changes (including redox compartmentation) affect mineral metabolism is unknown.
Superoxide Dismutase, Mitochondrial Dysfunction, and Neurodegenerative Diseases
Shamim I. Ahmad in Handbook of Mitochondrial Dysfunction, 2019
ALS prognosis correlates with the extent of IC-CuZnSOD aggregation into fibrils, much like amyloid-β fibrils in Alzheimer’s disease [70]. Fibrils form by IC-CuZnSOD misfolding and self-association into high-order, low energy structures. Examples are metal-deficient variants that destabilize the tertiary structure of the protein, and non-native interactions are energetically favored [116]. Other variants, affecting monomer structure and stability of the dimer interface, are thought to aggregate into fibrils as a result of lower thermostability and increased disorder [70,116]. Protein fold stability is further compromised through a lack of disulfide bond formation. Despite the cytosol being a strong reducing environment, IC-CuZnSOD harbors an oxidized disulfide bond that is buried near the dimer interface in WT. Variants that decrease dimer stability and expose the bond are more susceptible to disulfide reduction by glutathione/glutaredoxin systems [134]. A lack of Cu or Zn metallation also decreases dimer stability, meaning variants that compromise metal binding also expose the disulfide bond for reduction. The propensity for IC-CuZnSOD to form fibrils is related to factors compromising its stability, such as mutations and reduced disulfide bonds.
Novel mutation (R192C) in CYB5R3 gene causing NADH-cytochrome b5 reductase deficiency in eight Indian patients associated with autosomal recessive congenital methemoglobinemia type-I
Published in Hematology, 2018
Prabhakar S. Kedar, Vinod Gupta, Prashant Warang, Ashish Chiddarwar, Manisha Madkaikar
The crystal structure of the NADH-binding domain of CYTB5R has been elucidated and is extremely useful for understanding the relationship between CYTB5R structure and function. The NADH-binding domain of CYTB5R displays an α/β fold, with a central β-sheet formed by five parallel and one anti-parallel β-strands flanked by Nα1-helices on each side. The arrangement of the α−helices and the β strands in the NADH-binding domain is to form three β1−α−β2 motifs: Νβ1−Να1−Νβ2 (Ser173-Asn209), Νβ2−Να3−Νβ3 (Val202-Leu238) and Νβ5−Να6−Νβ6 (Leu269-Phe300) [13]. The first motif is typical of the NAD+-binding motif, in which the glycine-rich loop (Gly179–Gly182) forms a 310-turn and is in close contact with the ADP part of NAD+ in human CYTB5R. The codon 192 is localized in the β-strand at the amino edge of the β-sheet and forms a salt bridge with the buried isoleucine (I) codon 97 localized at the bottom of the Nβ2-strand (Figure 3). This contributes to the stabilization of the salt-bridge network elaborated between some of the charged amino acids of the NADH-binding domain. Within extracellular proteins, cysteine is frequently involved in disulfide bonds, where pairs of cysteine are oxidized to form a covalent bond. If one half of a disulfide bond pair is lost, then the protein may not fold properly. As suggested by Bando et al. [13], some of these salt bridges are important elements of folding, and therefore the mutation of R192C involved in a salt bridge may induce the abnormal folding of the Nβ2-strand.
Evaluation of the role of thiol / disulfide homeostasis in the etiology of idiopathic male infertility with a novel and automated assay
Published in Systems Biology in Reproductive Medicine, 2022
Uygar Micoogullari, Mehmet Caglar Cakici, Furkan Umut Kilic, Erdem Kisa, Burak Ozcift, Alper Caglayan, Salim Neselioglu, Omer Faruk Karatas, Ozcan Erel
Thiol-disulfide homeostasis is vital because it is involved in numerous functions such as detoxification, apoptosis, antioxidant protection, signal transduction, as well as regulation of transcription factors, enzymatic activity, and signaling mechanisms (Circu and Aw 2010; Erel and Neselioglu 2014; Cabrillana et al. 2016; Alsalman et al. 2018). Thiols have strong antioxidant capacities in their sulfhydryl groups, containing sulfur and hydrogen atoms covalently bonded to a carbon atom. This disulfide bond is also called a disulfide bridge or SS-bond. Total thiol represents the total number of reducible/oxidizable sulfur atoms in serum. It denotes both the two sulfides in each disulfide and the sum of one sulfur atom in each native thiol. Thiols play a crucial role in protecting cells from the harmful effects of ROS (Erel and Neselioglu 2014; Eren et al. 2015). The majority of the plasma thiol pool consists primarily of albumin and other proteins. A small portion consists of low molecular weight thiols such as glutathione, cysteine, cysteinyl glycine, γ-glutamylcysteine, and homocysteine (Erel and Neselioglu 2014). ROS oxidize thiol groups of proteins, cysteine residues, and low molecular weight compounds to form reversible disulfide bond structures. The thiol-disulfide balance can be reached by reducing those disulfide bonds to thiols again. Thiols in the blood are oxidized first in response to an increase in ROS (Erel and Neselioglu 2014).
Antibody markup language (AbML) — a notation language for antibody-based drug formats and software for creating and rendering AbML (abYdraw)
Published in mAbs, 2022
James Sweet-Jones, Maham Ahmad, Andrew C.R. Martin
Users may draw antibody-based drugs from scratch or begin with a template design of common formats (including MsAbs) that may be manipulated by the user. To draw domains, a user must select a specificity and any modifications for that domain and then place it on the canvas. Both specificities and modifications can be updated whilst on the canvas by selecting a specificity or modification, but not a domain type. Once drawn, domains may be moved to a space where they interact with other domains to be paired. VH and VL domains must face each other to be considered as interacting. Users can right-click newly drawn domains to change the direction they are facing. Nanobody domains cannot be paired with other domains, as these are single-domain VHH fragments. Standard connectors between domains are drawn by starting on the N-terminal domain of the pair and ending on the C-terminal domain of the pair. Disulfide bonds can be drawn starting from either of the interacting domains (including linkers and hinges). To insert a comment (e.g., NOTE, TYPE, ANTI, MOD), the appropriate comment-type button is clicked and, in the case of TYPE and MOD, which have restricted allowed values, the required value is selected from a drop-down list. If the desired comment is not available, comment text is typed into the text entry box and the required domain is clicked to associate the comment with that domain. Clicking the ‘Tidy’ button will then relocate the comment to the bottom of the canvas, but the comment will still be associated with the specified domain.
Related Knowledge Centers
- Biochemistry
- Cysteine
- Functional Group
- Inorganic Chemistry
- Protein
- Thiol
- Persulfide
- Ion
- Dihedral Angle
- Diphenyl Disulfide