The role of a disulfide bridge in the stability and folding kinetics of Arabidopsis thaliana cytochrome c6A

Mason, Jody M.; Bendall, Derek S.; Howe, Christopher J.; Worrall, J.A.R.

doi:10.1016/j.bbapap.2011.10.015

Cited by 12 publications

(8 citation statements)

References 62 publications

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“…All titrations were carried out in triplicate. The fraction unfolded (F u ) at any given [GuHCl] was determined, 45 and the free energy of unfolding (ΔG unf ) and dependence of ΔG unf on denaturant concentration (m value) were calculated using an equation for a two-state unfolding process. 46 The pH dependence of the 695 nm band in the UV−visible spectrum was monitored at 20 °C by determining its absorbance at various values of pH for a solution of 100 μM oxidized Cc in a quartz cuvette (Hellma) with a small aliquot of K 3 [Fe(CN) 6 ] present to maintain an oxidizing environment.…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

Naturally Occurring Disease-Related Mutations in the 40–57 Ω-Loop of Human Cytochrome c Control Triggering of the Alkaline Isomerization

et al. 2018

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Naturally occurring mutations found in one of the two Ω-loop substructures in human cytochrome c are associated with low blood platelet count (thrombocytopenia). Both Ω-loops participate in the formation of conformers associated with cytochrome c peroxidase activity and apoptotic function. At alkaline pH values, the Met80 ligand to the ferric heme iron dissociates, and a lysine residue in the 71-85 Ω-loop coordinates to the iron. The alkaline isomerization has been the focus of extensive kinetic studies, and it is established that a deprotonation triggers the release of the Met80 ligand (p K). A second deprotonation stabilizes a pentacoordinate heme form (p K). In this study, site-directed variants at the 41 and 48 positions in the 40-57 Ω-loop and at the 81 and 83 positions in the 71-85 Ω-loop reveal that conformational transitions in the 71-85 Ω-loop, leading to the alkaline or peroxidatic conformers, are controlled by the 40-57 Ω-loop. We find that the variants causing thrombocytopenia, G41S and Y48H, lower the p K and increase p K. Our results are presented in a mechanistic framework, depicted by a cube, that accounts for the pH dependencies of the equilibrium and kinetic parameters governing the alkaline transition of the native protein and Ω-loop variants. The data are most consistent with the trigger for Met80 replacement by a lysine being a deprotonation within a hydrogen bonded unit that links the two Ω-loops rather than an individual group. Such a proposal aligns with the entatic contribution made by the same unit in controlling the Met80-Fe(III) bond strength.

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Section: ■ Materials and Methodsmentioning

confidence: 99%

Naturally Occurring Disease-Related Mutations in the 40–57 Ω-Loop of Human Cytochrome c Control Triggering of the Alkaline Isomerization

et al. 2018

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“…∆Gunf is the free energy of denaturation in water and m represents the dependence of the free energy of denaturation on [GuHCl]. To represent the data in terms of fraction denatured (FD) at any given [GuHCl] equation(2)was used [47].…”

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confidence: 99%

Comparison of the structural dynamic and mitochondrial electron-transfer properties of the proapoptotic human cytochrome c variants, G41S, Y48H and A51V

Deacon

White

Moore

et al. 2020

Journal of Inorganic Biochemistry

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Mitochondrial cytochrome c is associated with electron transfer in the respiratory chain and in apoptosis. Four cytochrome c variants have been identified in families that suffer from mild autosomal dominant thrombocytopenia, a platelet disorder associated with increased apoptosis. Three out of the four substitutions, G41S, Y48H and A51V are located on the 40-57 Ω-loop. The G41S and Y48H variants perturb key physicochemical and dynamic properties that result in enhanced functional features associated with apoptotic activity. Herein we characterise the ferric A51V variant. We show by chemical denaturation that this variant causes the native state to be destabilized. Through azide binding kinetics, the population of a pentacoordinate heme form, whereby the Met80 axial ligand is dissociated, is estimated to be of equal magnitude to that found in the Y48H variant. This pentacoordinate form gives rise to peroxidase activity, which despite the similar pentacoordinate population of the A51V variant to that of the Y48H variant, the peroxidase activity of the A51V variant is supressed. Far-UV circular dichroism spectroscopy and pH jump studies, suggest that a combination of structural and dynamic features in addition to the population of the pentacoordinate form regulate peroxidase activity in these disease variants. Additionally, the steady-state ratio of ferric/ferrous cytochrome c when in turnover with cytochrome c oxidase has been investigated for all 40-57 Ω-loop variants. These studies show that the lower pKa of the alkaline transition for the disease causing variants increases the ferric to ferrous heme ratio, indicating a possible influence on respiration in vivo.

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“…Next, we can also argue that folding of VEGFR1D2 may be considered a hierarchic process where global folding is ignited by small‐sized local structures (e.g., the disulfide bridge) that interact to generate intermediate states of increasing complexity leading, ultimately, to the native structure [36]. Intriguingly, there are other known examples (e.g., insulin and pro‐insulin) where protein folding is driven by an oxidatively induced disulfide pairing [37], thus supporting the hypothesis that a disulfide bond can act as folding nucleus and ease the folding process [38].…”

Section: Discussionmentioning

confidence: 95%

Structural characterization of the thermal unfolding pathway of human VEGFR1 D2 domain

et al. 2021

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Folding stability is a crucial feature of protein evolution and is essential for protein functions. Thus, the comprehension of protein folding mechanisms represents an important complement to protein structure and function, crucial to determine the structural basis of protein misfolding. In this context, thermal unfolding studies represent a useful tool to get a molecular description of the conformational transitions governing the folding/unfolding equilibrium of a given protein. Here, we report the thermal folding/unfolding pathway of VEGFR1D2, a member of the immunoglobulin superfamily by means of a high‐resolution thermodynamic approach that combines differential scanning calorimetry with atomic‐level unfolding monitored by NMR. We show how VEGFR1D2 folding is driven by an oxidatively induced disulfide pairing: the key event in the achievement of its functional structure is the formation of a small hydrophobic core that surrounds a disulfide bridge. Such a ‘folding nucleus’ induces the cooperative transition to the properly folded conformation supporting the hypothesis that a disulfide bond can act as a folding nucleus that eases the folding process.

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The role of a disulfide bridge in the stability and folding kinetics of Arabidopsis thaliana cytochrome c6A

Cited by 12 publications

References 62 publications

Naturally Occurring Disease-Related Mutations in the 40–57 Ω-Loop of Human Cytochrome c Control Triggering of the Alkaline Isomerization

Naturally Occurring Disease-Related Mutations in the 40–57 Ω-Loop of Human Cytochrome c Control Triggering of the Alkaline Isomerization

Comparison of the structural dynamic and mitochondrial electron-transfer properties of the proapoptotic human cytochrome c variants, G41S, Y48H and A51V

Structural characterization of the thermal unfolding pathway of human VEGFR1 D2 domain

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