Thermal stability of <i>Clostridium pasteurianum</i> rubredoxin: Deconvoluting the contributions of the metal site and the protein

Bonomi, Francesco; Fessas, Dimitrios; Iametti, Stefania; Kurtz, Donald M.; Mazzini, Stefania

doi:10.1110/ps.9.12.2413

Cited by 31 publications

(40 citation statements)

References 37 publications

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“…2) [19,23], were differentially affected by denaturants. Six molar urea led to a nearly complete loss of the far-UV CD features of apoCpRd, whereas this concentration of urea had no effect on the far-UV CD features of apoPfRd.…”

Section: Holord But Not Apord Structures Are Stable Towards High Denamentioning

confidence: 98%

“…Metal contents of the reconstituted Rds were determined by inductively coupled plasma mass spectrometry (MS) at the Chemical Analysis Laboratory, University of Georgia, Athens, GA, USA. Accessible and total thiols were determined using dithiobisnitrobenzoate in the absence or in the presence of 6 M guanidine, respectively [19].…”

Section: Iron Uptake By Apords and Refolding Of Holordsmentioning

confidence: 99%

“…Both instruments were equipped with computer-controlled, Peltier-driven cell holders for temperature control. 1 H NMR experiments were performed as reported previously [19,20,23] …”

Section: Spectroscopymentioning

confidence: 99%

“…3) [19,23]. The visible CD spectral changes following addition of iron salts required 30-60 min to reach their end points (i.e., no further spectral changes) at room temperature.…”

Section: Recoveries Of Holords From Apords Are Incomplete In the Absementioning

confidence: 99%

“…Investigations of thermally induced unfolding of CpRd showed that loss of the metal ion immediately precedes formation of the (apparently) unstructured apoRd [19], but that Fe(SCys) 4 site stability does not control the thermostability of the native folded structure [20]. Engineered V ↔ A exchanges of residue 44, which forms an N-H…S hydrogen bond to one of the ironcoordinating Cys residues, significantly raised and lowered the respective stabilities of the Fe(SCys) 4 sites in CpRd and PfRd [21][22][23][24] but had only minor effects on the thermal stabilities of the folded holoRds.…”

Section: Introductionmentioning

confidence: 99%

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“Iron priming” guides folding of denatured aporubredoxins

Bonomi¹,

Iametti²,

Ferranti³

et al. 2008

J Biol Inorg Chem

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The relationship between iron uptake by aporubredoxins (apoRds) and formation of native holorubredoxins (holoRd), including their Fe(SCys) 4 sites, was studied. In the absence of denaturants, apoRds exhibited spectroscopic features consistent with structures very similar to those of the folded holoRds. However, additions of either ferric or ferrous salts to the apoRds in the absence of denaturants gave less than 40% recovery of the native holoRd circular dichroism and UV-vis spectroscopic features. In the presence of either 6 M urea or 6 M guanidine hydrochloride, the nativelike structural features of the apoRds were absent. Nevertheless, nearly quantitative recoveries of the native holoRd spectroscopic features were achieved by addition of either ferric or ferrous salts to the denatured apoRds without diluting the denaturant. Consistent with this observation, the native spectroscopic features were unaffected by addition of the same denaturant concentrations to the as-isolated holoRds. Denaturing concentrations of urea or guanidine hydrochloride also increased the rates of holoRd recoveries from apoRds and ferrous salts. Mass spectrometry confirmed that ferric iron binding to the denatured apoRds precedes the recoveries of protein secondary structures and Fe(SCys) 4 sites. Thus, iron binding to the apoRds guides, both kinetically and thermodynamically, refolding to the native holoRd structures. Our results imply that the ferrous oxidation state would more efficiently drive formation of the native holoRd structure from the nascent apoprotein in vivo, but that the Fe(SCys) 4 site must attain the ferric state in order to achieve its native structure.Correspondence to: Francesco Bonomi. Electronic supplementary material The online version of this article

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Section: Holord But Not Apord Structures Are Stable Towards High Denamentioning

confidence: 98%

Section: Iron Uptake By Apords and Refolding Of Holordsmentioning

confidence: 99%

“…Both instruments were equipped with computer-controlled, Peltier-driven cell holders for temperature control. 1 H NMR experiments were performed as reported previously [19,20,23] …”

Section: Spectroscopymentioning

confidence: 99%

“…3) [19,23]. The visible CD spectral changes following addition of iron salts required 30-60 min to reach their end points (i.e., no further spectral changes) at room temperature.…”

Section: Recoveries Of Holords From Apords Are Incomplete In the Absementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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“Iron priming” guides folding of denatured aporubredoxins

Bonomi¹,

Iametti²,

Ferranti³

et al. 2008

J Biol Inorg Chem

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Dispersion Interactions Govern the Strong Thermal Stability of a Protein

Vondrášek

Kubař

Jenney

et al. 2007

Chemistry A European J

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Rubredoxin from the hyperthermophile Pyrococcus furiosus (Pf Rd) is an extremely thermostable protein, which makes it an attractive subject of protein folding and stability studies. A fundamental question arises as to what the reason for such extreme stability is and how it can be elucidated from a complex set of interatomic interactions. We addressed this issue first theoretically through a computational analysis of the hydrophobic core of the protein and its mutants, including the interactions taking place inside the core. Here we show that a single mutation of one of phenylalanine's residues inside the protein's hydrophobic core results in a dramatic decrease in its thermal stability. The calculated unfolding Gibbs energy as well as the stabilization energy differences between a few core residues follows the same trend as the melting temperature of protein variants determined experimentally by microcalorimetry measurements. NMR spectroscopy experiments have shown that the only part of the protein affected by mutation is the reasonably rearranged hydrophobic core. It is hence concluded that stabilization energies, which are dominated by London dispersion, represent the main source of stability of this protein.

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Protein interactions in the biological assembly of iron–sulfur clusters in Escherichia coli: Molecular and mechanistic aspects of the earliest assembly steps

2022

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This contribution focuses on the earliest steps of the assembly of FeS clusters and their insertion into acceptor apoproteins, that call for transient formation of a 2Fe2S cluster on a scaffold protein from sulfide and iron salts. For the sake of simplicity, this report is essentially limited to the Escherichia coli iscencoded proteins and does not take into account agents that modulate the enzymatic synthesis of sulfide by protein in the same operon or the redox events associated with both sulfide generation and conversion of 2Fe2S structures in clusters of higher nuclearity. Therefore, the results discussed here are based on chemical reconstitution systems using inorganic sulfide, ferric salts, and excess thiols. This simplification offers the possibility to address some mechanistic issues related to the role of protein/protein interaction as for modulating: (a) the rate of cluster assembly on scaffold proteins; (b) the stability of the cluster on the scaffold protein; and (c) the rate of transfer to acceptor apoproteins as also influenced by the acceptor concentration. The emerging picture highlights the mechanistic versatility of the systems, that is discussed in terms of the capability of such an apparently simple combination of proteins to cope with various physiological situation. The hypothetical mechanism presented here may represent an additional way of modulating the rate and outcome of the overall process while avoiding potential toxicity issues. K E Y W O R D S chaperones, cluster assembly and transfer, co-chaperones, iron-sulfur clusters, IscA, IscU, scaffold proteins 1 | INTRODUCTION Sulfide-bridged structures containing iron and other metals are considered among the earliest point of contact between inorganic chemistry and living beings.

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Thermal stability of Clostridium pasteurianum rubredoxin: Deconvoluting the contributions of the metal site and the protein

Cited by 31 publications

References 37 publications

“Iron priming” guides folding of denatured aporubredoxins

“Iron priming” guides folding of denatured aporubredoxins

Dispersion Interactions Govern the Strong Thermal Stability of a Protein

Protein interactions in the biological assembly of iron–sulfur clusters in Escherichia coli: Molecular and mechanistic aspects of the earliest assembly steps

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