Dataset Information

Contributions of the C-terminal helix to the structural stability of a hyperthermophilic Fe-superoxide dismutase (TcSOD).

ABSTRACT: Hyperthermophilic superoxide dismutases (SODs) are of particular interest due to their potential industrial importance and scientific merit in studying the molecular mechanisms of protein folding and stability. Compared to the mesophilic SODs, the hyperthermostable Fe-SODs (TcSOD and ApSOD) have an extended C-terminal helix, which forms an additional ion-pairing network. In this research, the role of the extended C-terminus in the structural stability of TcSOD was studied by investigating the properties of two deletion mutants. The results indicated that the ion-pairing network at the C-terminus had limited contributions to the stability of TcSOD against heat- and GdnHCl-induced inactivation. The intactness of the C-terminal helix had dissimilar impact on the two stages of TcSOD unfolding induced by guanidinium chloride. The mutations slightly decreased the Gibbs free energy of the dissociation of the tetrameric enzymes, while greatly affected the stability of the molten globule-like intermediate. These results suggested that the additional ion-pairing network mainly enhanced the structural stability of TcSOD by stabilizing the monomers.

SUBMITTER: Wang S

PROVIDER: S-EPMC2802007 | biostudies-literature | 2009 Dec

REPOSITORIES: biostudies-literature

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Contributions of the C-terminal helix to the structural stability of a hyperthermophilic Fe-superoxide dismutase (TcSOD).

Wang Sha S Yan Yong-Bin YB Dong Zhi-Yang ZY

International journal of molecular sciences 20091223 12

Hyperthermophilic superoxide dismutases (SODs) are of particular interest due to their potential industrial importance and scientific merit in studying the molecular mechanisms of protein folding and stability. Compared to the mesophilic SODs, the hyperthermostable Fe-SODs (TcSOD and ApSOD) have an extended C-terminal helix, which forms an additional ion-pairing network. In this research, the role of the extended C-terminus in the structural stability of TcSOD was studied by investigating the pr ...[more]

PMID: 20054483

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Project description:We have prepared and thoroughly characterized, using X-ray crystallographic, spectroscopic, and computational methods, the diazide adduct of [Fe(III)(dapsox)(H2O)2](+) [dapsox = 2,6-diacetylpyridinebis(semioxamazide)], (1), a low-molecular weight, functional analogue of iron superoxide dismutase (FeSOD). The X-ray crystal structure of the dimeric form of 1, (Na[Fe(III)(dapsox)(N3)2]·DMF)2 (2) shows two axially coordinated, symmetry inequivalent azides with differing Fe-N3 bond lengths and Fe-N-N2 bond angles. This inequivalence of the azide ligands likely reflects the presence of an interdimer hydrogen bonding interaction between a dapsox NH group and the coordinated nitrogen of one of the two azide ligands. Resonance Raman (rR) data obtained for frozen aqueous solution and solid-state samples of 2 indicate that the azides remain inequivalent in solution, suggesting that one of the azide ligands of 1 engages in an intermolecular hydrogen bonding interaction with a water molecule. Density functional theory (DFT) and time-dependent DFT calculations have been used to study two different computational models of 1, one using coordinates taken from the X-ray crystal structure of 2, and the other generated via DFT geometry optimization. An evaluation of these models on the basis of electronic absorption, magnetic circular dichroism, and rR data indicates that the crystal structure based model yields a more accurate electronic structure description of 1, providing further support for the proposed intermolecular hydrogen bonding of 1 in the solid state and in solution. An analysis of the experimentally validated DFT results for this model reveals that the azides have both ?- and ?-bonding interactions with the Fe(III) center and that more negative charge is located on the Fe-bound, rather than on the terminal, nitrogen atom of each azide. These observations are reminiscent of the results previously reported for the azide adduct of FeSOD and provide clues regarding the origin of the high catalytic activity of Fe-dapsox for superoxide disproportionation.

Project description:Background:Characterizing the structure and function of superoxide dismutase (SOD), as an antioxidant enzyme providing opportunities for its application in food supplements. Objectives:In this study, the features of the Manganese-SOD of Lactococcus Lactis (SDLL), subsp. Cremoris MG1363, as probiotic bacteria, were determined on the basis of the computational methods. Materials and Methods:The protein's physicochemical properties and the prediction of its secondary structure were determined via the ProtParam server and the GOR program respectively. Moreover, the 3D structures of the proteins were constructed via the MODELLER on the basis of the homology method and the threading algorithm MUSTER. On the other hand, the structural stability of the models was assayed under the quasi-physiological conditions by the GROMACS program via the GROMOS96 43a1 force field in Linux system. Finally, using the Molecular Docking Studies, the functionality features of the models were predicted through their affinity with the corresponding substrates. Results:The results revealed the physicochemical properties of the SDLL and a 3D model of a chain of the enzyme being similar to the SOD from the Bacillus Subtilis (SDBS). The model of the SDLL was checked for quality control purposes including the Ramachandran plot, the ERRAT and the Verifiy3D. The model was suggestive of the structural stability in quasi-physiological conditions; yet, less than that of the SDBS. Assessing the cause of the instability in the SDLL model was indicative of two unstable regions in the area far from the enzyme's active position, they were considered suitable for mutagenesis. Accordingly, the loop substitution for the corresponding region of SDBS and the deletion of the loop positioned at the C terminal of SDLL resulted in a mutant of SDLL with more stability and appropriate affinity with the corresponding substrate. Conclusion:In general, the study provides a new model of SDLL with certain thermostable features, and a new mutant with suitable stability and functionality on the basis of the direct mutagenesis being used in different applications.

Dataset Information

Contributions of the C-terminal helix to the structural stability of a hyperthermophilic Fe-superoxide dismutase (TcSOD).

Publications

Contributions of the C-terminal helix to the structural stability of a hyperthermophilic Fe-superoxide dismutase (TcSOD).

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