Guanidine-induced unfolding of the Sso7d protein from the hyperthermophilic archaeon Sulfolobus solfataricus

Granata, Vincenzo; Vecchio, Pompea Del; Barone, G.; Shehi, Erlet; Fusi, Paola; Tortora, Paolo; Graziano, Giuseppe

doi:10.1016/j.ijbiomac.2004.04.002

Cited by 10 publications

(3 citation statements)

References 53 publications

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“…34,35 Sso7d (T m 98°C) is the most thermostable protein possessing the SH3-fold. 36 Mutagenesis of multiple residues in a protein, as in the case of generating binding proteins through Wild type Sso7d and Sso7d mutants were incubated in 20 mM sodium phosphate buffer with various concentrations of Gdn-HCl for 12-16 h at 4°C and then the CD spectra were recorded. The fraction of folded protein was estimated as, f = (θ -θ max )/( θ min -θ max ), where θ is the baseline-corrected observed molar ellipticity at 222 nm, and θ max and θ min are the maximum and minimum values of molar ellipticity for a given protein, respectively.…”

Section: Discussionmentioning

confidence: 99%

Highly Stable Binding Proteins Derived from the Hyperthermophilic Sso7d Scaffold

Gera

Hussain

Wright

et al. 2011

Journal of Molecular Biology

150

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Section: Discussionmentioning

confidence: 99%

Highly Stable Binding Proteins Derived from the Hyperthermophilic Sso7d Scaffold

Gera

Hussain

Wright

et al. 2011

Journal of Molecular Biology

150

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“…The fold of Sso7d is highly similar to those of the chromo domain4 and the SH3 domain,5 small structural modules which mediate the protein–protein interactions in eukaryotic and bacterial cells. Sso7d is an amenable model for structural studies given its thermal, acid, and chemical stability 6. As the function of the protein is concerned, Sso7d is a multifunctional protein in in vitro assays.…”

Section: Introductionmentioning

confidence: 99%

Structural characterization of the functional regions in the archaeal protein Sso7d

et al. 2007

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Sso7d from the extreme thermophilic crenarchaeon Sulfolobus solfataricus is a multifunctional protein in in vitro assays, whose in vivo role is still puzzling. Crystals of Sso7d in complex with DNA elucidated the protein surface involved in the binding to the nucleic acid, whereas the locations of the Sso7d regions responsible for a chaperone activity in renaturing protein aggregates (i.e., the protein-binding surface and the site of ATPase activity) are still unknown. We identified the regions of Sso7d involved in protein-binding by limited proteolysis experiments associated to advanced mass spectrometric procedures performed on isolated Sso7d and Sso7d in complex with the peptide melittin. By affinity labeling of Sso7d with the ATP analogue 5'-p-fluorosulfonylbenzoyl adenosine and characterization of the labeled tryptic peptides by tandem mass spectrometry, we found that Y7 and K39 are residues involved in ATP binding/hydrolysis. Insights into the positions of the ligands melittin and ATP were achieved by a molecular modeling study; the models obtained were in agreement with most experimental data. A comparison among the complexes of Sso7d with DNA, with melittin, and with ATP showed that the DNA-binding surface and the protein-binding surface overlap, whereas the ATPase site is mostly independent of the binding sites for the nucleic acid and melittin.

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“…Sso7d and its aromatic mutants were denatured under extreme conditions of temperature, pressure, pH and solvents 2, 17, 27. In particular, temperature and pressure profiles indicated a strong destabilization of mutant F31A, in the order of 20 degrees and 10 kbar with respect to the Sso7d 2.…”

Section: Discussionmentioning

confidence: 99%

Structural determinants responsible for the thermostability of Sso7d and its single point mutants

et al. 2007

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Sso7d is a small protein especially attractive as a model for characterizing by NMR, the structural determinants responsible for thermal stability. With this aim, structural parameters of the Sso7d protein have been compared with those of single point mutants, in particular with K12L, more stable (T m around 1008C) and F31A, less stable (T m 618C) than the wild-type. In addition, a mutant lacking eight residues of the C-terminal helix (T m 508C) has been studied to investigate the role of the helix in structure stabilization. Melting temperatures, DDG's of unfolding, cavities, electrostatic interactions, solvent accessible areas, hydrophobic cores, and aromatic cluster geometries have been analyzed in order to gain insights into the key structural factors determining the thermostability differences. Our data suggest that absence of cavities and a larger salt bridge network represent the major determinants contributing to the enhanced stability of K12L relative to the Sso7d protein and its mutant F31A.Introduction. The interest for thermophilic proteins has increased during the last years for several reasons: first of all they can open a wide field of microbiological and technical industrial applications and processes, furthermore, they provide excellent models for understanding the molecular mechanisms underlying protein folding and stabilization, and for describing their functional properties. The stability of proteins from organisms living under extreme conditions is a long standing issue and different authors attributed several factors affecting their greater structural stability with respect to the mesophilic counterpart. 1 It has been pointed out that there is not a unique structural requirement for making a protein thermostable. Several factors have been found to play a relevant role in the protein stability, such as increased hydrophobic and aromatic contacts, optimization of charge-charge interactions, and side chain packing 1 ; however, the mechanisms by which these proteins attain thermostability remain unclear.In the last years we have studied the recombinant form of Sso7d, initially isolated from the thermoacidophilic archeaobacterium Sulfolobus solfataricus living in volcanic hot springs at 878C and displaying a pressure resistance up to 14 kbar. 2 The protein is composed of 62

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Guanidine-induced unfolding of the Sso7d protein from the hyperthermophilic archaeon Sulfolobus solfataricus

Cited by 10 publications

References 53 publications

Highly Stable Binding Proteins Derived from the Hyperthermophilic Sso7d Scaffold

Highly Stable Binding Proteins Derived from the Hyperthermophilic Sso7d Scaffold

Structural characterization of the functional regions in the archaeal protein Sso7d

Structural determinants responsible for the thermostability of Sso7d and its single point mutants

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