Denaturant-Induced Unfolding of the Acetyl-Esterase from <i>Escherichia coli</i>

Vecchio, Pompea Del; Graziano, Giuseppe; Granata, Vincenzo; Farias, Tiziana; Barone, G.; Mandrich, Luigi; Rossi, Mosè; Manco, Giuseppe

doi:10.1021/bi0400507

Cited by 4 publications

(5 citation statements)

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“…It is typically believed that the maximum size for a cooperative domain is about 200 residues (27)(28)(29). However, in recent years, several exceptions, especially among extremophilic proteins, have been found (27)(28)(29)(30)(31)(32). The data presented here suggest that even a large protein with a rather loose structure can exhibit a cooperative unfolding.…”

Section: Discussionmentioning

confidence: 51%

Chemical Denaturation of the Elongation Factor 1α Isolated from the Hyperthermophilic ArchaeonSulfolobus solfataricus

et al. 2005

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The stability against chemical denaturants of the elongation factor EF-1alpha (SsEF-1alpha), a protein isolated from the hyperthermophilic archaeon Sulfolobus solfataricus has been characterized in detail. Indeed, the atypical shape of the protein structure and the unusual living conditions of the host organism prompted us to analyze the effect of urea and guanidine hydrochloride (GuHCl) on the GDP complex of the enzyme (SsEF-1alpha x GDP) by fluorescence and circular dichroism. These studies were also extended to the nucleotide-free form of the protein (nfSsEF-1alpha). Interestingly, the experiments show that the denaturation curves of both SsEF-1alpha forms present a single inflection point, which is indicative of a cooperative unfolding process with no intermediate species. Moreover, the chemically induced unfolding process of both SsEF-1alpha x GDP and nfSsEF-1alpha is fully reversible. Both SsEF-1alpha forms exhibit remarkable stability against urea, but they do not display a strong resistance to the denaturing action of GuHCl. These findings suggest that electrostatic interactions significantly contribute to SsEF-1alpha stability.

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Section: Discussionmentioning

confidence: 51%

Chemical Denaturation of the Elongation Factor 1α Isolated from the Hyperthermophilic ArchaeonSulfolobus solfataricus

et al. 2005

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“…The three structures are closely similar: superimposing backbone atoms, the RMSD amounts to 1.11 Å for the pair EST2-AFEST, 0.79 Å for the pair EST2-Aes, and 1.26 Å for the pair AFEST-Aes [9]. The obtained results are reviewed below, except fluorescence data, for which interested readers are referred to the original articles [10,11]. The structure of EST2 is shown in Fig.…”

Section: Introductionmentioning

confidence: 85%

“…Denaturant-induced unfolding of AFEST, EST2 and Aes was investigated by means of both circular dichroism [10] and fluorescence measurements [11,19], in 20 mM phosphate buffer, pH 7.5 and 20 °C. The denaturation induced by urea or GuHCl was found to be reversible for all the three enzymes, as renaturation of completely unfolded samples by suitable dilution showed a full recovery of all the spectro-scopic signals of the enzymes in the native state.…”

Section: Stability Against Chemical Denaturantsmentioning

confidence: 99%

Conformational Stability of Esterase Enzymes from Different Sources

Grazianob

Vecchio²,

Barone³

2009

PPL

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In the last years we have performed a series of studies to characterize the conformational stability of three esterases from thermophilic and mesophilic sources: Aes esterase from Escherichia coli, EST2 from Alicyclobacillus acidocaldarius and AFEST from Archeoglobus fulgidus. These three esterases belong to the Hormone-sensitive lipase group of the superfamily of carboxylester hydrolases. The conformational stability of the three enzymes against temperature, urea and GuHCl has been determined by means of circular dichroism, fluorescence and differential scanning calorimetry measurements. Analysis of experimental data coupled with available structural information allowed us to suggest that the optimization of charge-charge interactions on the protein surface could one of the mechanisms to increase the thermal stability for the three esterases. This idea has been tested in the case of EST2, which shows a fully reversible thermal unfolding, by producing and studying variant forms of wild type enzyme in which a charged residue has been mutated. In the present article the obtained results are critically recollected in order to provide a clear and unified scenario.

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“…Moreover, these data are in agreement with some experimental evidence on the importance of salt bridges for protein stability. Recent findings demonstrate that thermal stability is not simply correlated with the number of ion pairs, but more interestingly, with optimized electrostatic interactions Del Vecchio et al, 2005). It is important to underline that not all the above-mentioned changes should be expected to occur in a single protein molecule, since proteins may adopt several strategies in altering their stability and activity in response to environmental variations (Mandrich and de Pascale, 2011).…”

Section: Proteinsmentioning

confidence: 99%