Protein engineering on enzymes of the peptide elongation cycle in Sulfolobus solfataricus

Bocchini, Vincenzo; Adinolfi, B.S.; Arcari, Paolo; Arcucci, Alessandro; Russo, Antonio Dello; Vendittis, Emmanuele De; Ianniciello, G.; Raimo, Gennaro

doi:10.1016/s0300-9084(00)88885-9

Cited by 7 publications

(10 citation statements)

References 16 publications

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“…In this work, we continue our investigation of the molecular mechanisms at the origin of extreme thermal stability by comparing the behavior of a representative pair of mesophilic/hyperthermophilic homologues: the G domains of the elongation factor thermo unstable (EF-Tu) protein from the mesophilic Escherichia coli bacterium and EF-1α protein from the hyperthermophilic archaeon Sulfolobus solfataricus . The thermal stabilities of the two domains differ by about 40 K , and are qualitatively well reproduced in silico . By using extensive MD simulations at high temperature ( T = 360 K), we previously probed the early steps of unfolding of the mesophilic domain.…”

Section: Introductionmentioning

confidence: 73%

Role of Internal Water on Protein Thermal Stability: The Case of Homologous G Domains

et al. 2014

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In this work we address the question whether the enhanced stability of thermophilic proteins has a direct connection with internal hydration. Our model systems are two homologues G-domains of different stability: the mesophilic G domain of the Elongation-Factor thermal unstable protein from E. coli and the hyperthermophilic G domain of the EF-α protein from S. solfataricus. Using molecular dynamics simulation at the microsecond time-scale we show that both proteins host water molecules in internal cavities and that these molecules exchange with the external solution in the nanosecond time scale. The hydration free energy of these sites evaluated via extensive calculations is found to be favourable for both systems, with the hyperthermophilic protein offering a slightly more favourable environment to host water molecules. We estimate that under ambient conditions, the free energy gain due to internal hydration is about 1.3 kcal/mol in favor of the hyperthermophilic variant. However, we also find that at the high working temperature of the hyperthermophile, the cavities are rather dehydrated, meaning that at extreme conditions other molecular factors secure the stability of the protein. Interestingly, we detect a clear correlation between the hydration of internal cavities and the protein conformational landscape. The emerging picture is that internal hydration is an effective observable to probe the conformational landscape of proteins. In the specific context of our investigation, the analysis confirms that the hyperthermophilic G-domain is characterized by multiple states and it has a more flexible structure than its mesophilic homologue.

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Section: Introductionmentioning

confidence: 73%

Role of Internal Water on Protein Thermal Stability: The Case of Homologous G Domains

et al. 2014

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“…Materials. Deuterium oxide (99.9% 2 H 2 O), 2 HCl, and NaO 2 H were purchased from Aldrich. GDP, Gpp(NH)p, and Tris were obtained from Sigma.…”

Section: Methodsmentioning

confidence: 99%

Salts Induce Structural Changes in Elongation Factor 1α from the Hyperthermophilic ArchaeonSulfolobus solfataricus: A Fourier Transform Infrared Spectroscopic Study^,

Tanfani¹,

Scirè²,

Raimo³

et al. 2001

Biochemistry

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Elongation factor 1alpha from the hyperthermophilic archaeon Sulfolobus solfataricus (SsEF-1alpha) carries the aminoacyl tRNA to the ribosome; it binds GDP or GTP, and it is also endowed with an intrinsic GTPase activity that is triggered in vitro by NaCl at molar concentrations [Masullo, M., De Vendittis, E., and Bocchini, V. (1994) J. Biol. Chem. 269, 20376-20379]. The structural properties of SsEF-1alpha were investigated by Fourier transform infrared spectroscopy. The estimation of the secondary structure of the SsEF-1alpha*GDP complex, made by curve fitting of the amide I' band or by factor analysis of the amide I band, indicated a content of 34-36% alpha-helix, 35-40% beta-sheet, 14-19% turn, and 7% unordered structure. The substitution of the GDP bound with the slowly hydrolyzable GTP analogue Gpp(NH)p induced a slight increase in the alpha-helix and beta-sheet content. On the other hand, the alpha-helix content of the SsEF-1alpha*GDP complex increased upon addition of salts, and the highest effect was produced by 5 M NaCl. The thermal stability of the SsEF-1alpha*GDP complex was significantly reduced when the GDP was replaced with Gpp(NH)p or in the presence of NaBr or NH4Cl, whereas a lower destabilizing effect was provoked by NaCl and KCl. Therefore, the extent of the destabilizing effect of salts depended on the nature of both the cation and the anion. The data suggested that the sodium ion was responsible for the induction of the GTPase activity, whereas the anion modulated the enzymatic activity through destabilization of particular regions of SsEF-1alpha. Finally, the infrared data suggested that, in particular region(s) of the polypeptide chain, the SsEF-1alpha*Gpp(NH)p complex possesses structural conformations which are different from those present in the SsEF-1alpha*GDP complex.

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“…The E. coli and SS G-domains are stable and functional at very different temperatures, e.g. half guanosine diphosphate (GDP) binding activity is measured at about 41 1C and 84 1C for the mesophile 16 and the hyperthermophile, 17 respectively. While as shown in Fig.…”

Section: And References Within)mentioning

confidence: 99%

“…The E.Coli and SS G-domains are stable and functional at very different temperatures, e.g. half guanosine diphosphate (GDP) binding activity is measured at about 41°C and 84 °C for the mesophile 16 and the hyperthermophile 17 , respectively. While as shown in Figure 1 the two G-domains share a similar structure, their sequence identity is rather low, 34% and the hyperthermophilic variant is enriched in charged (mostly basic) amino-acids, ~ 30% vs ~ 23%.…”

Section: Introductionmentioning

confidence: 99%

The effect of protein composition on hydration dynamics

Rahaman

Melchionna

Laage

et al. 2013

Phys. Chem. Chem. Phys.

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Water dynamics at the surface of two homologous proteins with different thermal resistances is found to be unaffected by the different underlying amino-acid compositions, and when proteins are folded it responds similarly to temperature variations. Upon unfolding the water dynamics slowdown with respect to bulk decreases by a factor of two. Our findings are explained by the dominant topological perturbation induced by the protein on the water hydrogen bond dynamics.

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Protein engineering on enzymes of the peptide elongation cycle in Sulfolobus solfataricus

Cited by 7 publications

References 16 publications

Role of Internal Water on Protein Thermal Stability: The Case of Homologous G Domains

Role of Internal Water on Protein Thermal Stability: The Case of Homologous G Domains

Salts Induce Structural Changes in Elongation Factor 1α from the Hyperthermophilic ArchaeonSulfolobus solfataricus: A Fourier Transform Infrared Spectroscopic Study^,

The effect of protein composition on hydration dynamics

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Protein engineering on enzymes of the peptide elongation cycle in Sulfolobus solfataricus

Cited by 7 publications

References 16 publications

Role of Internal Water on Protein Thermal Stability: The Case of Homologous G Domains

Role of Internal Water on Protein Thermal Stability: The Case of Homologous G Domains

Salts Induce Structural Changes in Elongation Factor 1α from the Hyperthermophilic ArchaeonSulfolobus solfataricus: A Fourier Transform Infrared Spectroscopic Study,

The effect of protein composition on hydration dynamics

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Salts Induce Structural Changes in Elongation Factor 1α from the Hyperthermophilic ArchaeonSulfolobus solfataricus: A Fourier Transform Infrared Spectroscopic Study^,