Sulfolobus solfataricus carboxypeptidase (CPSso) is a thermostable zinc-metalloenzyme, consisting of four identical subunits with a Mr of 43000. In a previous paper [Occhipinti et al. (2003) Biohys J. 85, 1165-1175] we developed a structure of the enzyme by molecular modeling and validated it by site-directed mutagenesis and small angle X-ray scattering. Here, we report investigations aimed at further validating the model, as well as at identifying molecular determinants responsible for thermostability. To this end, we took advantage of mass spectrometry techniques, notably LC-MS/MS. The structure was confirmed by such approaches, in that they lead to the identification of a disulfide bridge formed by Cys286 and Cys293, whose location in the model is well suited for giving rise to the cross link. More notably, we also identified a protease-resistant core, consisting of the N- and C-terminal antiparallel alpha-helices, which in the model are predicted to interact with each other via hydrophobic quadrants. Based on the model, we also tentatively identified the most tightly interacting residues as Leu7, Ala380 and Leu376. Whereas the replacement of Ala380 by serine did not detectably impair protein stability, a dramatic drop in thermostability was observed when the two leucines were replaced by either aspartate (L7D; L376D) or asparagine (L7N; L376N). We then investigated the kinetic thermal stability of the wild type and the mutants by determining the thermodynamic activation parameters, deltaG++, deltaH++ and deltaS++. Besides highlighting the key role of the hydrophobic core in thermostability, these results suggest clearly different mechanisms of destabilization by the single mutations, depending on whether the leucines are replaced by asparagines or aspartates.

Sommaruga, S., De Palma, A., Mauri, P., Trisciani, M., Basilico, F., Martelli, P., et al. (2008). A combined approach of mass spectrometry, molecular modeling, and site-directed mutagenesis highlights key structural features responsible for the thermostability of Sulfolobus solfataricus carboxypeptidase. PROTEINS, 71(4), 1843-1852 [10.1002/prot.21868].

A combined approach of mass spectrometry, molecular modeling, and site-directed mutagenesis highlights key structural features responsible for the thermostability of Sulfolobus solfataricus carboxypeptidase

TORTORA, PAOLO;
2008

Abstract

Sulfolobus solfataricus carboxypeptidase (CPSso) is a thermostable zinc-metalloenzyme, consisting of four identical subunits with a Mr of 43000. In a previous paper [Occhipinti et al. (2003) Biohys J. 85, 1165-1175] we developed a structure of the enzyme by molecular modeling and validated it by site-directed mutagenesis and small angle X-ray scattering. Here, we report investigations aimed at further validating the model, as well as at identifying molecular determinants responsible for thermostability. To this end, we took advantage of mass spectrometry techniques, notably LC-MS/MS. The structure was confirmed by such approaches, in that they lead to the identification of a disulfide bridge formed by Cys286 and Cys293, whose location in the model is well suited for giving rise to the cross link. More notably, we also identified a protease-resistant core, consisting of the N- and C-terminal antiparallel alpha-helices, which in the model are predicted to interact with each other via hydrophobic quadrants. Based on the model, we also tentatively identified the most tightly interacting residues as Leu7, Ala380 and Leu376. Whereas the replacement of Ala380 by serine did not detectably impair protein stability, a dramatic drop in thermostability was observed when the two leucines were replaced by either aspartate (L7D; L376D) or asparagine (L7N; L376N). We then investigated the kinetic thermal stability of the wild type and the mutants by determining the thermodynamic activation parameters, deltaG++, deltaH++ and deltaS++. Besides highlighting the key role of the hydrophobic core in thermostability, these results suggest clearly different mechanisms of destabilization by the single mutations, depending on whether the leucines are replaced by asparagines or aspartates.
Articolo in rivista - Articolo scientifico
Protein engineering; protein thermostability; Archaeon; hydrophobic effect; molecular modeling
English
2008
71
4
1843
1852
none
Sommaruga, S., De Palma, A., Mauri, P., Trisciani, M., Basilico, F., Martelli, P., et al. (2008). A combined approach of mass spectrometry, molecular modeling, and site-directed mutagenesis highlights key structural features responsible for the thermostability of Sulfolobus solfataricus carboxypeptidase. PROTEINS, 71(4), 1843-1852 [10.1002/prot.21868].
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/9036
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