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13 results on '"dimeric protein"'

1. Intersite communication in dimeric enzymes highlighted by structural and thermodynamic analysis of didansyltyrosine binding to thymidylate synthases.

Author

Venturelli, Alberto, Guaitoli, Giambattista, Vanossi, Davide, Saitta, Francesca, Fessas, Dimitrios, Vitiello, Simone, Malpezzi, Giulia, Aiello, Daniele, Ferrari, Stefania, Tondi, Donatella, Ponterini, Glauco, and Paola Costi, Maria

Subjects
*THYMIDYLATE synthase, *BINDING sites, *ISOTHERMAL titration calorimetry, *LACTOBACILLUS casei, *SYNTHASES
Abstract

A dansyl derivative binding triggers negative cooperativity between the active sites of the hTS dimer and allows the characterization of different TS features. [Display omitted] • Intersite communication in dimeric enzymes represents a challenge of underestimated importance in enzyme inhibition strategy. • Fluorometric and calorimetric evidence consistently reveal different binding thermodynamics of a dansyl derivative to the active site of two monomers of hTS. • A ligand induced negative cooperativity between the communicating active sites, avenues a more efficient enzyme inhibition with a lower inhibitor dose. Intersite communication in dimeric enzymes, triggered by ligand binding, represents both a challenge and an opportunity in enzyme inhibition strategy. Though often understestimated, it can impact on the in vivo biological mechansim of an inhibitor and on its pharmacokinetics. Thymidylate synthase (TS) is a homodimeric enzyme present in almost all living organisms that plays a crucial role in DNA synthesis and cell replication. While its inhibition is a valid strategy in the therapy of several human cancers, designing specific inhibitors of bacterial TSs poses a challenge to the development of new anti-infective agents. N,O-didansyl- l -tyrosine (DDT) inhibits both Escherichia coli TS (Ec TS) and Lactobacillus casei TS (Lc TS). The available X-ray structure of the DDT:dUMP: Ec TS ternary complex indicated an unexpected binding mode for DDT to EcTS, involving a rearrangement of the protein and addressing the matter of communication between the two active sites of an enzyme dimer. Combining molecular-level information on DDT binding to Ec TS and Lc TS extracted from structural and FRET-based fluorometric evidence with a thermodynamic characterization of these events obtained by fluorometric and calorimetric titrations, this study unveiled a negative cooperativity between the DDT bindings to the two monomers of each enzyme dimer. This result, complemented by the species-specific thermodynamic signatures of the binding events, implied that communication across the protein dimer was triggered by the first DDT binding. These findings could challenge the conventional understanding of TS inhibition and open the way for the development of novel TS inhibitors with a different mechanism of action and enhanced efficacy and specificity. [ABSTRACT FROM AUTHOR]

Published
2024
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2. A Novel Dimeric Exoglucanase (GH5_38): Biochemical and Structural Characterisation towards its Application in Alkyl Cellobioside Synthesis

Author

Mpho S. Mafa, Heinrich W. Dirr, Samkelo Malgas, Rui W. M. Krause, Konanani Rashamuse, and Brett I. Pletschke

Subjects
alkyl cellobiosides, dimeric protein, exoglucanase, gh5_38, transglycosylation, Organic chemistry, QD241-441
Abstract

An exoglucanase (Exg-D) from the glycoside hydrolase family 5 subfamily 38 (GH5_38) was heterologously expressed and structurally and biochemically characterised at a molecular level for its application in alkyl glycoside synthesis. The purified Exg-D existed in both dimeric and monomeric forms in solution, which showed highest activity on mixed-linked β-glucan (88.0 and 86.7 U/mg protein, respectively) and lichenin (24.5 and 23.7 U/mg protein, respectively). They displayed a broad optimum pH range from 5.5 to 7 and a temperature optimum from 40 to 60 °C. Kinetic studies demonstrated that Exg-D had a higher affinity towards β-glucan, with a Km of 7.9 mg/mL and a kcat of 117.2 s−1, compared to lichenin which had a Km of 21.5 mg/mL and a kcat of 70.0 s−1. The circular dichroism profile of Exg-D showed that its secondary structure consisted of 11% α-helices, 36% β-strands and 53% coils. Exg-D performed transglycosylation using p-nitrophenyl cellobioside as a glycosyl donor and several primary alcohols as acceptors to produce methyl-, ethyl- and propyl-cellobiosides. These products were identified and quantified via thin-layer chromatography (TLC) and liquid chromatography−mass spectrometry (LC-MS). We concluded that Exg-D is a novel and promising oligomeric glycoside hydrolase for the one-step synthesis of alkyl glycosides with more than one monosaccharide unit.

Published
2020
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3. Combined fcs and pch analysis to quantify protein dimerization in living cells

Author

Nederveen-Schippers, Laura M., Pathak, Pragya, Keizer-Gunnink, Ineke, Westphal, Adrie H., van Haastert, Peter J.M., Borst, Jan Willem, Kortholt, Arjan, Skakun, Victor, Nederveen-Schippers, Laura M., Pathak, Pragya, Keizer-Gunnink, Ineke, Westphal, Adrie H., van Haastert, Peter J.M., Borst, Jan Willem, Kortholt, Arjan, and Skakun, Victor

Abstract

Protein dimerization plays a crucial role in the regulation of numerous biological processes. However, detecting protein dimers in a cellular environment is still a challenge. Here we present a methodology to measure the extent of dimerization of GFP-tagged proteins in living cells, using a combination of fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) analysis of single-color fluorescence fluctuation data. We named this analysis method brightness and diffusion global analysis (BDGA) and adapted it for biological purposes. Using cell lysates containing different ratios of GFP and tandem-dimer GFP (diGFP), we show that the average brightness per particle is proportional to the fraction of dimer present. We further adapted this methodology for its application in living cells, and we were able to distinguish GFP, diGFP, as well as ligand-induced dimerization of FKBP12 (FK506 binding protein 12)-GFP. While other analysis methods have only sporadically been used to study dimerization in living cells and may be prone to errors, this paper provides a robust approach for the investigation of any cytosolic protein using single-color fluorescence fluctuation spectroscopy

Published
2021

4. Combined FCS and PCH analysis to quantify protein dimerization in living cells

Author

Laura M Nederveen-Schippers, Jan Willem Borst, Adrie H. Westphal, Arjan Kortholt, Victor V. Skakun, Peter J.M. van Haastert, Pragya Pathak, Ineke Keizer-Gunnink, and Cell Biochemistry

Subjects
0301 basic medicine, Dimer, FK506 binding protein 12, Ligands, Biochemistry, Dictyostelium discoideum, Green fluorescent protein, fluorescence fluctuation spectroscopy, Diffusion, chemistry.chemical_compound, Cytosol, 0302 clinical medicine, Dictyostelium, Biology (General), Protein Dimerization, Cells, Cultured, Spectroscopy, biology, General Medicine, Brightness and diffusion global analysis, Fluorescence, Computer Science Applications, Chemistry, FKBP, 030220 oncology & carcinogenesis, Dimerization, QH301-705.5, Green Fluorescent Proteins, Biochemie, Photon counting histogram, Fluorescence correlation spectroscopy, GFP, Article, Catalysis, Inorganic Chemistry, 03 medical and health sciences, Dimeric protein, Physical and Theoretical Chemistry, Molecular Biology, QD1-999, Photons, Organic Chemistry, Proteins, biology.organism_classification, Spectrometry, Fluorescence, 030104 developmental biology, chemistry, Biophysics, Protein Multimerization, Fluorescence fluctuation spec-troscopy
Abstract

Protein dimerization plays a crucial role in the regulation of numerous biological processes. However, detecting protein dimers in a cellular environment is still a challenge. Here we present a methodology to measure the extent of dimerization of GFP-tagged proteins in living cells, using a combination of fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) analysis of single-color fluorescence fluctuation data. We named this analysis method brightness and diffusion global analysis (BDGA) and adapted it for biological purposes. Using cell lysates containing different ratios of GFP and tandem-dimer GFP (diGFP), we show that the average brightness per particle is proportional to the fraction of dimer present. We further adapted this methodology for its application in living cells, and we were able to distinguish GFP, diGFP, as well as ligand-induced dimerization of FKBP12 (FK506 binding protein 12)-GFP. While other analysis methods have only sporadically been used to study dimerization in living cells and may be prone to errors, this paper provides a robust approach for the investigation of any cytosolic protein using single-color fluorescence fluctuation spectroscopy.

Published
2021
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5. Probing the equilibrium unfolding of ketosteroid isomerase through xenon-perturbed 1H–15N multidimensional NMR spectroscopy.

Author

Lee, Hyeong, Moon, Hye, Jang, Do, Cha, Hyung, Hong, Bee, Choi, Kwan, and Lee, Hee

Abstract

We used xenon-perturbed 1H–15N multidimensional NMR to investigate the structural changes in the urea-induced equilibrium unfolding of the dimeric ketosteroid isomerase (KSI) from Pseudomonas putida biotype B. Three limited regions located on the β3-, β5- and β6-strands of dimeric interface were significantly perturbed by urea in the early stage of KSI unfolding, which could lead to dissociation of the dimer into structured monomers at higher denaturant concentration as the interactions in these regions are weakened. The results indicate that the use of xenon as an indirect probe for multidimensional NMR can be a useful method for the equilibrium unfolding study of protein at residue level. [ABSTRACT FROM AUTHOR]

Published
2008
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6. The solution structure of reduced dimeric copper zinc superoxide dismutase.

Author

Banci, Lucia, Bertini, Ivano, Cramaro, Fiorenza, Del Conte, Rebecca, and Viezzoli, Maria Silvia

Subjects
*SUPEROXIDE dismutase, *COPPER compounds, *AMINO acids
Abstract

The solution structure of homodimeric Cu2 Zn2 superoxide dismutase (SOD) of 306 aminoacids was determined on a 13 C, 15 N and 70% 2 H labeled sample. Two-thousand eight-hundred and five meaningful NOEs were used, of which 96 intersubunit, and 115 dihedral angles provided a family of 30 conformers with an rmsd from the average of 0.78 ± 0.11 and 1.15 ± 0.09 Å for the backbone and heavy atoms, respectively. When the rmsd is calculated for each subunit, the values drop to 0.65 ± 0.09 and 1.08 ± 0.11 Å for the backbone and heavy atoms, respectively. The two subunits are identical on the NMR time scale, at variance with the X-ray structures that show structural differences between the two subunits as well as between different molecules in the unit cell. The elements of secondary structure, i.e. eight β sheets, are the same as in the X-ray structures and are well defined. The odd loops (I, III and V) are well resolved as well as loop II located at the subunit interface. On the contrary, loops IV and VI show some disorder. The residues of the active cavity are well defined whereas within the various subunits of the X-ray structure some are disordered or display different orientation in different X-ray structure determinations. The copper(I) ion and its ligands are well defined. This structure thus represents a well defined model in solution relevant for structure–function analysis of the protein. The comparison between the solution structure of monomeric mutants and the present structure shows that the subunit–subunit interactions increase the order in loop II. This has the consequences of inducing the structural and dynamic properties that are optimal for the enzymatic function of the wild-type enzyme. The regions 37–43 and 89–95, constituting loops III and V and the initial part of the β barrel and showing several mutations in... [ABSTRACT FROM AUTHOR]

Published
2002
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7. Tuning the local solvent composition at a drug carrier surface: the effect of dimethyl sulfoxide/water mixture on the photofunctional properties of hypericin–β-lactoglobulin complexes

Author

Nonell, Santi, Rodríguez Amigo, Beatriz, Agut Bonsfills, Montserrat, Delcanale, P., Juárez Jiménez, Jordi, Luque, Francisco Javier, Abbruzzetti, S., Viappiani, C., and Universitat Ramon Llull. IQS

Subjects
Dimeric protein, Antibacterial treatment, Fotosensibilització (Biologia), Hydrophobic photosensitizers, Sulfòxids, 54 - Química
Abstract

Aggregation is a major problem for the anti-microbial photodynamic applications of hydrophobic photosensitizers since it strongly reduces the amount of singlet oxygen generated in aqueous solutions. Binding of hypericin (Hyp) to the milk whey protein β-lactoglobulin (βLG), occurring at the two hydrophobic cavities located at the interface of the protein homodimer, can be exploited to confer water-solubility and biocompatibility to the photosensitizer. The introduction of a small amount of the organic cosolvent dimethyl sulfoxide (DMSO) leads to a remarkable improvement of the photophysical properties of the complex Hyp–βLG by increasing its fluorescence emission and singlet oxygen photosensitization quantum yields. Surprisingly, the ability of the complex to photo-inactivate bacteria of the strain Staphylococcus aureus is strongly reduced in the presence of DMSO, despite the higher yield of photosensitization. The reasons for this apparently contradictory behavior are investigated, providing new insights into the use of carrier systems for hydrophobic photosensitizers.

Published
2017

8. A Novel Dimeric Exoglucanase (GH5_38): Biochemical and Structural Characterisation towards its Application in Alkyl Cellobioside Synthesis.

Author

Mafa, Mpho S., Dirr, Heinrich W., Malgas, Samkelo, Krause, Rui W. M., Rashamuse, Konanani, Pletschke, Brett I., Musmarra, Dino, and Capasso, Sante

Subjects
GLUCANS, LIQUID chromatography-mass spectrometry, BETA-glucans, CIRCULAR dichroism
Abstract

An exoglucanase (Exg-D) from the glycoside hydrolase family 5 subfamily 38 (GH5_38) was heterologously expressed and structurally and biochemically characterised at a molecular level for its application in alkyl glycoside synthesis. The purified Exg-D existed in both dimeric and monomeric forms in solution, which showed highest activity on mixed-linked β-glucan (88.0 and 86.7 U/mg protein, respectively) and lichenin (24.5 and 23.7 U/mg protein, respectively). They displayed a broad optimum pH range from 5.5 to 7 and a temperature optimum from 40 to 60 °C. Kinetic studies demonstrated that Exg-D had a higher affinity towards β-glucan, with a Km of 7.9 mg/mL and a kcat of 117.2 s−1, compared to lichenin which had a Km of 21.5 mg/mL and a kcat of 70.0 s−1. The circular dichroism profile of Exg-D showed that its secondary structure consisted of 11% α-helices, 36% β-strands and 53% coils. Exg-D performed transglycosylation using p-nitrophenyl cellobioside as a glycosyl donor and several primary alcohols as acceptors to produce methyl-, ethyl- and propyl-cellobiosides. These products were identified and quantified via thin-layer chromatography (TLC) and liquid chromatography–mass spectrometry (LC-MS). We concluded that Exg-D is a novel and promising oligomeric glycoside hydrolase for the one-step synthesis of alkyl glycosides with more than one monosaccharide unit. [ABSTRACT FROM AUTHOR]

Published
2020
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10. Functional and structural study of the dimeric inner membrane protein SbmA

Author

Paula Andrea Vincent, Doriano Lamba, Giulia Runti, Natalia Soledad Corbalan, Marco Scocchi, Konstantinos Beis, Sonia Covaceuszach, Robert C. Ford, Conrado Adler, Corbalan, N, Runti, Giulia, Adler, C, Covaceuszach, S, Ford, Rc, Lamba, D, Beis, K, Scocchi, Marco, and Vincent, Pa

Subjects
Models, Molecular, Protein Conformation, structure-base homology modelling, Mutant, Molecular Sequence Data, Peptide, medicine.disease_cause, Microbiology, purl.org/becyt/ford/1 [https], Ciencias Biológicas, Protein structure, inner membrane protein, SbmA, dimeric protein, peptide trasport, site directed mutagenesis, medicine, Escherichia coli, Homology modeling, Amino Acid Sequence, Function, purl.org/becyt/ford/1.6 [https], Molecular Biology, Peptide sequence, chemistry.chemical_classification, biology, Membrane transport protein, Escherichia coli Proteins, Structure, Membrane Transport Proteins, Biological Transport, Articles, Gene Expression Regulation, Bacterial, Bioquímica y Biología Molecular, Amino acid, Dimer, chemistry, Biochemistry, EM, Mutation, biology.protein, CIENCIAS NATURALES Y EXACTAS, secondary transporter
Abstract

SbmA protein has been proposed as a dimeric secondary transporter. The protein is involved in the transport of microcins B17 and J25, bleomycin, proline-rich antimicrobial peptides, antisense peptide phosphorodiamidate morpholino oligomers, and peptide nucleic acids into the Escherichia coli cytoplasm. The sbmA homologue is found in a variety of bacteria, though the physiological role of the protein is hitherto unknown. In this work, we carried out a functional and structural analysis to determine which amino acids are critical for the transport properties of SbmA. We created a set of 15 site-directed sbmA mutants in which single conserved amino acids were replaced by glycine residues. Our work demonstrated that strains carrying the site-directed mutants V102G, F219G, and E276G had a null phenotype for SbmA transport functions. In contrast, strains carrying the single point mutants W19G, W53G, F60G, S69G, N155G, R190, L233G, A344G, T255G, N308G, and R385G showed transport capacities indistinguishable from those of strains harboring a wild-type sbmA. The strain carrying the Y116G mutant exhibited mixed phenotypic characteristics. We also demonstrated that those sbmA mutants with severely impaired transport capacity showed a dominant negative phenotype. Electron microscopy data and in silico three-dimensional (3D) homology modeling support the idea that SbmA forms a homodimeric complex, closely resembling the membrane-spanning region of the ATP-binding cassette transporter family. Direct mapping of the sbmA single point mutants on the protein surface allowed us to explain the observed phenotypic differences in transport ability. Fil: Corbalan, Natalia Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina Fil: Runti, Giulia. Università degli Studi di Trieste; Italia Fil: Adler, Conrado. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina Fil: Covaceuszach, Sonia. Consiglio Nazionale delle Ricerche; Italia Fil: Ford, Robert C.. University of Manchester; Reino Unido Fil: Lamba, Doriano. Consiglio Nazionale delle Ricerche; Italia Fil: Beis, Konstantinos. Imperial College London; Reino Unido Fil: Scocchi, Marco. Università degli Studi di Trieste; Italia Fil: Vincent, Paula Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina

Published
2013
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11. Dimer-monomer equilibrium of human thymidylate synthase monitored by fluorescence resonance energy transfer

Author

Genovese, Filippo, Ferrari, Stefania, Guaitoli, Giambattista, Caselli, Monica, Costi, Maria Paola, and Ponterini, Glauco

Subjects
Models, Molecular, FRET, protein protein interaction, dimeric protein, thymidylate synthase, Thymidylate Synthase, Article, Kinetics, Catalytic Domain, Fluorescence Resonance Energy Transfer, Humans, Protein Multimerization, Allosteric Site
Abstract

An ad hoc bioconjugation/fluorescence resonance energy transfer (FRET) assay has been designed to spectroscopically monitor the quaternary state of human thymidylate synthase dimeric protein. The approach enables the chemoselective engineering of allosteric residues while preserving the native protein functions through reversible masking of residues within the catalytic site, and is therefore suitable for activity/oligomerization dual assay screenings. It is applied to tag the two subunits of human thymidylate synthase at cysteines 43 and 43′ with an excitation energy donor/acceptor pair. The dimer–monomer equilibrium of the enzyme is then characterized through steady-state fluorescence determination of the intersubunit resonance energy transfer efficiency.

Published
2010

12. Myotoxic and cytolytic activities of dimeric Lys49 phospholipase A2 homologues are reduced, but not abolished, by a pH-induced dissociation

Author

Yamileth Angulo, Wonhwa Cho, José María Gutiérrez, Bruno Lomonte, and Andreimar M. Soares

Subjects
Snake venom, Myotoxin, Neurotoxins, Reptilian Proteins, Biology, Toxicology, medicine.disease_cause, Group II Phospholipases A2, Phospholipases A, Mice, Phospholipase A2, medicine, Animals, Creatine Kinase, Hydro-Lyases, chemistry.chemical_classification, Phospholipase A, Effector, Toxin, Cell Membrane, Hydrogen-Ion Concentration, Phospholipases A2, Enzyme, Biochemistry, chemistry, Toxicity, biology.protein, Dimeric Protein, Muscle, lipids (amino acids, peptides, and proteins), Dimerization, Snake Venoms
Abstract

Lys49 phospholipase A2 (PLA2) homologues are myotoxic proteins devoid of catalytic activity. Their toxic determinants map to the C-terminal region 115–129, which plays an effector role in membrane damage. The dimeric state was reported to be essential for a Lys49 PLA2 which lost its liposome-disrupting activity after dissociating into monomers at pH 5.0. This study, evaluated the effects of a pH-induced dissociation on the toxicity of four Lys49 PLA2s, using biological targets instead. Both their cytolytic and myotoxic activities were lower at pH 5.0 than at pH 7.2. However, in contrast with experiments using artificial bilayers, toxic effects upon biological targets were not abolished at pH 5.0. Importantly, C-terminal synthetic peptides of two Lys49 PLA2s also showed lower cytolytic action at pH 5.0 than at pH 7.2, indicating that factors other than the dimeric/monomeric state of the proteins may also be involved in these differences of toxicity. Results support the view that the dimeric state of Lys49 PLA2s could play an enhancing, although not essential role, in their C-terminal region-mediated mechanism of myotoxicity. International Foundation for Science/[F/2766-3]/IFS/Suecia Universidad de Costa Rica//UCR/Costa Rica NeTropica/[01-R-2003]//Suecia Fundação de Amparo à Pesquisa do Estado de São Paulo//FAPESP/Brasil UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)

Published
2005

13. Stability of aspartate aminotransferase from Sulfolobus solfataricus

Author

Maria Vittoria Cubellis, Maria Ina Arnone, Leila Birolo, Francesco Bossa, Stefano Pascarella, Giovanni Sannia, Gennaro Marino, Arnone, M. I., Birolo, L, Pascarella, S, Cubellis, MARIA VITTORIA, Bossa, F., Sannia, Giovanni, Marino, G., M. I., Arnone, Birolo, Leila, S., Pascarella, F., Bossa, and Marino, Gennaro

Subjects
Guanidinium chloride, Models, Molecular, Protein Denaturation, Protein Folding, Hot Temperature, Molecular model, ved/biology.organism_classification_rank.species, Size-exclusion chromatography, Molecular Sequence Data, Bioengineering, aspartate aminotransferase, chemical unfolding, dimeric protein, molecular modeling, thermostability, Biology, Biochemistry, Guanidines, Sulfolobus, chemistry.chemical_compound, Enzyme Stability, Denaturation (biochemistry), Amino Acid Sequence, Aspartate Aminotransferases, Guanidine, Molecular Biology, Binding Sites, ved/biology, Sulfolobus solfataricus, Crystallography, chemistry, Protein folding, Protein quaternary structure, Dimerization, Sequence Alignment, Biotechnology
Abstract

Aspartate aminotransferase from Sulfolobus solfataricus (SsAspAT) is an extremely thermophilic and thermostable dimeric enzyme which retains its structure and reaches maximal activity at 100 degrees C, The structural stability of this protein was investigated by coupling isothermally and thermally induced denaturation studies to molecular modeling, Gel filtration analysis indicated that SsAspAT unfolds with an N-2 reversible arrow 2D mechanism, In the molecular model, a cluster of hydrophobic residues was shown at the interface between the subunits of SsAspAT and suggested this cluster as a structural feature stabilizing the enzyme quaternary structure, At 25 degrees C, SsAspAT is less resistant to guanidinium chloride-induced denaturation than the cytosolic aspartate aminotransferase from pig heart (cpAspAT), which was chosen as a mesophilic counterpart in the thermodynamic analysis since it shares with SsAspAT the two-state unfolding mechanism, Therefore, in the case of aspartate aminotransferases, thermal stability does not correlate with the stability against chemical denaturants. Isothermal denaturation curves at 25 degrees C and melting profiles recorded in the presence of guanidinium chloride showed that the Delta G degrees(H2O) at 25 degrees C of SsAspAT exceeds that of cpAspAT by roughly 15 kJ/mol; the parameter bn, related to the number of binding sites for the denaturant differentially exposed in unfolded and folded states, is higher for SsAspAT than for cpAspAT; and Delta C-p is lower for the thermophilic enzyme than for the mesophilic one by 8 kJ/K.mol. These results are indicative of a less hydrophobic core for SsAspAT than cpAspAT, In agreement with this, the molecular model predicts that some charged side chains are buried in SsAspAT and interact to form an H-bond/ion-pair network

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