Pedro L. Mateo

Universidad de Granada

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Francisco Conejero‐Lara

Universidad de Granada

José C. Martínez

Universidad de Granada

Vladimir V. Filimonov

Institute of Protein Research

Miguel Cortijo

Universidad Complutense de Madrid

Javier Ruiz‐Sanz

Universidad de Granada

Ana I. Azuaga

Universidad de Granada

Eva S. Cobos

Universidad de Granada

Irene Luque

Universidad de Granada

José M. Sánchez‐Ruiz

Universidad de Granada

A. Cámara-Artigas

University of Almería

Cooperative Institutions

Universidad de Granada

Consejo Superior de Investigaciones Científicas

Centre National de la Recherche Scientifique

University of Oxford

Inserm

Universitat Autònoma de Barcelona

Instituto de Química Física Blas Cabrera

Institute of Protein Research

University of Groningen

Universidad Complutense de Madrid

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A Calorimetric Study of the Thermal Stability of Barnase and Its Interaction with 3'GMP

Biochemistry (1994)

José C. Martínez Mohamed El Harrous Vladimir V. Filimonov Pedro L. Mateo A.R. Fersht

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTA Calorimetric Study of the Thermal Stability of Barnase and Its Interaction with 3'GMPJose C. Martinez, Mohamed El Harrous, Vladimir V. Filimonov, Pedro L. Mateo, and Alan R. FershtCite this: Biochemistry 1994, 33, 13, 3919–3926Publication Date (Print):April 5, 1994Publication History Published online1 May 2002Published inissue 5 April 1994https://pubs.acs.org/doi/10.1021/bi00179a018https://doi.org/10.1021/bi00179a018research-articleACS PublicationsRequest reuse permissionsArticle Views181Altmetric-Citations39LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts

Barnase

Thermal Stability

10.1021/bi00179a018

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Citations (47)

Characterization of Single-Tryptophan Mutants of Histidine-Containing Phosphocarrier Protein: Evidence for Local Rearrangements during Folding from High Concentrations of Denaturant

Biochemistry (2003)

Ana I. Azuaga Denis Canet G. Smeenk Roel Berends Fritz Titgemeijer

We have used site-directed mutagenesis in combination with a battery of biophysical techniques to probe the stability and folding behavior of a small globular protein, the histidine-containing phosphocarrier protein (HPr). Specifically, the four phenylalanine residues (2, 22, 29, and 48) of the wild-type protein were individually replaced by single tryptophans, thus introducing site-specific probes for monitoring the behavior of the protein. The folding of the tryptophan mutants was investigated by NMR, DSC, CD, intrinsic fluorescence, fluorescence anisotropy, and fluorescence quenching. The heat-induced denaturation of all four mutants, and the GdnHCl-induced unfolding curves of F2W, F29W, and F48W, can be fitted adequately to a two-state model, in agreement with the observations for the wild-type protein. The GdnHCl unfolding transitions of F22W, however, showed the accumulation of an intermediate state at low concentrations of denaturant. Kinetic refolding studies of F2W, F29W, and F48W showed a major single phase, independent of the probe used (CD, fluorescence, and fluorescence anisotropy) and similar to that of the wild-type protein. In contrast, F22W showed two phases in the fluorescence experiments corresponding to the two phases previously observed in ANS binding studies of the wild-type protein [Van Nuland et al. (1998) Biochemistry 37, 622−637]. Residue 22 was found from NMR studies to be part of the binding interface on HPr for ANS. These observations indicate that the second slow phase reflects a local, rather than a global, rearrangement from a well-structured highly nativelike intermediate state to the fully folded native state that has less hydrophobic surface exposed to the solvent. The detection of the second slow phase by the use of selective labeling of different regions of the protein with fluorophores illustrates the need for an integrated approach in order to understand the intricate details of the folding reactions of even the simplest proteins.

Denaturation (fissile materials)

Site-directed mutagenesis

Folding (DSP implementation)

Guanidine

Equilibrium unfolding

Globular protein

10.1021/bi027182p

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Citations (11)

Differential scanning calorimetry of lobster haemocyanin

European Journal of Biochemistry (1990)

Mercedes GUZMÁ‐CASADO Antonio Parody‐Morreale Pedro L. Mateo José M. Sánchez‐Ruiz

Differential scanning calorimetry has been performed with Palinurus vulgaris haemocyanin monomers and hexamers. The denaturation of the protein is irreversible. Both the temperature of the transition maximum and the enthalpy are lower for the monomer than for the hexamer. A scan rate dependence of the temperature of the maxima is found for both the monomer and the hexamer; for the hexamer at least, this can be explained in terms of a two-state kinetic model. Some comments are made as to the use of equilibrium thermodynamics in the analysis of irreversible scanning calorimetric traces.

Random hexamer

Endotherm

10.1111/j.1432-1033.1990.tb15386.x

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Citations (57)

Thermodynamic Analysis of the Structural Stability of Phage 434 Cro Protein

Biochemistry (1999)

S. Padmanabhan Douglas V. Laurents Ana María Fernández Montserrat Elías‐Arnanz Javier Ruiz‐Sanz

Thermodynamic parameters describing the phage 434 Cro protein have been determined by calorimetry and, independently, by far-UV circular dichroism (CD) measurements of isothermal urea denaturations and thermal denaturations at fixed urea concentrations. These equilibrium unfolding transitions are adequately described by the two-state model. The far-UV CD denaturation data yield average temperature-independent values of 0.99 +/- 0.10 kcal mol(-)(1) M(-)(1) for m and 0.98 +/- 0.05 kcal mol(-)(1) K(-)(1) for DeltaC(p)()(,U), the heat capacity change accompanying unfolding. Calorimetric data yield a temperature-independent DeltaC(p)()(,U) of 0.95 +/- 0.30 kcal mol(-)(1) K(-)(1) or a temperature-dependent value of 1.00 +/- 0.10 kcal mol(-)(1) K(-)(1) at 25 degrees C. DeltaC(p)()(,U) and m determined for 434 Cro are in accord with values predicted using known empirical correlations with structure. The free energy of unfolding is pH-dependent, and the protein is completely unfolded at pH 2.0 and 25 degrees C as judged by calorimetry or CD. The stability of 434 Cro is lower than those observed for the structurally similar N-terminal domain of the repressor of phage 434 (R1-69) or of phage lambda (lambda(6)(-)(85)), but is close to the value reported for the putative monomeric lambda Cro. Since a protein's structural stability is important in determining its intracellular stability and turnover, the stability of Cro relative to the repressor could be a key component of the regulatory circuit controlling the levels and, consequently, the functions of the two proteins in vivo.

Equilibrium unfolding

Denaturation (fissile materials)

Chemical Stability

Sedimentation equilibrium

10.1021/bi991757+

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Citations (19)

Presence of a Slow Dimerization Equilibrium on the Thermal Unfolding of the 205−316 Thermolysin Fragment at Neutral pH

Biochemistry (1996)

Francisco Conejero‐Lara Pedro L. Mateo

Differential scanning calorimetry and size-exclusion chromatography have been used to characterize the dimerization and unfolding of the 205−316 C-terminal fragment of thermolysin at pH 7.5. We show that the folded fragment dimerizes at low temperature with a moderate affinity and undergoes thermal unfolding according to a N2 ⇄ 2N ⇄ 2U model. This behavior has already been observed at acid pH, where a similar dissociation equilibrium has been found [Azuaga, A., Conejero-Lara, F., Rivas G., De Filippis, V., Fontana, A., & Mateo, P. L. (1995) Biochim. Biophys. Acta 1252, 95−102]. Nevertheless, at pH 7.5 the dimerization equilibrium slows down below about 30 °C, with virtually no interconversion between the monomeric and the dimeric states of the fragment. We have studied the kinetics of interconversion between monomer and dimer by size-exclusion chromatography experiments and have shown that a very high energy barrier (83.8 kJ/mol at 26.5 °C) exists between either state. A mathematical analysis of the DSC thermograms on the basis of the proposed model has allowed us to obtain the thermodynamic characterization of the dimerization and the unfolding processes of the fragment and confirms the kinetic parameters obtained in the chromatographic experiments. The thermodynamic functions for the unfolding of the fragment are compatible with some degree of disorder in the structures of both the monomer and the dimer. According to circular dichroism measurements, the dimerization of the fragment seems to be linked to some conformational change in the subunits, most probably due to a rearrangement of the existing secondary-structure elements. This fragment displays several features already observed in folding intermediates, such as the partial disorder of the polypeptidic chain, association processes, and kinetic barriers between different regions in the conformational space.

Thermolysin

Fragment (logic)

10.1021/bi952358r

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Citations (19)

Thermodynamic Analysis of the Activation of Glycogen Phosphorylase b Over a Range of Temperatures

Journal of Biological Chemistry (1989)

Carmen Barón Juan F. González Pedro L. Mateo Miguel Cortijo

Abstract Equilibrium dialysis and isothermal microcalorimetry experiments have been carried out to characterize the thermodynamics of the binding of AMP to glycogen phosphorylase b (EC 2.4.1.1) at pH 6.9 over the temperature range of 25-35 degrees C. Thermal titrations were performed at each temperature in various buffer systems, which have afforded the calculation of the number of protons exchanged when the AMP binds to each site in the protein. Thermodynamic parameters were obtained for the binding of AMP to the two nucleotide and the two inhibitor sites of the dimeric enzyme. The former show positive cooperativity while the latter behave as independent binding sites. A positive delta Cp value was obtained for the AMP binding to the two N sites (1.3 and 1.4 kJ K-1 mol-1), while the delta Cp was negative for the binding to the I sites (-1.9 kJ K-1 mol-1). The application of Sturtevant's method to our data (Sturtevant, J. M. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 2236-2240) and their comparison with a similar analysis undertaken with phosphorylase a (Mateo, P. L., Gonzalez, J. F., Baron, C., Lopez-Mayorga, O., and Cortijo, M. (1986) J. Biol. Chem. 261, 17067-17072) has opened the way to some understanding of the thermodynamics of the allosteric transition in the protein.

Phosphorylase kinase

10.1016/s0021-9258(18)51568-4

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Citations (23)

Thermodynamic analysis of the chemotactic protein from Escherichia coli, CheY

Biochemistry (1993)

Vladimir V. Filimonov Jesús Prìeto José C. Martínez Marta Bruix Pedro L. Mateo

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTThermodynamic analysis of the chemotactic protein from Escherichia coli, CheYVladimir V. Filimonov, Jesus Prieto, Jose C. Martinez, Marta Bruix, Pedro L. Mateo, and Luis SerranoCite this: Biochemistry 1993, 32, 47, 12906–12921Publication Date (Print):November 1, 1993Publication History Published online1 May 2002Published inissue 1 November 1993https://doi.org/10.1021/bi00210a045RIGHTS & PERMISSIONSArticle Views194Altmetric-Citations47LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (4 MB) Get e-Alertsclose Get e-Alerts

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10.1021/bi00210a045

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Citations (58)

Thermodynamic Study of the Binding of Amp to Glycogen Phosphorylase a and b

Birkhäuser Basel eBooks (1987)

Juan F. González Carmen Barón Sergio Úbeda Álvarez Pedro L. Mateo Miguel Cortijo

The binding of the allosteric activator AMP to both glycogen phosphorylase a and b from rabbit skeletal muscle has been studied by isothermal microcalorimetry and equilibrium dialysis over a certain temperature range. Two binding sites were obtained per monomer of phosphorylase b (activator and inhibitor sites) and one per monomer of phosphorylase a (activator site). Equilibrium constants and the changes in enthalpy, entropy and Gibbs energy were calculated at each temperature. The heat capacity change was obtained from the temperature dependence of the enthalpy values. The structural interpretation of the thermodynamic data has been arrived at from their hydrophobic and vibrational contributions, obtained as described by Sturtevant 1977.

Isothermal microcalorimetry

Phosphorylase kinase

Isothermal process

Atmospheric temperature range

10.1007/978-3-0348-9308-4_52

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Citations (0)

The DSC data analysis for small, single-domain proteins. Application to the SH3 domain

Reactive and Functional Polymers (1998)

José C. Martínez Ana Rosa Viguera Luís Serrano Vladimir V. Filimonov Pedro L. Mateo

Single domain

10.1016/s1381-5148(97)00088-6