Thermodynamic and Kinetic Analysis of the SH3 Domain of Spectrin Shows a Two-State Folding Transition

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

doi:10.1021/bi00174a022

Cited by 281 publications

(304 citation statements)

References 24 publications

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“…The high unfolding temperature is remarkable, considering that the molecule is a protein fragment. This high stability has also been reported for the smaller 255-316 thermolysin fragment under the same experimental conditions (Dalzoppo et al, 1985;Conejero-Lara et al, 1994) as well as for other submolecular domains (Viguera et al, 1994;Azuaga, 1995).…”

Section: Differential Scanning Calorimetrysupporting

confidence: 78%

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

Conejero‐Lara

Mateo

1996

Biochemistry

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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 N 2 a 2N a 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. 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.It is well established that the folding pathways of proteins can occur via one or more intermediates, which can range from relatively unordered, pre-molten globules and moltenglobule states to highly ordered, near-native intermediate states (Ptitsyn, 1995, and references therein). These intermediate states sometimes have been well characterized, showing the early-forming persistent structural elements in specific domains of the protein, which later lead to the complete folding of the rest of the chain (Radford et al., 1992;Dobson et al., 1994). Isolated fragments from globular proteins are being widely used as models to provide insight into the folding problem (Vita et al., 1979(Vita et al., , 1984(Vita et al., , 1989Fontana, 1990;Chaffotte et al., 1991;Peng & Kim, 1994;Rico et al., 1994;Conejero-Lara et al., 1994). These submolecular domains can provide information about local events which could well be of importance during the folding of the complete polypeptide chain.In previous studies Fontana and co-workers have isolated and characterized a set of thermolysin fragments, most of them from the C-terminal part of the protein, using mainly circular dich...

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Section: Differential Scanning Calorimetrysupporting

confidence: 78%

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

Conejero‐Lara

Mateo

1996

Biochemistry

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“…DSC curves were fit to equations that could be applied to the two-state transition model. Two different approaches were used to define ∆H U and ∆S U : (a) in terms of ∆H m and T m (eqs 5-7, [Ur] ) 0) as described in detail elsewhere (24)(25)(26)31) or (b) in terms of T h and T s (eqs 8-10) as described below. Both approaches were used for individual or multiple-curve fitting under the assumption that ∆C p,U is temperature-dependent since, from a consideration of the calorimetric curves, it is clear that C p,N -(T) and C p,U (T) have different slopes so that ∆C p,U is not constant but decreases with temperature in the transition temperature range.…”

Section: Discussionmentioning

confidence: 99%

Thermodynamic Analysis of the Structural Stability of Phage 434 Cro Protein

et al. 1999

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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 ∆C p,U , the heat capacity change accompanying unfolding. Calorimetric data yield a temperatureindependent ∆C 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°C. ∆C 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°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 λ (λ 6-85 ), but is close to the value reported for the putative monomeric λ 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.An understanding of the physical interactions underlying the structure, folding, and function of a protein requires a complete thermodynamic description of its conformational stability. Such a description relies on quantitative analyses of thermally or chemically induced folding-unfolding transitions (monitored by calorimetry or spectroscopically), followed by a data extrapolation to given conditions of temperature, pH, etc. To extrapolate thermal denaturation data, the change in heat capacity (∆C p,U ) and its temperature dependence must be known. These are best determined by calorimetry, although other methods have also been developed. Extrapolation of data from chemical denaturation (e.g., urea and guanidine hydrochloride) is based either on the linear free energy model (LEM) 1 found to be valid for a number of proteins (1, 2) or on the denaturant-protein binding model (DBM) (3, 4). Combined analyses of chemical and thermal denaturation data which assume a temperatureindependent ∆C p,U and the thermodynamic equivalence of thermally and chemically denatured states have been reported for various proteins (2,(5)(6)(7)(8)(9)(10)(11)(12).The conformational stability and the principal thermodynamic parameters (∆G U , ∆H U , ∆S U , T m , and ∆C p,U ) which describe the folding transitions of bacteriophage 434 Cro protein are examined here. In vivo, Cro and the repressor proteins regulate the switch between phage lysis and lysogeny by sequence-specific DNA binding (13). 434 Cro has several properties that are desirable for physical studie...

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“…The temperature dependencies of the molar partial heat capacity, C p , of the proteins were calculated from the DSC data as shown by Privalov and Potekhin using a partial specific volume of 0.73 mL/g (23). The thermal unfolding of all the mutant proteins occurred under equilibrium conditions; the C p curves were fitted using a two-state model as described previously (24). All folding data described in this paper were fitted using the software package Origin (OriginLab, Northampton, MA) unless stated otherwise.…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2003

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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.

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Thermodynamic and Kinetic Analysis of the SH3 Domain of Spectrin Shows a Two-State Folding Transition

Cited by 281 publications

References 24 publications

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

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

Thermodynamic Analysis of the Structural Stability of Phage 434 Cro Protein

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

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