A thermodynamic analysis of a family of small globular proteins: SH3 domains

Filimonov, Vladimir V.; Azuaga, Ana I.; Viguera, Ana Rosa; Serrano, Luís; Mateo, Pedro L.

doi:10.1016/s0301-4622(99)00025-3

Cited by 55 publications

(59 citation statements)

References 38 publications

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“…C m values which are more accurately determined appear to have some temperature dependence, while ∆G U (T,0) values, the zerourea value at temperatures between 5 and 25°C, range within the error of the measurement. As for some other small proteins (7,10), the change in the number of denaturant binding sites, ∆n, is small and relatively invariant with temperature between 5 and 25°C with a mean value of 35 (T,0) from DBM and LEM differ by about 0.5 to 0.8 kcal mol -1 , as observed for some other proteins (5,10,26). Figure 2 illustrates the effects of urea on thermostability; as the fixed urea concentration increases, high-temperature unfolding (heat denaturation) occurs at lower temperatures, and low-temperature unfolding (cold denaturation) is apparent by 3 M urea.…”

Section: Isothermal Urea Denaturations By Far-uv CDsupporting

confidence: 56%

“…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%

“…Such effects are typically entropic (electrostatic) in origin. Small heat effects that might accompany unfolding in the acidic pH range as a result of proton transfer are nearly fully offset by the approximate equivalence of the ionization enthalpies of the buffers and the protein acidic groups (19,30,41,42 (5,10,26). The small size and the relatively low stability of 434 Cro allow reliable determination of ∆H m over only a narrow temperature range.…”

Section: Thermal Unfolding Of 434 Cro As Determined By Differential Smentioning

confidence: 99%

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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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Section: Isothermal Urea Denaturations By Far-uv CDsupporting

confidence: 56%

Section: Discussionmentioning

confidence: 99%

Section: Thermal Unfolding Of 434 Cro As Determined By Differential Smentioning

confidence: 99%

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Thermodynamic Analysis of the Structural Stability of Phage 434 Cro Protein

et al. 1999

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“…Indeed, although numerous thorough studies have been performed on the SH3 model system for protein folding [29][30][31][32][33] , most of those have so far been focused on residues forming the conserved structural core, either via their involvement in the hydrophobic core packing or through their contributions to the hydrogen bonding network.…”

Section: The L180p Mutation Which Causes Nephronophtisis Strongly Dementioning

confidence: 99%

Solution NMR structure of the SH3 domain of human nephrocystin and analysis of a mutation‐causing juvenile nephronophthisis

et al. 2005

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Human nephrocystin is a protein associated with juvenile NPH, an autosomal recessive, inherited kidney disease responsible for chronic renal failure in children. It contains an SH3 domain involved in signaling pathways controlling cell adhesion and cytoskeleton organization. The solution structure of this domain was solved by triple resonance NMR spectroscopy. Within the core, the structure is similar to those previously reported for other SH3 domains but exhibits a number of specific noncanonical features within the polyproline ligand binding site. Some of the key conserved residues are missing, and the N‐Src loop exhibits an unusual twisted geometry, which results in a narrowing of the binding groove. This is induced by the replacement of a conserved Asp, Asn, or Glu residue by a Pro at one side of the N‐Src loop. A systematic survey of other SH3 domains also containing a Pro at this position reveals that most of them belong to proteins involved in cell adhesion or motility. A variant of this domain, which carries a point mutation causing NPH, was also analyzed. This change, L180P, although it corresponds to a nonconserved and solvent‐exposed position, causes a complete loss of the tertiary structure. Similar effects are also observed with the L180A variant. This could be a context‐dependent effect resulting from an interaction between neighboring charged side‐chains. Proteins 2005. © 2005 Wiley‐Liss, Inc.

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“…1 parameters as above to anticipate the entropic penalty of loop closure, the replacement of 14 residues by 43 residues should lead to a decrease in stability (⌬⌬G°) of ∼6.4 kJ/mole. The thermodynamic stability of SH3 domains is in the range of 12-21 kJ/mole (Lim et al 1994;Filimonov et al 1999;Martínez et al 1999), with PsaE at the lower limit (Mayer et al 1999). As a result, if the cytochrome b 5 loop destabilizes the PsaE scaffold by the calculated ⌬⌬G°, a noticeable effect should be detected in the equilibrium populations of molecules sampling the native and denatured conformations under native conditions.…”

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confidence: 99%

Insertion of the cytochrome b₅ heme‐binding loop into an SH3 domain. Effects on structure and stability, and clues about the cytochrome's architecture

2004

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Under native conditions, apocytochrome b 5 exhibits a stable core and a disordered heme-binding region that refolds upon association with the cofactor. The termini of this flexible region are in close proximity, suggesting that loop closure may contribute to the thermodynamic properties of the apocytochrome. A chimeric protein containing 43 residues encompassing the cytochrome loop was constructed using the cyanobacterial photosystem I accessory protein E (PsaE) from Synechococcus sp. PCC 7002 as a structured scaffold. PsaE has the topology of an SH3 domain, and the insertion was engineered to replace its 14-residue CD loop. NMR and optical spectroscopies showed that the hybrid protein (named EbE1) was folded under native conditions and that it retained the characteristics of an SH3 domain. NMR spectroscopy revealed that structural and dynamic differences were confined near the site of loop insertion. Variable-temperature 1D NMR spectra of EbE1 confirmed the presence of a kinetic unfolding barrier. Thermal and chemical denaturations of PsaE and EbE1 demonstrated cooperative, two-state transitions; the stability of the PsaE scaffold was found only moderately compromised by the insertion, with a ⌬T m of 8.3°C, a ⌬C m of 1.5 M urea, and a ⌬⌬G°of 4.2 kJ/mole. The data implied that the penalty for constraining the ends of the inserted region was lower than the ∼6.4 kJ/mole calculated for a self-avoiding chain. Extrapolation of these results to cytochrome b 5 suggested that the intrinsic stability of the folded portion of the apoprotein reflected only a small detrimental contribution from the large heme-binding domain.Keywords: protein structure/folding; stability and mutagenesis Supplemental material: see www.proteinscience.orgIn the fashion of many ligand-binding proteins, the watersoluble domain of rat hepatic cytochrome b 5 undergoes induced refolding upon association with its heme cofactor. In the holoprotein state, the three-dimensional structure is well represented by a narrow ensemble of conformations. As illustrated in Figure 1, the fold contains six helices and five strands (Mathews et al. 1979). The heme is fastened in its cavity by coordination bonds ligating the Fe ion to the imidazole N atoms of His 39 (end of ␣2) and His 63 (beginning of ␣4). Two hydrophobic regions, termed core 1 and core 2, are also apparent in the cytochrome structure. The residues of core 1 are located in the central part of the amino acid sequence; they establish most of the protein-heme contacts and therefore play a functional role. In contrast, the residues comprising core 2 are found within the N-and C-terminal segments. Core 2 does not require the heme Abbreviations: ⌬␦, change in chemical shift in ppm; bp, base pair; CD, circular dichroism; DQF-COSY, double-quantum-filtered correlated spectroscopy; EbE1, chimeric protein whose N-and C-terminal stretches are composed of residues from PsaE and whose intervening residues come from cytochrome b 5 ; EDTA, ethylenediaminetetraacetic acid; IPTG, isopropylthio-␤-D-galactoside;...

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A thermodynamic analysis of a family of small globular proteins: SH3 domains

Cited by 55 publications

References 38 publications

Thermodynamic Analysis of the Structural Stability of Phage 434 Cro Protein

Thermodynamic Analysis of the Structural Stability of Phage 434 Cro Protein

Solution NMR structure of the SH3 domain of human nephrocystin and analysis of a mutation‐causing juvenile nephronophthisis

Insertion of the cytochrome b₅ heme‐binding loop into an SH3 domain. Effects on structure and stability, and clues about the cytochrome's architecture

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A thermodynamic analysis of a family of small globular proteins: SH3 domains

Cited by 55 publications

References 38 publications

Thermodynamic Analysis of the Structural Stability of Phage 434 Cro Protein

Thermodynamic Analysis of the Structural Stability of Phage 434 Cro Protein

Solution NMR structure of the SH3 domain of human nephrocystin and analysis of a mutation‐causing juvenile nephronophthisis

Insertion of the cytochrome b5 heme‐binding loop into an SH3 domain. Effects on structure and stability, and clues about the cytochrome's architecture

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Insertion of the cytochrome b₅ heme‐binding loop into an SH3 domain. Effects on structure and stability, and clues about the cytochrome's architecture