Hyperthermophile Protein Folding Thermodynamics: Differential Scanning Calorimetry and Chemical Denaturation of Sac7d

McCrary, Bradford S.; Edmondson, Stephen P.; Shriver, John W.

doi:10.1006/jmbi.1996.0677

Cited by 141 publications

(161 citation statements)

References 84 publications

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“…The thermodynamic stability of the five variants studied compares well with several other proteins from thermophiles (23) and is close to that of native Sac7d (25). Sac7*39, with 15 residues substituted (23%), has the same stability and unfolding properties as Sac7d.…”

Section: Discussionsupporting

confidence: 55%

Remodeling a DNA-binding protein as a specific in vivo inhibitor of bacterial secretin PulD

Mouratou

Schaeffer

Guilvout

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

116

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We engineered a class of proteins that binds selected polypeptides with high specificity and affinity. Use of the protein scaffold of Sac7d, belonging to a protein family that binds various ligands, overcomes limitations inherent in the use of antibodies as intracellular inhibitors: it lacks disulfide bridges, is small and stable, and can be produced in large amounts. An in vitro combinatorial/ selection approach generated specific, high-affinity (up to 140 pM) binders against bacterial outer membrane secretin PulD. When exported to the Escherichia coli periplasm, they inhibited PulD oligomerization, thereby blocking the type II secretion pathway of which PulD is part. Thus, high-affinity inhibitors of protein function can be derived from Sac7d and can be exported to, and function in, a cell compartment other than that in which they are produced.calorimetry ͉ intrabody ͉ ribosome display ͉ Sac7d ͉ type II secretion system

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Section: Discussionsupporting

confidence: 55%

Remodeling a DNA-binding protein as a specific in vivo inhibitor of bacterial secretin PulD

Mouratou

Schaeffer

Guilvout

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

116

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“…If a protein folds via a cooperative two-state equilibrium process: (i) the DSC endotherm will fit well to this two state model (single cooperative folding unit [29][30][31] ) and (ii) the DH vH produced from the model-dependent equation will equal the model-independent DH cal (as shown by many studies on small globular proteins). 32,33 When the CTPRa protein endotherms were fit to the two-state model it was found that qualitatively between two and six repeats fit well (CTPRa2-CTPRa6). In contrast, CTPRa8, CTPRa10 and the CTPR proteins did not produce satisfactory fits [ Fig.…”

Section: Thermal Denaturation Of Ctpra and Ctpr Proteinsmentioning

confidence: 99%

Modulation of the multistate folding of designed TPR proteins through intrinsic and extrinsic factors

et al. 2012

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Tetratricopeptide repeats (TPRs) are a class of all alpha-helical repeat proteins that are comprised of 34-aa helix-turn-helix motifs. These stack together to form nonglobular structures that are stabilized by short-range interactions from residues close in primary sequence. Unlike globular proteins, they have few, if any, long-range nonlocal stabilizing interactions. Several studies on designed TPR proteins have shown that this modular structure is reflected in their folding, that is, modular multistate folding is observed as opposed to two-state folding. Here we show that TPR multistate folding can be suppressed to approximate two-state folding through modulation of intrinsic stability or extrinsic environmental variables. This modulation was investigated by comparing the thermodynamic unfolding under differing buffer regimes of two distinct series of consensus-designed TPR proteins, which possess different intrinsic stabilities. A total of nine proteins of differing sizes and differing consensus TPR motifs were each thermally and chemically denatured and their unfolding monitored using differential scanning calorimetry (DSC) and CD/fluorescence, respectively. Analyses of both the DSC and chemical denaturation data show that reducing the total stability of each protein and repeat units leads to observable two-state unfolding. These data highlight the intimate link between global and intrinsic repeat stability that governs whether folding proceeds by an observably two-state mechanism, or whether partial unfolding yields stable intermediate structures which retain sufficient stability to be populated at equilibrium.

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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 CDmentioning

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%

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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Hyperthermophile Protein Folding Thermodynamics: Differential Scanning Calorimetry and Chemical Denaturation of Sac7d

Cited by 141 publications

References 84 publications

Remodeling a DNA-binding protein as a specific in vivo inhibitor of bacterial secretin PulD

Remodeling a DNA-binding protein as a specific in vivo inhibitor of bacterial secretin PulD

Modulation of the multistate folding of designed TPR proteins through intrinsic and extrinsic factors

Thermodynamic Analysis of the Structural Stability of Phage 434 Cro Protein

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