Thermal stability and DNA binding activity of a variant form of the Sso7d protein from the archeon Sulfolobus solfataricus truncated at leucine 54

Sso7d is a 62-residue, basic protein from the hyperthermophilic archaeon Sulfolobus solfataricus. Around neutral pH, it exhibits a denaturation temperature close to 100 degrees C and a non-sequence-specific DNA binding activity. Here, we report the characterization by circular dichroism and fluorescence measurements of a variant form of Sso7d truncated at leucine 54 (L54Delta). It is shown that L54Delta has a folded conformation at neutral pH and that its thermal unfolding is a reversible process, represented well by the two-state N <=> D transition model, with a denaturation temperature of 53 degrees C. Fluorescence titration experiments indicate that L54Delta binds tightly to calf thymus DNA, even though the binding parameters are smaller than those of the wild-type protein. Therefore, the truncation of eight residues at the C-terminus of Sso7d markedly affects the thermal stability of the protein, which nevertheless retains a folded structure and DNA binding activity.     

Structural and dynamic effects of alpha-helix deletion in Sso7d: implications for protein thermal stability

Sso7d is a 62-residue protein from the hyperthemophilic archaeon Sulfolobus solfataricus with a denaturation temperature close to 100 degrees C around neutral pH. An engineered form of Sso7d truncated at leucine 54 (L54Delta) is significantly less stable, with a denaturation temperature of 53 degrees C. Molecular dynamics (MD) studies of Sso7d and its truncated form at two different temperatures have been performed. The results of the MD simulations at 300 K indicate that: (1) the flexibility of Sso7d chain at 300 K agrees with that detected from X-ray and NMR structural studies; (2) L54Delta remains stable in the native folded conformation and possesses an overall dynamic behavior similar to that of the parent protein. MD simulations performed at 500 K, 10 ns long, indicate that, while Sso7d is in-silico resistant to high temperature, the truncated variant partially unfolds, revealing the early phases of the thermal unfolding pathway of the protein. Analysis of the trajectories of L54Delta suggests that the unzipping of the N-terminal and C-terminal beta-strands should be the first event of the unfolding pathway, and points out the regions more resistant to thermal unfolding. These findings allow one to understand the role played by specific interactions connecting the two ends of the chain for the high thermal stability of Sso7d, and support recent hypotheses on its folding mechanism emerged from site-directed mutagenesis studies.     

The role of the salt concentration, proton, and phosphate binding on the thermal stability of wild and cloned DNA-binding protein Sso7d from Sulfolobus solfataricus

The acidic pH (1.5-7.0) and ionic strength (0.005-0.2M) dependence of thermodynamic functions of protein Sso7d from Sulfolobus solfataricus, cloned (c-Sso7d) and N-heptapeptide deleted [c-des(1-7)Sso7d] in glycine, and phosphate buffers was studied by means of adiabatic scanning calorimetry. The difference of proton binding was estimated from deltaHcal(pH), Td(pH), and (deltaTd/deltapH). It was found that a single group non-co-operative ionization with apparent pKa = 3.25 for both cloned and deleted proteins govern the thermal unfolding of two different (protonated and unprotonated) forms. deltaH degrees is found to be pH-independent and the changes in stability (deltaG degrees ) originate from changes in entropy terms. The apparent pKa measured at high salt concentrations decreases with 0.5 pH units from glycine to phosphate and the free energy of transfer at high ionic strength is 0.7 kcal/mol. The ionic strength dependence for the pH-dependent D-states is very different at pH 6.0 and 1.5. This is consistent with the property of denatured state to be more compacted or "closed" (Dc) at neutral or weak acidic pH and more random or "open" (Do) at acidic pH. From the Bjerrum's relation was found the number of screened charges important for the unfolding process. The main conclusions are: (1) the thermal stability of Sso7d has prominently entropic nature; (2) a single non-co-operative ionization controls the conformations in the D-state; and (3) pH-dependent conformational equilibrium could be functionally important in Sso7d-DNA recognition.     

Guanidine-induced unfolding of the Sso7d protein from the hyperthermophilic archaeon Sulfolobus solfataricus

The unfolding induced by guanidine hydrochloride of the small protein Sso7d from the hyperthermophilic archaeon Sulfolobus solfataricus has been investigated by means of circular dichroism and fluorescence measurements. At neutral pH and room temperature the midpoint of the transition occurred at 4M guanidine hydrochloride. Thermodynamic information was obtained by means of both the linear extrapolation model and the denaturant binding model, in the assumption of a two-state N<==>D transition. A comparison with thermodynamic data determined from the thermal unfolding of Sso7d indicated that the denaturant binding model has to be preferred. Finally, it is shown that Sso7d is the most stable against both temperature and guanidine hydrochloride among a set of globular proteins possessing a very similar 3D structure.     

Highly stable binding proteins derived from the hyperthermophilic Sso7d scaffold

We have shown that highly stable binding proteins for a wide spectrum of targets can be generated through mutagenesis of the Sso7d protein from the hyperthermophilic archaeon Sulfolobus solfataricus. Sso7d is a small (∼ 7 kDa, 63 amino acids) DNA-binding protein that lacks cysteine residues and has a melting temperature of nearly 100 °C. We generated a library of 10⁸ Sso7d mutants by randomizing 10 amino acid residues on the DNA-binding surface of Sso7d, using yeast surface display. Binding proteins for a diverse set of model targets could be isolated from this library; our chosen targets included a small organic molecule (fluorescein), a 12 amino acid peptide fragment from the C-terminus of β-catenin, the model proteins hen egg lysozyme and streptavidin, and immunoglobulins from chicken and mouse. Without the application of any affinity maturation strategy, the binding proteins isolated had equilibrium dissociation constants in the nanomolar to micromolar range. Further, Sso7d-derived binding proteins could discriminate between closely related immunoglobulins. Mutant proteins based on Sso7d were expressed at high yields in the Escherichia coli cytoplasm. Despite extensive mutagenesis, Sso7d mutants have high thermal stability; five of six mutants analyzed have melting temperatures > 89 °C. They are also resistant to chemical denaturation by guanidine hydrochloride and retain their secondary structure after extended incubation at extreme pH values. Because of their favorable properties, such as ease of recombinant expression, and high thermal, chemical and pH stability, Sso7d-derived binding proteins will have wide applicability in several areas of biotechnology and medicine.     

Thermodynamics of core hydrophobicity and packing in the hyperthermophile proteins Sac7d and Sso7d

The influence of core hydrophobicity and packing on the structure and stability of the hyperthermophile proteins Sac7d and Sso7d have been studied by calorimetry, circular dichroism, and NMR. Valine 30 is positioned in Sac7d to allow a cavity-filling Val --> Ile substitution which occurs naturally in the homologous more thermostable Sso7d. The cavity-filling mutation in Sac7d has been characterized and compared to the reciprocal Ile --> Val mutation in Sso7d. A detailed analysis of the stability of the proteins was obtained by globally fitting the variation of DSC parameters and circular dichroism intensities as a function of temperature (0-100 degrees C), salt (0-0.3 M), and pH (0-8). A global analysis over such a range of conditions permitted an unusually precise measure of the thermodynamic parameters, as well as the separation of the thermodynamics of the intrinsic unfolding reaction from the linked effects of protonation and chloride binding associated with acid-induced folding. The results indicate differences in the energetics of unfolding Sac7d and Sso7d that would not be apparent from an analysis of DSC data alone using conventional methods. The sign and magnitude of the changes in DeltaG, DeltaH, TDeltaS, and DeltaC(P) of unfolding resulting from core Ile/Val substitutions in the two proteins were consistent with differences in hydrophobicity of Val and Ile and negligible changes in packing (van der Waals) interactions. The benefit of increased hydrophobicity of the core increased with temperature, with maximal effect around 116 degrees C. Increased hydrophobicity of the core achieved not only an increase in the free energy of unfolding, but also a lateral shift of the temperature of maximal stability to higher temperature.     

Role of hydrophobic core on the thermal stability of proteins-molecular dynamics simulations on a single point mutant of sso7d

Abstract The role of salt bridges in chromatin protein Sso7d, from S. solfataricus has previously been shown to be crucial for its unusual high thermal stability. Experimental studies have shown that single site mutation of Sso7d (F31A) leads to a substantial decrease in the thermal stability of this protein due to distortion of the hydrophobic core. In the present study, we have performed a total of 0.2 μs long molecular dynamics (MD) simulations on F31A at room temperature, and at 360 K, close to the melting temperature of the wild type (WT) protein to investigate the role of hydrophobic core on protein stability. Sso7d-WT was shown to be stable at both 300 and 360 K; however, F31A undergoes denaturation at 360 K, consistent with experimental results. The structural and energetic properties obtained using the analysis of MD trajectories indicate that the single mutation results in high flexibility of the protein, and loosening of intramolecular interactions. Correlation between the dynamics of the salt bridges with the structural transitions and the unfolding pathway indicate the importance of both salt bridges and hydrophobic in effecting thermal stability of proteins in general.     

Role of hydrophobic core on the thermal stability of proteins - molecular dynamics simulations on a single point mutant of Sso7d abstract

The role of salt bridges in chromatin protein Sso7d, from S. solfataricus has previously been shown to be crucial for its unusual high thermal stability. Experimental studies have shown that single site mutation of Sso7d (F31A) leads to a substantial decrease in the thermal stability of this protein due to distortion of the hydrophobic core. In the present study, we have performed a total of 0.2 s long molecular dynamics (MD) simulations on F31A at room temperature, and at 360 K, close to the melting temperature of the wild type (WT) protein to investigate the role of hydrophobic core on protein stability. Sso7d-WT was shown to be stable at both 300 and 360 K; however, F31A undergoes denaturation at 360 K, consistent with experimental results. The structural and energetic properties obtained using the analysis of MD trajectories indicate that the single mutation results in high flexibility of the protein, and loosening of intramolecular interactions. Correlation between the dynamics of the salt bridges with the structural transitions and the unfolding pathway indicate the importance of both salt bridges and hydrophobic in effecting thermal stability of proteins in general.     

pH dependence of the reversible and irreversible thermal denaturation of gamma interferons

Heated at pH 6.0 and at 50 degrees C, human interferon gamma (HuIFN-gamma) is inactivated via the formation of insoluble aggregates. At pH 6.0, the aggregation rate increases with temperature from 40 to 65 degrees C. There is a temperature-dependent time lag to aggregate formation observed in the generation of light-scattering particles at pH 6.0, and this correlates with the fast phase observed in the kinetics of reversible thermal unfolding. In addition, the dependence of aggregation kinetics on temperature closely follows the reversible melting curve. These observations suggest that at pH 6.0 irreversible thermal denaturation and aggregation depend on partial or complete unfolding of the molecule. At pH 5.0, also at 50 degrees C, the molecule is stable to irreversible aggregation. In reversible unfolding in 0.25 M guanidine hydrochloride, the Tm for HuIFN-gamma increases from 30.5 degrees C at pH 4.75 to 41.8 degrees C at pH 6.25, in analogy to the behavior of other globular proteins. These observations suggest that the relative instability of HuIFN-gamma to irreversible denaturation via aggregation at pH 6.0 compared to pH 5.0 is not due to an increased stability toward unfolding at the lower pH. Alternatively, stability at pH 5.0 must be due either to the improved solution properties of the unfolded state or to the improved solubility/decreased kinetic lifetime of an unfolding intermediate. Aggregation of HuIFN-gamma at 50 degrees C is half-maximal at pH 5.7, suggesting that protonation of one or both of the histidine residues may be involved in this stabilization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermal unfolding of small proteins with SH3 domain folding pattern

The thermal unfolding of three SH3 domains of the Tec family of tyrosine kinases was studied by differential scanning calorimetry and CD spectroscopy. The unfolding transition of the three protein domains in the acidic pH region can be described as a reversible two-state process. For all three SH3 domains maximum stability was observed in the pH region 4.5 < pH < 7.0 where these domains unfold at temperatures of 353K (Btk), 342K (Itk), and 344K (Tec). At these temperatures an enthalpy change of 196 kJ/mol, 178 kJ/mol, and 169 kJ/mol was measured for Btk-, Itk-, and Tec-SH3 domains, respectively. The determined changes in heat capacity between the native and the denatured state are in an usual range expected for small proteins. Our analysis revealed that all SH3 domains studied are only weakly stabilized and have free energies of unfolding which do not exceed 12–16 kJ/mol but show quite high melting temperatures. Comparing unfolding free energies measured for eukaryotic SH3 domains with those of the topologically identical Sso7d protein from the hyperthermophile Sulfolobus solfataricus, the increased melting temperature of the thermostable protein is due to a broadening as well as a significant lifting of its stability curve. However, at their physiological temperatures, 310K for mesophilic SH3 domains and 350K for Sso7d, eukaryotic SH3 domains and Sso7d show very similar stabilities. Proteins 31:309–319, 1998. © 1998 Wiley-Liss, Inc.     

pH dependence of the stability of barstar to chemical and thermal denaturation.

Equilibrium unfolding of barstar with guanidine hydrochloride (GdnHCl) and urea as denaturants as well as thermal unfolding have been carried out as a function of pH using fluorescence, far-UV and near-UV CD, and absorbance as probes. Both GdnHCl-induced and urea-induced denaturation studies at pH 7 show that barstar unfolds through a two-state F<->U mechanism and yields identical values for delta GU, the free energy difference between the fully folded (F) and unfolded (U) forms, of 5.0 +/- 0.5 kcal.mol-1 at 25 degrees C. Thermal denaturation of barstar also follows a two-state F<->U unfolding transition at pH 7, and the value of delta GU at 25 degrees C is similar to that obtained from chemical denaturation. The pH dependence of denaturation by GdnHCl is complex. The Cm value (midpoint of the unfolding transition) has been used as an index for stability in the pH range 2-10, because barstar does not unfold through a two-state transition on denaturation by GdnHCl at all pH values studied. Stability is maximum at pH 2-3, where barstar exists in a molten globule-like form that forms a large soluble oligomer. The stability decreases with an increase in pH to 5, the isoelectric pH of the protein. Above pH 5, the stability increases as the pH is raised to 7. Above pH 8, it again decreases as the pH is raised to 10. The decrease in stability from pH 7 to 5 in wild-type (wt) barstar, which is shown to be characterized by an apparent pKa of 6.2 +/- 0.2, is not observed in H17Q, a His 17-->Gln 17 mutant form of barstar. This decrease in stability has therefore been correlated with the protonation of His 17 in barstar. The decrease in stability beyond pH 8 in wt barstar, which is characterized by an apparent pKa of 9.2 +/- 0.2, is not detected in BSCCAA, the Cys 40 Cys 82-->Ala 40 Ala 82 double mutant form of barstar. Thus, this decrease in stability has been correlated with the deprotonation of at least one of the two cysteines present in wt barstar. The increase in stability from pH 5 to 3 is characterized by an apparent pKa of 4.6 +/- 0.2 for wt barstar and BSCCAA, which is similar to the apparent pKa that characterizes the structural transition leading to the formation of the A form. The use of Cm as an index of stability has been supported by thermal denaturation studies.(ABSTRACT TRUNCATED AT 400 WORDS)     

Role of Hydrophobic Core on the Thermal Stability of Proteins—Molecular Dynamics Simulations on a Single Point Mutant of Sso7d

Abstract

The role of salt bridges in chromatin protein Sso7d, from S. solfataricus has previously been shown to be crucial for its unusual high thermal stability. Experimental studies have shown that single site mutation of Sso7d (F31A) leads to a substantial decrease in the thermal stability of this protein due to distortion of the hydrophobic core. In the present study, we have performed a total of 0.2 μs long molecular dynamics (MD) simulations on F31A at room temperature, and at 360 K, close to the melting temperature of the wild type (WT) protein to investigate the role of hydrophobic core on protein stability. Sso7d-WT was shown to be stable at both 300 and 360 K; however, F31A undergoes denaturation at 360 K, consistent with experimental results. The structural and energetic properties obtained using the analysis of MD trajectories indicate that the single mutation results in high flexibility of the protein, and loosening of intramolecular interactions. Correlation between the dynamics of the salt bridges with the structural transitions and the unfolding pathway indicate the importance of both salt bridges and hydrophobic in effecting thermal stability of proteins in general.     

Stability and physicochemical properties of the bovine brain phosphatidylethanolamine-binding protein.

The equilibrium behaviour of the bovine phosphatidylethanolamine-binding protein (PEBP) has been studied under various conditions of pH, temperature and urea concentration. Far-UV and near-UV CD, fluorescence and Fourier transform infrared spectroscopies indicate that, in its native state, PEBP is mainly composed of beta-sheets, with Trp residues mostly localized in a hydrophobic environment; these results suggest that the conformation of PEBP in solution is similar to the three-dimensional structure determined by X-ray crystallography. The pH-induced conformational changes show a transition midpoint at pH 3.0, implying nine protons in the transition. At neutral pH, the thermal denaturation is irreversible due to protein precipitation, whereas at acidic pH values the protein exhibits a reversible denaturation. The thermal denaturation curves, as monitored by CD, fluorescence and differential scanning calorimetry, support a two-state model for the equilibrium and display coincident values with a melting temperature Tm = 54 degrees C, an enthalpy change DeltaH = 119 kcal.mol-1 and a free energy change DeltaG(H2O, 25 degrees C) = 5 kcal.mol-1. The urea-induced unfolding profiles of PEBP show a midpoint of the two-state unfolding transition at 4.8 M denaturant, and the stability of PEBP is 4.5 kcal.mol-1 at 25 degrees C. Moreover, the surface active properties indicate that PEBP is essentially a hydrophilic protein which progressively unfolds at the air/water interface over the course of time. Together, these results suggest that PEBP is well-structured in solution but that its conformation is weakly stable and sensitive to hydrophobic conditions: the PEBP structure seems to be flexible and adaptable to its environment.     

Thermal versus guanidine-induced unfolding of ubiquitin. An analysis in terms of the contributions from charge-charge interactions to protein stability.

We have characterized the guanidine-induced unfolding of both yeast and bovine ubiquitin at 25 degrees C and in the acidic pH range on the basis of fluorescence and circular dichroism measurements. Unfolding Gibbs energy changes calculated by linear extrapolation from high guanidine unfolding data are found to depend very weakly on pH. A simple explanation for this result involves the two following assumptions: (1) charged atoms of ionizable groups are exposed to the solvent in native ubiquitin (as supported by accessible surface area calculations), and Gibbs energy contributions associated with charge desolvation upon folding (a source of pK shifts) are small; (2) charge-charge interactions (another source of pK shifts upon folding) are screened out in concentrated guanidinium chloride solutions. We have also characterized the thermal unfolding of both proteins using differential scanning calorimetry. Unfolding Gibbs energy changes calculated from the calorimetric data do depend strongly on pH, a result that we attribute to the pH dependence of charge-charge interactions (not eliminated in the absence of guanidine). In fact, we find good agreement between the difference between the two series of experimental unfolding Gibbs energy changes (determined from high guanidine unfolding data by linear extrapolation and from thermal denaturation data in the absence of guanidine) and the theoretical estimates of the contribution from charge-charge interactions to the Gibbs energy change for ubiquitin unfolding obtained by using the solvent-accessibility-corrected Tanford-Kirkwood model, together with the Bashford-Karplus (reduced-set-of-sites) approximation. This contribution is found to be stabilizing at neutral pH, because most charged groups on the native protein interact mainly with groups of the opposite charge, a fact that, together with the absence of large charge-desolvation contributions, may explain the high stability of ubiquitin at neutral pH. In general, our analysis suggests the possibility of enhancing protein thermal stability by adequately redesigning the distribution of solvent-exposed, charged residues on the native protein surface.     

Strong Enrichment of Aromatic Residues in Binding Sites from a Charge-neutralized Hyperthermostable Sso7d Scaffold Library

The Sso7d protein from the hyperthermophilic archaeon Sulfolobus solfataricus is an attractive binding scaffold because of its small size (7 kDa), high thermal stability (T_m of 98 °C), and absence of cysteines and glycosylation sites. However, as a DNA-binding protein, Sso7d is highly positively charged, introducing a strong specificity constraint for binding epitopes and leading to nonspecific interaction with mammalian cell membranes. In the present study, we report charge-neutralized variants of Sso7d that maintain high thermal stability. Yeast-displayed libraries that were based on this reduced charge Sso7d (rcSso7d) scaffold yielded binders with low nanomolar affinities against mouse serum albumin and several epitopes on human epidermal growth factor receptor. Importantly, starting from a charge-neutralized scaffold facilitated evolutionary adaptation of binders to differentially charged epitopes on mouse serum albumin and human epidermal growth factor receptor, respectively. Interestingly, the distribution of amino acids in the small and rigid binding surface of enriched rcSso7d-based binders is very different from that generally found in more flexible antibody complementarity-determining region loops but resembles the composition of antibody-binding energetic hot spots. Particularly striking was a strong enrichment of the aromatic residues Trp, Tyr, and Phe in rcSso7d-based binders. This suggests that the rigidity and small size of this scaffold determines the unusual amino acid composition of its binding sites, mimicking the energetic core of antibody paratopes. Despite the high frequency of aromatic residues, these rcSso7d-based binders are highly expressed, thermostable, and monomeric, suggesting that the hyperstability of the starting scaffold and the rigidness of the binding surface confer a high tolerance to mutation.     

Hydrogen-exchange kinetics of the indole NH proton of the buried tryptophan in the constant fragment of the immunoglobulin light chain

The constant fragment of the immunoglobulin light chain (type lambda) has two tryptophyl residues at positions 150 and 187. Trp-150 is buried in the interior, and Trp-187 lies on the surface of the molecule. The hydrogen-deuterium exchange kinetics of the indole NH proton of Trp-150 were studied at various pH values at 25 degrees C by 1H nuclear magnetic resonance. Exchange rates were approximately first order in hydroxyl ion dependence above pH 8, were relatively independent of pH between pH 7 and 8, and decreased below pH 7. On the assumption that the exchange above pH 8 proceeds through local fluctuations of the protein molecule, the exchange rates between pH 7 and 8 through global unfolding were estimated. The exchange rate constant within this pH range at 25 degrees C thus estimated was consistent with that of the global unfolding of the constant fragment under the same conditions as those reported previously [Kikuchi, H., Goto, Y., & Hamaguchi, K. (1986) Biochemistry 25, 2009-2013]. The activation energy for the exchange process at pH 7.8 was the same as that for the unfolding process by 2 M guanidine hydrochloride. The exchange rates of backbone NH protons were almost the same as that of the indole NH proton of Trp-150 at pH 7.1. These observations also indicated that the exchange between pH 7 and 8 occurs through global unfolding of the protein molecule and is rate-limited by the unfolding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermal denaturation of Micrococcus lysodeikticus adenosine triphosphatase. Influence of temperature on the circular dichroism, fluroescence and enzymic activity of the protein.

The soluble ATPase (adenosine triphosphatase) from Micrococcus lysodeikticus underwent a major unfolding transition when solutions of the enzyme at pH 7.5 were heated. The midpoint occurred at 46 degrees C when monitored by changes in enzymic activity and intrinsic fluorescence, and at 49 degrees C when monitored by circular dichroism. The products of thermal denaturation retained much secondary structure, and no evidence of subunit dissociation was detected after cooling at 20 degrees C. The thermal transition was irreversible, and thiol groups were not involved in the irreversibility. The presence of ATP, adenylyl imidodiphosphate, CaCl2 or higher concentrations of ATPase conferred stability against thermal denaturation, but did not prevent the irreversibility one denaturation had taken place. In the presence of guanidinium chloride, thermal denaturation occurred at lower temperatures. The midpoints of the transition were 45 degrees C in 0.25 M-, 38 degrees C in 0.5 M-and 30 degrees C in 0.75 M-denaturant. In the highest concentration of guanidinium chloride a similar unfolding transition induced by cooling was observed. Its midpoint was 9 degrees C, and the temperature of maximum stability of the protein was 20 degrees C. The discontinuities occurring the the Arrhenius plots of the activity of this enzyme had no counterpart in variations in the far-u.v. circular dichroism or intrinsic fluorescence of the protein at the same temperature.     

In vitro DNA binding of the archaeal protein Sso7d induces negative supercoiling at temperatures typical for thermophilic growth.

The topological state of DNA in hyperthermophilic archaea appears to correspond to a linking excess in comparison with DNA in mesophilic organisms. Since DNA binding proteins often contribute to the control of DNA topology by affecting DNA geometry in the presence of DNA topoisomerases, we tested whether the histone-like protein Sso7d from the hyperthermophilic archaeon Sulfolobus solfataricus alters DNA conformation. In ligase-mediated supercoiling assays carried out at 37, 60, 70, 80 and 90 degrees C we found that DNA binding of increasing amounts of Sso7d led to a progressive decrease in plasmid linking number (Lk), producing negative supercoiling. Identical unwinding effects were observed when recombinant non-methylated Sso7d was used. For a given Sso7d concentration the DNA unwinding induced was augmented with increasing temperature. However, after correction for the overwinding effect of high temperature on DNA, plasmids ligated at 60-90 degrees C exhibited similar sigma values at the highest Sso7d concentrations assayed. These results suggest that Sso7d may play a compensatory role in vivo by counteracting the overwinding effect of high temperature on DNA. Additionally, Sso7d unwinding could be involved in the topological changes observed during thermal stress (heat and cold shock), playing an analogous role in crenarchaeal cells to that proposed for HU in bacteria.     

Effect of pH on the structure and stability of Bacillus circulans ssp. alkalophilus phosphoserine aminotransferase: thermodynamic and crystallographic studies

pH is one of the key parameters that affect the stability and function of proteins. We have studied the effect of pH on the pyridoxal-5'-phosphate-dependent enzyme phosphoserine aminotransferase produced by the facultative alkaliphile Bacillus circulans ssp. alkalophilus using thermodynamic and crystallographic analysis. Enzymatic activity assay showed that the enzyme has maximum activity at pH 9.0 and relative activity less than 10% at pH 7.0. Differential scanning calorimetry and circular dichroism experiments revealed variations in the stability and denaturation profiles of the enzyme at different pHs. Most importantly, release of pyridoxal-5'-phosphate and protein thermal denaturation were found to occur simultaneously at pH 6.0 in contrast to pH 8.5 where denaturation preceded cofactor's release by approximately 3 degrees C. To correlate the observed differences in thermal denaturation with structural features, the crystal structure of phosphoserine aminotransferase was determined at 1.2 and 1.5 A resolution at two different pHs (8.5 and 4.6, respectively). Analysis of the two structures revealed changes in the vicinity of the active site and in surface residues. A conformational change in a loop involved in substrate binding at the entrance of the active site has been identified upon pH change. Moreover, the number of intramolecular ion pairs was found reduced in the pH 4.6 structure. Taken together, the presented kinetics, thermal denaturation, and crystallographic data demonstrate a potential role of the active site in unfolding and suggest that subtle but structurally significant conformational rearrangements are involved in the stability and integrity of phosphoserine aminotransferase in response to pH changes.     

Structural stability of the PsbQ protein of higher plant photosystem II

We have characterized the stability and folding behavior of the isolated extrinsic PsbQ protein of photosystem II (PSII) from a higher plant, Spinacia oleracea, using intrinsic protein fluorescence emission and near- and far-UV circular dichroism (CD) spectroscopy in combination with differential scanning calorimetry (DSC). Experimental results reveal that both chemical denaturation using guanidine hydrochloride (GdnHCl) and thermal unfolding of PsbQ proceed as a two-state reversible process. The denaturation free-energy changes (DeltaG(D)) at 20 degrees C extrapolated from GdnHCl (4.0 +/- 0.6 kcal mol(-1)) or thermal unfolding (4.4 +/- 0.8 kcal mol(-1)) are very close. Moreover, the far-UV CD spectra of the denatured PsbQ registered at 90 degrees C in the absence and presence of 6.0 M GdnHCl superimpose, leading us to conclude that both denatured states of PsbQ are structurally and energetically similar. The thermal unfolding of PsbQ has been also characterized by CD and DSC over a wide pH range. The stability of PsbQ is at its maximum at pH comprised between 5 and 8, being wider than the optimal pH for oxygen evolution in the lumen of thylakoid membranes. In addition, no significant structural changes were detected in PsbQ between 50 and 55 degrees C in the pH range of 3-8, suggesting that PsbQ behaves as a soluble and stable particle in the lumen when it detaches from PSII under physiological stress conditions such as high temperature (45-50 degrees C) or low pH (<5.0). Sedimentation experiments showed that, in solution at 20 degrees C, the PsbQ protein is a monomer with an elongated shape.