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.     

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.     

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.     

Linkage of proton binding to the thermal unfolding of Sso7d from the hyperthermophilic archaebacterium Sulfolobus solfataricus.

In this study the pH dependence of the thermal stability of Sso7d from Sulfolobus solfataricus is analyzed. This small globular protein of 63 residues shows a very marked dependence of thermal stability on pH: the denaturation temperature passes from 65.2 degrees C at pH 2.5 to 97.9 degrees C at pH 4.5. Analysis of the data points out that the binding of at least two protons is coupled to the thermal unfolding. By linking the proton binding to the conformational unfolding equilibrium, a thermodynamic model, which is able to describe the dependence upon the solution pH of both the excess heat capacity function and the denaturation Gibbs energy change for Sso7d, is developed. The decreased stability in very acid conditions is due to the binding of two protons on identical and noninteracting sites of the unfolded state. Actually, such sites are two carboxyl groups possessing very low pKa values in the native structure, probably involved in salt-bridges on the protein surface.     

Insights into the thermal stabilization and conformational transitions of DNA by hyperthermophile protein Sso7d: molecular dynamics simulations and MM-PBSA analysis

In the assembly of DNA-protein complex, the DNA kinking plays an important role in nucleoprotein structures and gene regulation. Molecular dynamics (MD) simulations were performed on specific protein-DNA complexes in this study to investigate the stability and structural transitions of DNA depending on temperature. Furthermore, we introduced the molecular mechanics/Poisson–Boltzmann surface area (MM-PBSA) approach to analyze the interactions between DNA and protein in hyperthermophile. Focused on two specific Sso7d-DNA complexes (PDB codes: 1BNZ and 1BF4), we performed MD simulations at four temperatures (300, 360, 420, and 480?K) and MM-PBSA at 300 and 360?K to illustrate detailed information on the changes of DNA. Our results show that Sso7d stabilizes DNA duplex over a certain temperature range and DNA molecules undergo B-like to A-like form transitions in the binary complex with the temperature increasing, which are consistent with the experimental data. Our work will contribute to a better understanding of protein-DNA interaction.     

Insights into the thermal stabilization and conformational transitions of DNA by hyperthermophile protein Sso7d: molecular dynamics simulations and MM-PBSA analysis

In the assembly of DNA-protein complex, the DNA kinking plays an important role in nucleoprotein structures and gene regulation. Molecular dynamics (MD) simulations were performed on specific protein-DNA complexes in this study to investigate the stability and structural transitions of DNA depending on temperature. Furthermore, we introduced the molecular mechanics/Poisson-Boltzmann surface area (MM-PBSA) approach to analyze the interactions between DNA and protein in hyperthermophile. Focused on two specific Sso7d-DNA complexes (PDB codes: 1BNZ and 1BF4), we performed MD simulations at four temperatures (300, 360, 420, and 480?K) and MM-PBSA at 300 and 360?K to illustrate detailed information on the changes of DNA. Our results show that Sso7d stabilizes DNA duplex over a certain temperature range and DNA molecules undergo B-like to A-like form transitions in the binary complex with the temperature increasing, which are consistent with the experimental data. Our work will contribute to a better understanding of protein-DNA interaction.     

Thermal stability and unfolding pathways of Sso7d and its mutant F31A: insight from molecular dynamics simulation

The thermo-stability and unfolding behaviors of a small hyperthermophilic protein Sso7d as well as its single-point mutation F31A are studied by molecular dynamics simulation at temperatures of 300 K, 371 K and 500 K. Simulations at 300 K show that the F31A mutant displays a much larger flexibility than the wild type, which implies that the mutation obviously decreases the protein's stability. In the simulations at 371 K, although larger fluctuations were observed, both of these two maintain their stable conformations. High temperature simulations at 500 K suggest that the unfolding of these two proteins evolves along different pathways. For the wild-type protein, the C-terminal alpha-helix is melted at the early unfolding stage, whereas it is destroyed much later in the unfolding process of the F31A mutant. The results also show that the mutant unfolds much faster than its parent protein. The deeply buried aromatic cluster in the F31A mutant dissociates quickly relative to the wild-type protein at high temperature. Besides, it is found that the triple-stranded antiparallel β-sheet in the wild-type protein plays an important role in maintaining the stability of the entire structure.     

Increasing temperature accelerates protein unfolding without changing the pathway of unfolding

We have traditionally relied on extremely elevated temperatures (498K, 225 degrees C) to investigate the unfolding process of proteins within the timescale available to molecular dynamics simulations with explicit solvent. However, recent advances in computer hardware have allowed us to extend our thermal denaturation studies to much lower temperatures. Here we describe the results of simulations of chymotrypsin inhibitor 2 at seven temperatures, ranging from 298K to 498K. The simulation lengths vary from 94ns to 20ns, for a total simulation time of 344ns, or 0.34 micros. At 298K, the protein is very stable over the full 50ns simulation. At 348K, corresponding to the experimentally observed melting temperature of CI2, the protein unfolds over the first 25ns, explores partially unfolded conformations for 20ns, and then refolds over the last 35ns. Above its melting temperature, complete thermal denaturation occurs in an activated process. Early unfolding is characterized by sliding or breathing motions in the protein core, leading to an unfolding transition state with a weakened core and some loss of secondary structure. After the unfolding transition, the core contacts are rapidly lost as the protein passes on to the fully denatured ensemble. While the overall character and order of events in the unfolding process are well conserved across temperatures, there are substantial differences in the timescales over which these events take place. We conclude that 498K simulations are suitable for elucidating the details of protein unfolding at a minimum of computational expense.     

Thermal Stability and Unfolding Pathways of Sso7d and its Mutant F31A: Insight from Molecular Dynamics Simulation

Abstract

The thermo-stability and unfolding behaviors of a small hyperthermophilic protein Sso7d as well as its single-point mutation F31A are studied by molecular dynamics simulation at temperatures of 300 K, 371 K and 500 K. Simulations at 300 K show that the F31A mutant displays a much larger flexibility than the wild type, which implies that the mutation obviously decreases the protein's stability. In the simulations at 371 K, although larger fluctuations were observed, both of these two maintain their stable conformations. High temperature simulations at 500 K suggest that the unfolding of these two proteins evolves along different pathways. For the wild-type protein, the C-terminal alpha-helix is melted at the early unfolding stage, whereas it is destroyed much later in the unfolding process of the F31A mutant. The results also show that the mutant unfolds much faster than its parent protein. The deeply buried aromatic cluster in the F31A mutant dissociates quickly relative to the wild-type protein at high temperature. Besides, it is found that the triple-stranded antiparallel β-sheet in the wild-type protein plays an important role in maintaining the stability of the entire structure.     

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.     

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.     

Selective excitation of native fluctuations during thermal unfolding simulations: horse heart cytochrome c as a case study

The effect of temperature on the activation of native fluctuation motions during molecular dynamics unfolding simulations of horse heart cytochrome c has been studied. Essential dynamics analysis has been used to analyze the preferred directions of motion along the unfolding trajectories obtained by high temperature simulations. The results of this study have evidenced a clear correlation between the directions of the deformation motions that occur in the first stage of the unfolding process and few specific essential motions characterizing the 300 K dynamics of the protein. In particular, one of those collective motions, involved in the fluctuation of a loop region, is specifically excited in the thermal denaturation process, becoming progressively dominant during the first 500 ps of the unfolding simulations. As further evidence, the essential dynamics sampling performed along this collective motion has shown a tendency of the protein to promptly unfold. According to these results, the mechanism of thermal induced denaturation process involves the selective excitation of one or few specific equilibrium collective motions.     

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.     

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.     

Probing the energy landscape of protein folding/unfolding transition states

Previous molecular dynamics (MD) simulations of the thermal denaturation of chymotrypsin inhibitor 2 (CI2) have provided atomic-resolution models of the transition state ensemble that is well supported by experimental studies. Here, we use simulations to further investigate the energy landscape around the transition state region. Nine structures within approximately 35 ps and 3 A C(alpha) RMSD of the transition state ensemble identified in a previous 498 K thermal denaturation simulation were quenched under the quasi-native conditions of 335 K and neutral pH. All of the structures underwent hydrophobically driven collapse in response to the drop in temperature. Structures less denatured than the transition state became structurally more native-like, while structures that were more denatured than the transition state tended to show additional loss of native structure. The structures in the immediate region of the transition state fluctuated between becoming more and less native-like. All of the starting structures had the same native-like topology and were quite similar (within 3.5 A C(alpha) RMSD). That the structures all shared native-like topology, yet diverged into either more or less native-like structures depending on which side of the transition state they occupied on the unfolding trajectory, indicates that topology alone does not dictate protein folding. Instead, our results suggest that a detailed interplay of packing interactions and interactions with water determine whether a partially denatured protein will become more native-like under refolding conditions.     

Phi-analysis at the experimental limits: mechanism of beta-hairpin formation

The 37-residue Formin-binding protein, FBP28, is a canonical three-stranded beta-sheet WW domain. Because of its small size, it is so insensitive to chemical denaturation that it is barely possible to determine accurately a denaturation curve, as the transition spans 0-7 M guanidinium hydrochloride (GdmCl). It is also only marginally stable, with a free energy of denaturation of just 2.3 kcal/mol at 10 degrees Celsius so only small changes in energy upon mutation can be tolerated. But these properties and relaxation times for folding of 25 micros-400 micros conspire to allow the rapid acquisition of accurate and reproducible kinetic data for Phi-analysis using classical temperature-jump methods. The transition state for folding is highly polarized with some regions having Phi-values of 0 and others 1, as readily seen in chevron plots, with Phi-values of 0 having the refolding arms overlaying and those of 1 the unfolding arms superimposable. Good agreement is seen with transition state structures identified from independent molecular dynamics (MD) simulations at 60, 75, and 100 degrees Celsius, which allows us to explore further the details of the folding and unfolding pathway of FBP28. The first beta-turn is near native-like in the transition state for folding (experimental) and unfolding (MD and experiment). The simulations show that there are transient contacts between the aromatic side-chains of the beta-strands in the denatured state and that these interactions provide the driving force for folding of the first beta-hairpin of this three-stranded sheet. Only after the backbone hydrogen bonds are formed between beta1 and beta2 does a hydrogen bond form to stabilize the intervening turn, or the first beta-turn.     

Molecular dynamics simulations as a tool for improving protein stability

Haloalkane dehalogenase (DhlA) was used as a model protein to explore the possibility to use molecular dynamics (MD) simulations as a tool to identify flexible regions in proteins that can serve as a target for stability enhancement by introduction of a disulfide bond. DhlA consists of two domains: an alpha/beta-hydrolase fold main domain and a cap domain composed of five alpha-helices. MD simulations of DhlA showed high mobility in a helix-loop-helix region in the cap domain, involving residues 184-211. A disulfide cross-link was engineered between residue 201 of this flexible region and residue 16 of the main domain. The mutant enzyme showed substantial changes in both thermal and urea denaturation. The oxidized form of the mutant enzyme showed an increase of the apparent transition temperature from 47.5 to 52.5 degrees C, whereas the T(m,app) of the reduced mutant decreased by more than 8 degrees C compared to the wild-type enzyme. Urea denaturation results showed a similar trend. Measurement of the kinetic stability showed that the introduction of the disulfide bond caused a decrease in activation free energy of unfolding of 0.43 kcal mol(-1) compared to the wild-type enzyme and also indicated that the helix-loop-helix region was involved early in the unfolding process. The results show that MD simulations are capable of identifying mobile protein domains that can successfully be used as a target for stability enhancement by the introduction of a disulfide cross-link.     

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.     

Role of the N-terminal region for the conformational stability of esterase 2 from Alicyclobacillus acidocaldarius

In order to clarify the role played by the N-terminal region for the conformational stability of the thermophilic esterase 2 (EST2) from Alicyclobacillus acidocaldarius, two mutant forms have been investigated: a variant obtained by deleting the first 35 residues at the N-terminus (EST2-36del), and a variant obtained by mutating Lys102 to Gln (K102Q) to perturb the N-terminus by destroying the salt bridge E43-K102. The temperature- and denaturant-induced unfolding of EST2 and the two mutant forms have been studied by means of circular dichroism (CD), differential scanning calorimetry (DSC) and fluorescence measurements. In line with its thermophilic origin, the denaturation temperature of EST2 is high: T(d)=91 degrees C and 86 degrees C if detected by recording the CD signal at 222 nm and 290 nm, respectively. This difference suggests that the thermal denaturation process, even though reversible, is more complex than a two-state Nright arrow over left arrowD transition. The non-two-state behaviour is more pronounced in the case of the two mutant forms. The complex DSC profiles of EST2 and both mutant forms have been analysed by means of a deconvolution procedure. The thermodynamic parameters characterizing the two transitions obtained in the case of EST2 are: T(d,1)=81 degrees C, Delta(d)H(1)=440 kJ mol(-1), Delta(d)C(p,1)=7 kJ K(-1)mol(-1), T(d,2)=86 degrees C, Delta(d)H(2)=710 kJ mol(-1), and Delta(d)C(p,2)=9 kJ K(-1)mol(-1). The first transition occurs at lower temperatures in the two mutant forms, whereas the second transition is always centred at 86 degrees C. The results indicate that EST2 possesses two structural domains whose coupling is tight in the wild-type protein, but markedly weakens in the two mutant forms as a consequence of the perturbations in the N-terminal region.