Osmolyte effect on the stability and folding of a hyperthermophilic protein

Proteins are known to be stabilized by naturally occurring osmolytes such as amino acids, sugars, and methylamines. Here, we examine the effect of trimethylamine-N-oxide (TMAO) on the conformational stability of ribonuclease HII from a hyperthermophile, Thermococcus kodakaraensis (Tk-RNase HII), which inherently possesses high conformational stability. Heat- and guanidine hydrochloride-induced unfolding experiments demonstrated that the conformational stability of Tk-RNase HII in the presence of 0.5M TMAO was higher than that in the absence of TMAO at all examined temperatures. TMAO affected the unfolding and refolding kinetics of Tk-RNase HII to a similar extent. These results indicate that proteins are universally stabilized by osmolytes, regardless of their robustness, and suggest a stabilization mechanism by osmolytes, caused by the unfavorable interaction of osmolytes with protein backbones in the denatured state. Our results also imply that the basic protein folding principle is not dependent on protein stability and evolution.     

Hydrophobic effect on the stability and folding of a hyperthermophilic protein

Ribonuclease HII from hyperthermophile Thermococcus kodakaraensis (Tk-RNase HII) is a kinetically robust monomeric protein. The conformational stability and folding kinetics of Tk-RNase HII were measured for nine mutant proteins in which a buried larger hydrophobic side chain is replaced by a smaller one (Leu/Ile to Ala). The mutant proteins were destabilized by 8.9 to 22.0 kJ mol^− 1 as compared with the wild-type protein. The removal of each -CH₂- group burial decreased the stability by 5.1 kJ mol^− 1 on average in the mutant proteins of Tk-RNase HII examined. This is comparable with the value of 5.3 kJ mol^− 1 obtained from experiments for proteins from organisms growing at moderate temperature. We conclude that the hydrophobic residues buried inside protein molecules contribute to the stabilization of hyperthermophilic proteins to a similar extent as proteins at normal temperature. In the folding experiments, the mutant proteins of Tk-RNase HII examined exhibited faster unfolding compared with the wild-type protein. These results indicate that the buried hydrophobic residues strongly contribute to the kinetic robustness of Tk-RNase HII. This is the first report that provides a practical cause of slow unfolding of hyperthermostable proteins.     

Maltose-binding protein from the hyperthermophilic bacterium Thermotoga maritima: stability and binding properties

Recombinant maltose-binding protein from Thermotoga maritima (TmMBP) was expressed in Escherichia coli and purified to homogeneity, applying heat incubation of the crude extract at 75 degrees C. As taken from the spectral, physicochemical and binding properties, the recombinant protein is indistinguishable from the natural protein isolated from the periplasm of Thermotoga maritima. At neutral pH, TmMBP exhibits extremely high intrinsic stability with a thermal transition >105 degrees C. Guanidinium chloride-induced equilibrium unfolding transitions at varying temperatures result in a stability maximum at approximately 40 degrees C. At room temperature, the thermodynamic analysis of the highly cooperative unfolding equilibrium transition yields DeltaG(N-->U)=100(+/-5) kJ mol(-1 )for the free energy of stabilization. Compared to mesophilic MBP from E. coli as a reference, this value is increased by about 60 kJ mol(-1). At temperatures around the optimal growth temperature of T. maritima (t(opt) approximately 80 degrees C), the yield of refolding does not exceed 80 %; the residual 20 % are misfolded, as indicated by a decrease in stability as well as loss of the maltose-binding capacity. TmMBP is able to bind maltose, maltotriose and trehalose with dissociation constants in the nanomolar to micromolar range, combining the substrate specificities of the homologs from the mesophilic bacterium E. coli and the hyperthermophilic archaeon Thermococcus litoralis. Fluorescence quench experiments allowed the dissociation constants of ligand binding to be quantified. Binding of maltose was found to be endothermic and entropy-driven, with DeltaH(b)=+47 kJ mol(-1) and DeltaS(b)=+257 J mol(-1) K(-1). Extrapolation of the linear vant'Hoff plot to t(opt) resulted in K(d) approximately 0.3 microM. This result is in agreement with data reported for the MBPs from E. coli and T. litoralis at their respective optimum growth temperatures, corroborating the general observation that proteins under their specific physiological conditions are in corresponding states.     

Alcohol dehydrogenase from the hyperthermophilic archaeon Pyrobaculum aerophilum: stability at high temperature

The structural and stability properties of a novel zinc-dependent alcohol dehydrogenase from the hyperthermophilic archaeon Pyrobaculum aerophilum (PyAeADHII) were investigated by Fourier transformed infrared spectroscopy (FTIR). This enzyme is a thermostable homo-tetramer belonging to the GroES-fold motif proteins characterized by an irregular β-barrel conformation. Our results revealed a protein with a secondary structure rich in β-sheet (32% of the total secondary elements) and it showed a three-step thermal unfolding pathway. The complete enzyme denaturation was preceded by the formation of a relaxed tertiary/quaternary structure and previously by an excited native-like conformation. Two-dimensional correlation spectroscopy analysis (2D-COS) and differential scanning calorimetry (DSC) experiments supported these data and allowed us to determine the protein melting temperature at 96.9 °C as well as to suggest the sequence of the events that occurred during the protein denaturation process.     

Comparative analyses of the conformational stability of a hyperthermophilic protein and its mesophilic counterpart.

Comparison of the conformational stability of an O(6)-methylguanine-DNA methyltransferase (MGMT) from the hyperthermophilic archaeon Thermococcus kodakaraensis strain KOD1 (Tk-MGMT), and its mesophilic counterpart C-terminal Ada protein from Escherichia coli (Ec-AdaC) was performed in order to obtain information about the relationship between thermal stability and other factors, such as thermodynamic parameters, thermodynamic stability and other unfolding conditions. Tk-MGMT unfolded at Tm = 98.6 degrees C, which was 54.8 degrees C higher than the unfolding temperature of Ec-AdaC. The maximum free energy (DeltaG(max)) of the proteins were different; the value of Tk-MGMT (42.9 kJ.mol-1 at 29.5 degrees C) was 2.6 times higher than that of Ec-AdaC (16.6 kJ.mol-1 at 7.4 degrees C). The high conformational stability of Tk-MGMT was attributed to a 1.6-fold higher enthalpy value than that of Ec-AdaC. In addition, the DeltaG(max) temperature of Tk-MGMT was considerably higher (by 22.1 degrees C). The apparent heat capacity of denaturation (DeltaC(p)) of Tk-MGMT was 0.7-fold lower than that of Ec-AdaC. These three synergistic effects, increasing DeltaGmax, shifted DeltaG vs. temperature curve, and low DeltaC(p), give Tk-MGMT its thermal stability. Unfolding profiles of the two proteins, tested with four alcohols and three denaturants, showed that Tk-MGMT possessed higher stability than Ec-AdaC in all conditions studied. These results indicate that the high stability of Tk-MGMT gives resistance to chemical unfolding, in addition to thermal unfolding.     

A thermodynamic study of mesophilic, thermophilic, and hyperthermophilic L-arabinose isomerases: the effects of divalent metal ions on protein stability at elevated temperatures

To gain insight into the structural stability of homologous homo-tetrameric l-arabinose isomerases (AI), we have examined the isothermal guanidine hydrochloride (GdnHCl)-induced unfolding of AIs from mesophilic Bacillus halodurans (BHAI), thermophilic Geobacillus stearothermophilus (GSAI), and hyperthermophilic Thermotoga maritima (TMAI) using circular dichroism spectroscopy. The GdnHCl-induced unfolding of the AIs can be well described by a two-state reaction between native tetramers and unfolded monomers, which directly confirms the validity of the linear extrapolation method to obtain the intrinsic stabilities of these proteins. The resulting unfolding free energy (DeltaGU) values of the AIs as a function of temperature were fit to the Gibbs-Helmholtz equation to determine their thermodynamic parameters based on a two-state mechanism. Compared with the stability curves of BHAI in the presence and absence of Mn2+, those of holo GSAI and TMAI were more broadened than those of the apo enzymes at all temperatures, indicating increased melting temperatures (Tm) due to decreased heat capacity (DeltaGp). Moreover, the extent of difference in DeltaCp between the apo and holo thermophilic AIs is larger than that of BHAI. From these studies, we suggest that the metal dependence of the thermophilic AIs, resulting in the reduced DeltaCp, may play a significant role in structural stability compared to their mesophilic analogues, and that the extent of metal dependence of AI stability seems to be highly correlated to oligomerization.     

Electrostatic contributions to the stability of hyperthermophilic proteins.

Electrostatic contributions to the folding free energy of several hyperthermophilic proteins and their mesophilic homologs are calculated. In all the cases studied, electrostatic interactions are more favorable in the hyperthermophilic proteins. The electrostatic free energy is found not to be correlated with the number of ionizable amino acid residues, ion pairs or ion pair networks in a protein, but rather depends on the location of these groups within the protein structure. Moreover, due to the large free energy cost associated with burying charged groups, buried ion pairs are found to be destabilizing unless they undergo favorable interactions with additional polar groups, including other ion pairs. The latter case involves the formation of stabilizing ion pair networks as is observed in a number of proteins. Ion pairs located on the protein surface also provide stabilizing interactions in a number of cases. Taken together, our results suggest that many hyperthermophilic proteins enhance electrostatic interactions through the optimum placement of charged amino acid residues within the protein structure, although different design strategies are used in different cases. Other physical mechanisms are also likely to contribute, however optimizing electrostatic interactions offers a simple means of enhancing stability without disrupting the core residues characteristic of different protein families.     

The crystal structure of a liganded trehalose/maltose-binding protein from the hyperthermophilic Archaeon Thermococcus litoralis at 1.85 A

We report the crystallization and structure determination at 1.85 A of the extracellular, membrane-anchored trehalose/maltose-binding protein (TMBP) in complex with its substrate trehalose. TMBP is the substrate recognition site of the high-affinity trehalose/maltose ABC transporter of the hyperthermophilic Archaeon Thermococcus litoralis. In vivo, this protein is anchored to the membrane, presumably via an N-terminal cysteine lipid modification. The crystallized protein was N-terminally truncated, resulting in a soluble protein exhibiting the same binding characteristics as the wild-type protein. The protein shows the characteristic features of a transport-related, substrate-binding protein and is structurally related to the maltose-binding protein (MBP) of Escherichia coli. It consists of two similar lobes, each formed by a parallel beta-sheet flanked by alpha-helices on both sides. Both are connected by a hinge region consisting of two antiparallel beta-strands and an alpha-helix. As in MBP, the substrate is bound in the cleft between the lobes by hydrogen bonds and hydrophobic interactions. However, compared to maltose binding in MBP, direct hydrogen bonding between the substrate and the protein prevails while apolar contacts are reduced. To elucidate factors contributing to thermostability, we compared TMBP with its mesophilic counterpart MBP and found differences known from similar investigations. Specifically, we find helices that are longer than their structurally equivalent counterparts, and fewer internal cavities.     

Dps-like protein from the hyperthermophilic archaeon Pyrococcus furiosus

Oxidative stress is a universal phenomenon experienced by organisms in all domains of life. Proteins like those in the ferritin-like di-iron carboxylate superfamily have evolved to manage this stress. Here we describe the cloning, isolation, and characterization of a Dps-like protein from the hyperthermophilic archaeon Pyrococcus furiosus (PfDps-like). Phylogenetic analysis, primary structure alignments and higher order structural predictions all suggest that the P. furiosus protein is related to proteins within the broad superfamily of ferritin-like di-iron carboxylate proteins. The recombinant PfDps protein self-assembles into a 12 subunit quaternary structure with an outer shell diameter of approximately 10nm and an interior diameter of approximately 5 nm. Dps proteins functionally manage the toxicity of oxidative stress by sequestering intracellular ferrous iron and using it to reduce H(2)O(2) in a two electron process to form water. The iron is converted to a benign form as Fe(III) within the protein cage. This Dps-mediated reduction of hydrogen peroxide, coupled with the protein's capacity to sequester iron, contributes to its service as a multifunctional antioxidant.     

Solution structure and thermal stability of ribosomal protein L30e from hyperthermophilic archaeon Thermococcus celer

To understand the structural basis of thermostability, we have determined the solution structure of a thermophilic ribosomal protein L30e from Thermococcus celer by NMR spectroscopy. The conformational stability of T. celer L30e was measured by guanidine and thermal-induced denaturation, and compared with that obtained for yeast L30e, a mesophilic homolog. The melting temperature of T. celer L30e was 94 degrees C, whereas the yeast protein denatured irreversibly at temperatures >45 degrees C. The two homologous proteins also differ greatly in their stability at 25 degrees C: the free energy of unfolding was 45 kJ/mole for T. celer L30e and 14 kJ/mole for the yeast homolog. The solution structure of T. celer L30e was compared with that of the yeast homolog. Although the two homologous proteins do not differ significantly in their number of hydrogen bonds and the amount of solvent accessible surface area buried with folding, the thermophilic T. celer L30e was found to have more long-range ion pairs, more proline residues in loops, and better helix capping residues in helix-1 and helix-4. A K9A variant of T. celer L30e was created by site-directed mutagenesis to examine the role of electrostatic interactions on protein stability. Although the melting temperatures of the K9A variant is approximately 8 degrees C lower than that of the wild-type L30e, their difference in T(m) is narrowed to approximately 4.2 degrees C at 0.5 M NaCl. This salt-dependency of melting temperatures strongly suggests that electrostatic interactions contribute to the thermostability of T. celer L30e.     

Exploring the interplay of stability and function in protein evolution

A new split β‐lactamase assay promises experimental testing of the interplay of protein stability and function. Proteins are sufficiently stable to act effectively within cells. However, mutations generally destabilize structure, with effects on free energy that are comparable to the free energy of folding. Assays of protein functionality and stability in vivo enable a quick study of factors that influence these properties in response to targeted mutations. These assays can help molecular engineering but can also be used to target important questions, including why most proteins are marginally stable, how mutations alter structural makeup, and how thermodynamics, function, and environment shape molecular change. Processes of self‐organization and natural selection are determinants of stability and function. Non‐equilibrium thermodynamics provides crucial concepts, e.g., cells as emergent energy‐dissipating entities that do work and build their own parts, and a framework to study the sculpting role of evolution at different scales.     

Phosphorylation of the PTEN tail regulates protein stability and function

The PTEN gene is a tumor suppressor localized in the frequently altered chromosomal region 10q23. The tumor suppressor function of the PTEN protein (PTEN) has been linked to its ability to dephosphorylate the lipid second-messenger phosphatidylinositol 3,4, 5-trisphosphate and phosphatidylinositol 3,4-bisphosphate and, by doing so, to antagonize the phosphoinositide 3-kinase pathway. The PTEN protein consists of an amino-terminal phosphatase domain, a lipid binding C2 domain, and a 50-amino-acid C-terminal domain (the "tail") of unknown function. A number of studies have shown that the tail is dispensable for both phosphatase activity and blocking cell growth. Here, we show that the PTEN tail is necessary for maintaining protein stability and that it also acts to inhibit PTEN function. Thus, removing the tail results in a loss of stability but does not result in a loss of function because the resultant protein is more active. Furthermore, tail-dependent regulation of stability and activity is linked to the phosphorylation of three residues (S380, T382, and T383) within the tail. Therefore, the tail is likely to mediate the regulation of PTEN function through phosphorylation.     

A novel scoring function for discriminating hyperthermophilic and mesophilic proteins with application to predicting relative thermostability of protein mutants

Background  

The ability to design thermostable proteins is theoretically important and practically useful. Robust and accurate algorithms, however, remain elusive. One critical problem is the lack of reliable methods to estimate the relative thermostability of possible mutants.     

Exploring the influence of hyperthermophilic protein Ssh10b on the stability and conformation of RNA by molecular dynamics simulation

The hyperthermophilic Ssh10b from Sulfolobus shibatae is a member of the Sac10b family, which binds RNA in vivo as a physiological substrate, and it has been postulated to play a key role in chromosomal organization in Archaea. Even though the crystal structure of Ssh10b‐RNA was resolved successively by X‐ray diffraction (Protein Data Bank [PDB] code: 3WBM), the detailed dynamic characteristics of Ssh10b‐RNA are still unclear. In this study, molecular dynamics (MDs) simulations at 6 temperatures (300, 350, 375, 400, 450, and 500 K) and molecular mechanics Generalized‐Born surface area (MM‐GB/SA) free energy calculations were performed to investigate the mechanism of how Ssh10b protects and stabilizes RNA. The simulation results indicate that RNA is stabilized by Ssh10b when the temperature rises up to 375 K. RNA is found to undergo conformational transition between A‐RNA and A′‐RNA when Ssh10b binds to RNA at 3 different temperatures (300, 350, and 375 K). Salt bridges, hydrogen bonds and hydrophobic interactions are observed, and some residues have significant impact on the structural stability of the complex. This study increases our understanding of the dynamics and interaction mechanism of hyperthermophilic proteins and RNA at the atomic level, and offers a model for studying the structural biology of hyperthermophilic proteins and RNA.     

Thermodynamic stability and folding of proteins from hyperthermophilic organisms

Life grows almost everywhere on earth, including in extreme environments and under harsh conditions. Organisms adapted to high temperatures are called thermophiles (growth temperature 45-75 degrees C) and hyperthermophiles (growth temperature >or= 80 degrees C). Proteins from such organisms usually show extreme thermal stability, despite having folded structures very similar to their mesostable counterparts. Here, we summarize the current data on thermodynamic and kinetic folding/unfolding behaviors of proteins from hyperthermophilic microorganisms. In contrast to thermostable proteins, rather few (i.e. less than 20) hyperthermostable proteins have been thoroughly characterized in terms of their in vitro folding processes and their thermodynamic stability profiles. Examples that will be discussed include co-chaperonin proteins, iron-sulfur-cluster proteins, and DNA-binding proteins from hyperthermophilic bacteria (i.e. Aquifex and Theromotoga) and archea (e.g. Pyrococcus, Thermococcus, Methanothermus and Sulfolobus). Despite the small set of studied systems, it is clear that super-slow protein unfolding is a dominant strategy to allow these proteins to function at extreme temperatures.     

Equilibrium and kinetic stability of a hyperthermophilic protein, O6-methylguanine-DNA methyltransferase under various extreme conditions

In this work we have studied the equilibrium and kinetic stability of a hyperthermophilic protein, O(6)-methylguanine-DNA methyltransferase (Tk-MGMT), and its mesophilic counterpart AdaC, in various chemical solutions. In an unfolding experiment using guanidine hydrochloride (GdnHCl), the unfolding free-energy change of Tk-MGMT at 30 degrees C was 42.0 kJ mol(-1), and the half time for unfolding was 4.5 x 10(6) s, which is much slower than that of AdaC and representative mesophilic proteins. In unfolding experiments using methanol, ethanol, 2-propanol, trifluoroethanol (TFE), and sodium dodecyl sulfate (SDS), Tk-MGMT retained its native structure at high concentrations, despite the fact that these chemical solutions affect protein conformations in a number of different ways. Kinetic studies using TFE and SDS indicate that the unfolding rates of Tk-MGMT in these solutions are slow as in GdnHCl. Further, the results of a mutational experiment suggest that an ion-pair network plays a key role in this slow unfolding. This slow rate of unfolding under extreme conditions is a significant property that distinguishes Tk-MGMT from mesophilic proteins.     

Tyrosine phosphorylation controls PCNA function through protein stability

The proliferating cell nuclear antigen (PCNA) is an essential protein for DNA replication and damage repair. How its function is controlled remains an important question. Here, we show that the chromatin-bound PCNA protein is phosphorylated on Tyr 211, which is required for maintaining its function on chromatin and is dependent on the tyrosine kinase activity of EGF receptor (EGFR) in the nucleus. Phosphorylation on Tyr 211 by EGFR stabilizes chromatin-bound PCNA protein and associated functions. Consistently, increased PCNA Tyr 211 phosphorylation coincides with pronounced cell proliferation, and is better correlated with poor survival of breast cancer patients, as well as nuclear EGFR in tumours, than is the total PCNA level. These results identify a novel nuclear mechanism linking tyrosine kinase receptor function with the regulation of the PCNA sliding clamp.     

Topoisomerase activity of the hyperthermophilic replication initiator protein Rep75

The plasmid pGT5 from the hyperthermophilic archaeon Pyrococcus abyssi replicates via the rolling circle mechanism. pGT5 encodes the replication initiator protein Rep75 that exhibits a nicking–closing (NC) activity in vitro on single-stranded oligonucleotides containing the pGT5 double-stranded origin (dso) sequence. Some mesophilic Rep proteins present site-specific DNA topoisomerase-like activity on a negatively supercoiled plasmid harbouring the dso. We report here that Rep75 also exhibits topoisomerase activity on a negatively supercoiled DNA substrate. This DNA topoisomerase-like activity is dependent on the amino acids involved in NC activity of Rep75. However, in contrast with mesophilic Rep proteins, Rep75 topoisomerase activity is not dso dependent. Moreover, although pGT5 is known to be relaxed in vivo, Rep75 was not able to act on a relaxed plasmid in vitro, whether or not it contained the dso.