Thermostability of temperature-sensitive folding mutants of the P22 tailspike protein

Temperature-sensitive folding mutations (tsf) of the thermostable P22 tailspike protein prevent the mutant polypeptide chain from reaching the native state at the higher end of the temperature range of bacterial growth (37-42 degrees C). At lower temperatures the mutant polypeptide chains fold and associate into native proteins. The melting temperatures of the purified native forms of seven different tsf mutant proteins have been determined by differential scanning calorimetry. Under conditions in which the wild type protein had a melting temperature of 88.4 degrees C, the melting temperatures of the mutant proteins were all above 82 degrees C, more than 40 degrees C higher than the temperature for expression of the folding defect. Because the folding defects were observed in vivo, the thermostability of the native protein was also examined with infected cells. Once matured at 28 degrees C, intracellular tsf mutant tailspikes remained native when the cells were transferred to 42 degrees C, a temperature that prevents newly synthesized tsf chains from folding correctly. These results confirm that the failure of tsf polypeptide chains to reach their native state is not due to a lowered stability of the native state. Such mutants differ from the class of ts mutations which render the native state thermolabile. The intracellular folding defects must reflect decreased stabilities of folding intermediates or alteration in the off-pathway steps leading to aggregation and inclusion body formation. These results indicate that the stability of a native protein within the cells is not sufficient to insure the successful folding of the newly synthesized chains into the native state.     

Thermodynamic differences among homologous thermophilic and mesophilic proteins.

Here, we analyze the thermodynamic parameters and their correlations in families containing homologous thermophilic and mesophilic proteins which show reversible two-state folding <--> unfolding transitions between the native and the denatured states. For the proteins in these families, the melting temperatures correlate with the maximal protein stability change (between the native and the denatured states) as well as with the enthalpic and entropic changes at the melting temperature. In contrast, the heat capacity change is uncorrelated with the melting temperature. These and additional results illustrate that higher melting temperatures are largely obtained via an upshift and broadening of the protein stability curves. Both thermophilic and mesophilic proteins are maximally stable around room temperature. However, the maximal stabilities of thermophilic proteins are considerably greater than those of their mesophilic homologues. At the living temperatures of their respective source organisms, homologous thermophilic and mesophilic proteins have similar stabilities. The protein stability at the living temperature of the source organism does not correlate with the living temperature of the protein. We tie thermodynamic observations to microscopics via the hydrophobic effect and a two-state model of the water structure. We conclude that, to achieve higher stability and greater resistance to high and low temperatures, specific interactions, particularly electrostatic, should be engineered into the protein. The effect of these specific interactions is largely reflected in an increased enthalpy change at the melting temperature.     

Hsp70 from Cyprinion macrostomus macrostomus and Garra rufa obtuse: Stability and stability-dependent activity

Two fish species, Cyprinion macrostomus macrostomus and Garra rufa obtuse, tolerate adverse conditions in the Kangal hot springs and cope with multiple stressors such as food deprivation, extreme temperature, toxins, protein degradation, hypoxia, and microbial damage. These fish have evolved strategies to counteract the stressors including the induction of heat shock proteins (Hsps). Hsps play an essential role in maintaining cellular homeostasis, and one of the key proteins in the mechanism is Hsp70. Hsp70 itself is exposed to the same stressors as all other proteins, and, hence, the stability of Hsp70 was investigated. For this purpose, Hsp70 ATPase activity was determined at different urea concentrations. It was found that the protein maintains considerable ATP hydrolysis activity at higher denaturant conditions. Temperature effects on the substrate peptide binding showed that Hsp70s bind prominently at elevated temperatures. Furthermore, temperature effects on Hsp70 aggregation indicated that the presence of nucleotides decreases the aggregation process. The present work has determined the stability and activity of cmHsp70 and grHsp70 themselves under extreme conditions. The stability of the Hsp70 proteins maintains substrate proteins in the native state, which may aid in the adaptation of the fish species to the hot spring environment.     

A high-throughput assay of membrane protein stability

The preparation of purified, detergent-solubilized membrane proteins in a monodisperse and stable form is usually a prerequisite for investigation not only of their function but also for structural studies by X-ray crystallography and other approaches. Typically, it is necessary to explore a wide range of conditions, including detergent type, buffer pH, and the presence of additives such as glycerol, in order to identify those optimal for stability. Given the difficulty of expressing and purifying membrane proteins in large amounts, such explorations must ideally be performed on as small a scale as practicable. To achieve this objective in the UK Membrane Protein Structure Initiative, we have developed a rapid, economical, light-scattering assay of membrane protein aggregation that allows the testing of 48 buffer conditions in parallel on 6 protein targets, requiring less than 2 mg protein for each target. Testing of the assay on a number of unrelated membrane transporters has shown that it is of generic applicability. Proteins of sufficient purity for this plate-based assay are first rapidly prepared using simple affinity purification procedures performed in batch mode. Samples are then transferred by microdialysis into each of the conditions to be tested. Finally, attenuance at 340 nm is monitored in a 384-well plate using a plate reader. Optimal conditions for protein stability identified in the assay can then be exploited for the tailored purification of individual targets in as stable a form as possible.     

A FRET-based screening assay for nucleic acid ligands

Some of the most serious diseases are characterized by the presence of a specific secondary structure within DNA or RNA, often in the promoter or the coding region of the responsible gene, that enhances or disrupts expression of the protein. Structural elements that impact cellular function may also be formed in other genomic regions such as telomeres. Compounds that interact with such structural elements may be useful in diagnosis or treatment of patients. In this report, we present a FRET melting assay that allows testing of libraries of compounds against four different nucleic acid structures. Compounds are tested to determine whether they stabilize preformed secondary structures (i.e., whether they cause an increase in melting temperature (T(m))). This property is described by the ΔT(m) parameter, which is the difference between the T(m) of the compound-stabilized structure and the T(m) of the unbound structure. Model oligonucleotides are labeled with FAM as a fluorescent donor and TAMRA as an acceptor. The intensity of FAM fluorescence is recorded as a function of temperature. Melting temperatures are determined by the FRET method in 96-well plates; this assay could easily be converted into 384-well format.     

Thermal transitions of myosin and its helical fragments. I. Shifts in proton equilibria accompanying unfolding.

The thermal transitions of myosin and its helical fragments have been studied with pH as the observable. Heating unbuffered solutions of these proteins near their pI values causes an abrupt rise in pH at a characteristic temperature (the "melting temperature," Tm) which is due to structural changes within the protein. Since the pH shift turns out to be insensitive to the degree of protein aggregation, we have obtained acceptable melting curves even under conditions where the protein coagulates during melting. The melting profiles and Tm vlaues of myosin, myosin rod, and light meromyosin have been found to be remarkably similar (Tm equal to 40 plus or minus 1 degree, 0.5 M KCl, pH 5.9). Proton binding which occurs during melting coincides with the unfolding of a section of myosin rod. Taken in the context of other studies, the proton binding is thought to occur near the "hinge region."     

Screening for ligands using a generic and high-throughput light-scattering-based assay

Rapid identification of small molecules that interact with protein targets using a generic screening method greatly facilitates the development of therapeutic agents. The authors describe a novel method for performing homogeneous biophysical assays in a high-throughput format. The use of light scattering as a method to evaluate protein stability during thermal denaturation in a 384-well format yields a robust assay with a low frequency of false positives. This novel method leads to the identification of interacting small molecules without the addition of extraneous fluorescent probes. The analysis and interpretation of data is rapid, with sensitivity for protein stability comparable to differential scanning calorimetry. The authors propose potential uses in drug discovery, structural genomics, and functional genomics as a method to evaluate small-molecule interactions, identify natural cofactors that stabilize target proteins, and identify natural substrates and products for previously uncharacterized protein targets.     

Is arginine a protein-denaturant?

Arginine is a useful solvent additive for many applications, including refolding and solubilization of proteins from insoluble pellets, and suppression of protein aggregation and non-specific adsorption during formulation and purification. However, there is a concern that arginine may be a protein-denaturant, which may limit the expansion of its applications. Such concern arises from the facts that arginine decreases melting temperature and perturbs the spectroscopic properties of certain proteins and contains a guanidinium group, which is a critical chemical structure for denaturing activity of guanidine hydrochloride. Here, we show that although arginine does lower the melting temperatures of certain proteins, the extent is insufficient to cause denaturation of proteins at or below room temperature. The proteins described here show enzymatic activity and folded structure in the presence of arginine, although the local structure around aromatic amino acids is perturbed by arginine. Arginine differs from guandinine hydrochloride in the mode of interactions with proteins, which may be a primary reason why arginine is not a protein-denaturant.     

A high-throughput turbidometric assay for screening inhibitors of protein disulfide isomerase activity

Protein disulfide isomerase (PDI) plays a key role in protein folding by catalyzing rearrangements of disulfide bonds in substrate proteins following their synthesis in eukaryotic cells. Besides its major role in the processing and maturation of secretory proteins in the endoplasmic reticulum, this enzyme and its homologs have been implicated in multiple important cellular processes; however, they have not served as targets for the development of therapeutic agents. The authors developed a high-throughput screening assay for PDI and its homologous enzymes in 384-well microplates. The method is based on the enzyme-catalyzed reduction of insulin in the presence of dithiothreitol and measures the aggregation of reduced insulin chains at 650 nm. This kinetic assay was converted to an end-point assay by using hydrogen peroxide as a stop reagent. The feasibility of this high-throughput assay for screening chemical libraries was demonstrated in a pilot screen. The authors show that this homogenous turbidometric assay is robust and cost-effective and can be applied to identify PDI inhibitors from chemical libraries, opening this class of enzymes for therapeutic exploration.     

Temperature stability of proteins: Analysis of irreversible denaturation using isothermal calorimetry

The structural stability of proteins has been traditionally studied under conditions in which the folding/unfolding reaction is reversible, since thermodynamic parameters can only be determined under these conditions. Achieving reversibility conditions in temperature stability experiments has often required performing the experiments at acidic pH or other nonphysiological solvent conditions. With the rapid development of protein drugs, the fastest growing segment in the pharmaceutical industry, the need to evaluate protein stability under formulation conditions has acquired renewed urgency. Under formulation conditions and the required high protein concentration (～100 mg/mL), protein denaturation is irreversible and frequently coupled to aggregation and precipitation. In this article, we examine the thermal denaturation of hen egg white lysozyme (HEWL) under irreversible conditions and concentrations up to 100 mg/mL using several techniques, especially isothermal calorimetry which has been used to measure the enthalpy and kinetics of the unfolding and aggregation/precipitation at 12°C below the transition temperature measured by DSC. At those temperatures the rate of irreversible protein denaturation and aggregation of HEWL is measured to be on the order of 1 day^?1. Isothermal calorimetry appears a suitable technique to identify buffer formulation conditions that maximize the long term stability of protein drugs.     

Low thermodynamic but high kinetic stability of an antifreeze protein from Rhagium mordax

The equilibrium heat stability and the kinetic heat tolerance of a recombinant antifreeze protein (AFP) from the beetle Rhagium mordax (RmAFP1) are studied through differential scanning calorimetry and circular dichroism spectroscopy. In contrast to other insect AFPs studied with this respect, the RmAFP1 has only one disulfide bridge. The melting temperature, T_m, of the protein is determined to be 28.5°C (pH 7.4), which is much lower than most of those reported for AFPs or globular proteins in general. Despite its low melting temperature, both biophysical and activity measurements show that the protein almost completely refolds into the native state after repeated exposure of 70°C. RmAFP1 thus appears to be kinetically stable even far above its melting temperature. Thermodynamically, the insect AFPs seem to be dividable in three groups, relating to their content of disulfide bridges and widths of the ice binding motifs; high melting temperature AFPs (high disulfide content, TxT motifs), low melting temperature but high refolding capability AFPs (one disulfide bridge, TxTxTxT motifs) and irreversibly unfolded AFPs at low temperatures (no disulfide bridges, TxTxTxTxT motifs). The property of being able to cope with high temperature exposures may appear peculiar for proteins which strictly have their effect at subzero temperatures. Different aspects of this are discussed.     

The role of calcium in the conformational dynamics and thermal stability of the D-galactose/D-glucose-binding protein from Escherichia coli

We have characterized stability and conformational dynamics of the calcium depleted D-galactose/D-glucose-binding protein (GGBP) from Escherichia coli. The structural stability of the protein was investigated by steady state and time resolved fluorescence, and far-UV circular dichroism in the temperature range from 20 degrees C to 70 degrees C. We have found that the absence of the Ca(2+) ion results in a significant destabilization of the C-terminal domain of the protein. In particular, the melting temperature decreases by about 10 degrees C with the simultaneous loss of the melting cooperativity. Time resolved fluorescence quenching revealed significant loosening of the protein when highly shielded Trp residue(s) became accessible to acrylamide at higher temperatures. We have documented a significant stabilizing effect of glucose that mostly reverts the effect of calcium, that is, the thermal stability of the protein increases by about 10 degrees C and the melting cooperativity is restored. Moreover, the protein structure remains compact with low amplitude of the segmental mobility up to high temperatures. We have used molecular dynamics to identify the structural feature responsible for changes in the temperature stability. Disintegration of the Ca(2+)-binding loop seems to be responsible for the loss of the stability in the absence of calcium. The new insights on the structural properties and temperature stability of the calcium depleted GGBP contribute to better understanding of the protein function and constitute important information for the development of new biotechnological applications of this class of proteins.     

Effects of common cancer mutations on stability and DNA binding of full-length p53 compared with isolated core domains

Common cancer mutations of p53 tend either to lower the stability or distort the core domain of the protein or weaken its DNA binding affinity. We have previously analyzed in vitro the effects of mutations on the core domain of p53. Here, we extend those measurements to full-length p53, using either the wild-type protein or a biologically active superstable construct that is more amenable to accurate biophysical measurements to assess the possibilities of rescuing different types of mutations by anticancer drugs. The tetrameric full-length proteins had similar apparent melting temperatures to those of the individual domains, and the structural mutations lowered the melting temperature by similar amounts. The thermodynamic stability of tetrameric p53 is thus dictated by its core domain. We determined that the common contact mutation R273H weakened binding to the gadd45 recognition sequence by approximately 700-1000 times. Many mutants that have lowered melting temperatures should be good drug targets, although the common R273H mutant binds response elements too weakly for simple rescue.     

Fluorescence-based methods for screening writers and readers of histone methyl marks

The histone methyltransferase (HMT) family of proteins consists of enzymes that methylate lysine or arginine residues on histone tails as well as other proteins. Such modifications affect chromatin structure and play a significant regulatory role in gene expression. Many HMTs have been implicated in tumorigenesis and progression of multiple malignancies and play essential roles in embryonic development and stem cell renewal. Overexpression of some HMTs has been observed and is correlated positively with various types of cancer. Here the authors report development of a continuous fluorescence-based methyltransferase assay in a 384-well format and its application in determining kinetic parameters for EHMT1, G9a, PRMT3, SETD7, and SUV39H2 as well as for screening against libraries of small molecules to identify enzyme inhibitors. They also report the development of a peptide displacement assay using fluorescence polarization in a 384-well format to assay and screen protein peptide interactions such as those of WDR5 and EED, components of MLL and EZH2 methyltransferase complexes. Using these high-throughput screening methods, the authors have identified potent inhibitors and ligands for some of these proteins.     

Protein stability curves

The stability curve of a protein is defined as the plot of the free energy of unfolding as a function of temperature. For most proteins the change in heat capacity on denaturation, or unfolding, is large but approximately constant. When unfolding is s two-state process, most of the salient features of the stability curves of proteins can be derived from this fact. A number of relations are obtained, including the special features of low-temperature denaturation, the properties of the maximum in stability, and the interrelationships of the characteristic temperatures of the protein. The paper closes with a formula that permits one to calculate small changes in stabilization free energy from changes in the melting temperature of the protein.     

Stability Curve Prediction of Homologous Proteins Using Temperature-Dependent Statistical Potentials

The unraveling and control of protein stability at different temperatures is a fundamental problem in biophysics that is substantially far from being quantitatively and accurately solved, as it requires a precise knowledge of the temperature dependence of amino acid interactions. In this paper we attempt to gain insight into the thermal stability of proteins by designing a tool to predict the full stability curve as a function of the temperature for a set of 45 proteins belonging to 11 homologous families, given their sequence and structure, as well as the melting temperature () and the change in heat capacity () of proteins belonging to the same family. Stability curves constitute a fundamental instrument to analyze in detail the thermal stability and its relation to the thermodynamic stability, and to estimate the enthalpic and entropic contributions to the folding free energy. In summary, our approach for predicting the protein stability curves relies on temperature-dependent statistical potentials derived from three datasets of protein structures with targeted thermal stability properties. Using these potentials, the folding free energies () at three different temperatures were computed for each protein. The Gibbs-Helmholtz equation was then used to predict the protein''s stability curve as the curve that best fits these three points. The results are quite encouraging: the standard deviations between the experimental and predicted ''s, ''s and folding free energies at room temperature () are equal to 13 , 1.3 ) and 4.1 , respectively, in cross-validation. The main sources of error and some further improvements and perspectives are briefly discussed.     

High-throughput screening assay for sphingosine kinase inhibitors in whole blood using RapidFire® mass spectrometry

To facilitate discovery of compounds modulating sphingosine-1-phosphate (S1P) signaling, the authors used high-throughput mass spectrometry technology to measure S1P formation in human whole blood. Since blood contains endogenous sphingosine (SPH) and S1P, mass spectrometry was chosen to detect the conversion of an exogenously added 17-carbon-long variant of sphingosine, C17SPH, into C17S1P. The authors developed procedures to achieve homogeneous mixing of whole blood in 384-well plates and for a method requiring minimal manipulations to extract S1P from blood in 96- and 384-well plates prior to analyses using the RapidFire(?) mass spectrometry system.     

The congenital cataract-linked G61C mutation destabilizes γD-crystallin and promotes non-native aggregation

γD-crystallin is one of the major structural proteins in human eye lens. The solubility and stability of γD-crystallin play a crucial role in maintaining the optical properties of the lens during the life span of an individual. Previous study has shown that the inherited mutation G61C results in autosomal dominant congenital cataract. In this research, we studied the effects of the G61C mutation on γD-crystallin structure, stability and aggregation via biophysical methods. CD, intrinsic and extrinsic fluorescence spectroscopy indicated that the G61C mutation did not affect the native structure of γD-crystallin. The stability of γD-crystallin against heat- or GdnHCl-induced denaturation was significantly decreased by the mutation, while no influence was observed on the acid-induced unfolding. The mutation mainly affected the transition from the native state to the intermediate but not that from the intermediate to the unfolded or aggregated states. At high temperatures, both proteins were able to form aggregates, and the aggregation of the mutant was much more serious than the wild type protein at the same temperature. At body temperature and acidic conditions, the mutant was more prone to form amyloid-like fibrils. The aggregation-prone property of the mutant was not altered by the addition of reductive reagent. These results suggested that the decrease in protein stability followed by aggregation-prone property might be the major cause in the hereditary cataract induced by the G61C mutation.     

Maximal stabilities of reversible two-state proteins

The hydrophobic effect is the major force driving protein folding. Around room temperature, small organic solutes and hydrophobic amino acids have low solubilities in water and the hydrophobic effect is the strongest. These facts suggest that globular proteins should be maximally stable around room temperature. While this fundamental paradigm has been expected, it has not actually been shown to hold. Toward this goal, we have collected and analyzed experimental thermodynamic data for 31 proteins that show reversible two-state folding <--> unfolding transitions at or near neutral pH. Twenty-six of these are unique, and 20 of the 26 are maximally stable around room temperature irrespective of their structural properties, the melting temperature, or the living temperatures of their source organisms. Their average temperature of maximal stability is 293 +/- 8 K (20 +/- 8 degrees C). These proteins differ in size, fold, and number of domains, hydrophobic folding units, and oligomeric states. They derive from the cold-loving psychrophiles, from mesophiles, and from thermophiles. Analysis of the single-domain proteins present in this set shows that the variations in their thermodynamic parameters are correlated in a way which may explain the adaptation of the proteins to the living temperatures of the organisms from which they derive. The average energetic contribution of the individual amino acids toward protein stability decreases with an increase in protein size, suggesting that there may be an upper limit for protein maximal thermodynamic stability. For the remaining proteins, deviation of the maximal stability temperatures from room temperature may be due to greater uncertainties in their heat capacity change (DeltaC(p)) values, a weaker hydrophobic effect, and/or a stronger electrostatic contribution.     

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.