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PubMed1990

Regulation of red cell membrane deformability and stability by skeletal protein network.

The skeletal protein network of the red blood cell is thought to be important in regulating such membrane functions as deformability and stability. In the present study, we measured membrane deformability and stability of the resealed ghosts using an ektacytometer, a laser diffraction method, and identified the functional role of protein 4.1 and that of Ca2+ and calmodulin in maintaining membrane stability. To obtain direct evidence for a crucial role of protein 4.1 in maintaining membrane stability, we reconstituted protein 4.1-deficient membranes with purified protein 4.1. Although native membranes deficient in protein 4.1 had marked reduction in membrane stability, reconstitution with increasing concentrations of purified protein 4.1 resulted in progressive restoration of membrane stability, providing direct evidence that protein 4.1 is essential for normal membrane stability. To determine if Ca2+ and calmodulin could modulate membrane properties, we measured membrane stability and deformability of resealed ghosts prepared in the presence of varying concentrations of Ca2+ and physiologic concentrations of calmodulin. Our data show that Ca2+ concentrations in the range of 1 to 100 microM can markedly decrease membrane stability only in the presence of calmodulin, but not in its absence. In contrast, deformability decreased only at Ca2+ concentrations higher than 100 microM, and calmodulin had no effect. Examination of the the effects of Ca2+ and calmodulin on various membrane protein interactions has enabled us to suggest that the observed changes in membrane stability may be partly related to the effects of Ca2+ and calmodulin on spectrin-protein 4.1-actin interaction.

Calcium↗
PubMed2002

Effects of methylene blue stabilizer on in vitro viability and chemotaxis of 99mTc-exametazime-labeled leukocytes.

UNLABELLED: This study was conducted to evaluate the effects of methylene blue/phosphate buffer stabilizer on the labeling efficiency, cell membrane integrity, chemotaxis, and in vitro stability of leukocytes labeled with 99mTc-exametazime. METHODS: Whole blood (480 mL) was obtained from each of 3 healthy donors and divided equally into eight 60-mL aliquots. Two aliquots from each of the 3 subjects were used as a control (i.e., leukocytes were not labeled with either nonstabilized or stabilized 99mTc-exametazime) in this study. The remaining 6 aliquots from each of the 3 subjects were divided equally into the following formulation groups: (a) leukocytes labeled with nonstabilized 99mTc-exametazime, (b) leukocytes labeled with stabilized 99mTc-exametazime containing 250 microg methylene blue, and (c) leukocytes labeled with stabilized 99mTc-exametazime containing 500 microg methylene blue. Duplicate samples were evaluated to confirm the repeatability of the study. Six samples were studied under each experimental condition (i.e., control, a, b, and c groups). The cell membrane integrity and chemotatic capacity of leukocytes were measured at 1 and 3 h after preparation, whereas a complete blood count was obtained at 3 h after preparation. The labeling efficiency was calculated immediately on conclusion of cell labeling, whereas the in vitro stability of the radiolabeled leukocytes was evaluated at 3 h after radiolabeling. RESULTS: None of the samples showed trypan blue-stained cells. For the chemotactic index, no statistically significant differences were noted between the 3 labeling formulations at 1 h (P = 0.43) or 3 h (P = 0.10) after preparation. None of the labeling formulations differed significantly from the control values of the chemotactic index (all P > 0.25). For the complete blood count, no significant differences were observed between any of the groups, with the exception of platelet number in the control group (P = 0.004). The labeling efficiency (P = 0.10) and in vitro stability (P = 0.33) were also similar in our comparison of the 3 labeling formulations. CONCLUSION: With no major statistically significant difference noted either between the 3 labeling formulations or between the control and the 3 labeling formulations, we concluded that the methylene blue/phosphate buffer stabilizer (250 or 500 microg methylene blue) did not affect the cell membrane integrity, chemotactic capacity, labeling efficiency, in vitro stability, or complete blood count of leukocytes labeled with stabilized 99mTc-exametazime.

Cell Membrane↗
PubMed2004

Comparison of heart rate variability and heart rate between individuals with different emotional stability in two situations.

OBJECTIVE: To compare heart rate variability (HRV) and heart rate (HR) between individuals with different emotional stabilities in two situations and to discuss the relationship between situation, emotional stability and HRV. METHOD: Ninety-six healthy male flying students were assessed after 60 h flying exercises by senior pilots using Criterion Assessment Scale of Emotional Stability of Flying Cadets (CASESFC) compiled by our department. They were divided into groups of high emotional stability and low stability. ECG were recorded in situations of Sternberg Dual Task and Flying and HRV components and HR were analyzed and compared. RESULT: When subjects were exposed to Sternberg dual task situation, individuals with high emotional stability had lower LFnorm (P<0.05), HFnorm (P<0.05), LF/HF ratio and HR (P<0.05) than those with low emotional stability. When it comes to flying situation, individuals with high emotional stability had higher LFnorm and HFnorm, but LF/HF ratio and HR (P<0.05) were still lower than those with low emotional stability. CONCLUSION: We found that there is a tendency which implies the situational separation of fluctuation of autonomic nervous system related to emotional stability. It suggests that the ability of an individual to adjust to outer environment depends not only on personality, temperament and cognitive module, but also on a physiological basis.

Adolescent↗
PubMedDate not supplied

Investigation of potential RNA bulge stabilizing elements.

As a part of our interest in recognition and cleavage of RNA we carried out thermal melting studies with the aim of screening a number of simple oligonucleotide modifications for their potential as modifying elements for RNA bulge stabilizing oligonucleotides. A specific model system from our studies on oligonucleotide-based artificial nuclease (OBAN) systems was chosen and the bulge size was varied from one to five nucleotides. Introduction of single 2'-modified nucleoside moieties (2'-O-methyl, 2'-deoxy and 2'-deoxy-2'-amino) with different conformational preferences adjacent to the bulge revealed that a higher preference for the north conformers gave more stable bulges across the whole range of bulge sizes. Changing a bulge closing a G-U wobble base pair to a G-C pair resulted in the interesting observation that, although the fully complementary complex and small bulges were highly stabilized, there was little difference in the stability of the larger bulges. The wobble base pair even gave a slight stabilization of the 5 nt bulge system. Introduction of a uridine C-5 linker with a single ammonium group was clearly bulge stabilizing (DeltaT(m) + 4.6 to + 5.4 degrees C for the three most stabilized bulges), although with limited selectivity for different bulge sizes since the fully complementary duplex was also stabilized. Introduction of a naphthoyl group on a 2'-aminolinker mostly gave a destabilizing effect, while introduction of a 5-aminoneocuproine moiety on the same linker resulted in stabilization of all bulges, in particular those with two or four unpaired nucleotides (DeltaT(m) + 3.6 and + 2.9 degrees C respectively). The aromatic groups destabilize the fully complementary duplex, resulting in higher selectivity towards stabilization of bulges. A combination of the studied partial element should be suitable for future designs of modified oligonucleotides that, apart from standard base pairing, can also provide additional non-Watson-Crick recognition of RNA.

Base Pairing↗
PubMed2005

Mechanism of protein stabilization by sugars during freeze-drying and storage: native structure preservation, specific interaction, and/or immobilization in a glassy matrix?

The purpose of this study is to investigate the mechanism of protein stabilization by sugars in the solid state. That is, explore whether the stabilization is controlled by "glass dynamics" or by native structure preservation through "specific interaction" between sugars and protein. The IgG1 antibody (150 kD) and recombinant human serum albumin (rHSA) (65 kD) were formulated with sorbitol, trehalose, and sucrose. Degradation of lyophilized formulations was quantified using size exclusion (SEC) and ion-exchange chromatography (IEX). The secondary structure of the protein in these formulations was characterized using Fourier Transform Infrared (FTIR) spectroscopy. The molecular mobility, as measured by the stretched relaxation time (tau(beta)) was obtained by fitting the modified stretched exponential (MSE) equation to the calorimetric data from the Thermal Activity Monitor (TAM). Compared with sucrose and trehalose, sorbitol could only slightly protect the protein against aggregation and had no effect on chemical degradation. The chemical degradation and aggregation rates of the protein decreased when the weight ratio of sucrose to protein increased from 0 to 2:1. Storage stability of the proteins showed a reasonably good correlation with the degree of retention of native structure of protein during drying as measured by the spectral correlation coefficient for FTIR spectra. The plots of tau(beta) as a function of fraction of sucrose passed through a maximum at 1:1 weight ratio of sucrose to protein. That is, the molecular mobility did not correlate with the stability of protein at high levels of sucrose content. Although the glass transition appears to be an important parameter for stability, protein stabilization by sugars in the solid state cannot be wholly explained by the glass dynamics mechanism, at least as measured by tau(beta). However, it is possible that the beta-relaxations rather than the alpha-relaxations (i.e., the tau we measured) are critical to stability. The data show that storage stability correlates best with "structure" as determined by FTIR spectroscopy. However, while a specific interaction between stabilizer and protein might be responsible for the preservation of native structure, the evidence supporting this position is not compelling.

Algorithms↗
PubMed1992

Characterization of the stabilizing effect of point mutations of pyruvate oxidase from Lactobacillus plantarum: protection of the native state by modulating coenzyme binding and subunit interaction.

Point mutations in the gene of pyruvate oxidase from Lactobacillus plantarum, with proline residue 178 changed to serine, serine 188 to asparagine, and alanine 458 to valine, as well as a combination of the three single point mutations, lead to a significant functional stabilization of the protein. The enzyme is a tetrameric flavoprotein with tightly bound cofactors, FAD, TPP, and divalent metal ions. Thus, stabilization may be achieved either at the level of tertiary or quaternary interactions, or by enhanced cofactor binding. In order to discriminate between these alternatives, unfolding, dissociation, and cofactor binding of the mutant proteins were analyzed. The point mutations do not affect the secondary and tertiary structure, as determined by circular dichroism and protein fluorescence. Similarly, the amino acid substitutions neither modulate the enzymatic properties of the mutant proteins nor do they stabilize the structural stability of the apoenzymes. This holds true for both the local and the global structure with unfolding transitions around 2.5 M and 5 M urea, respectively. On the other hand, deactivation of the holoenzyme (by urea or temperature) is significantly decreased. The most important stabilizing effect is caused by the Ala-Val exchange in the C-terminal domain of the molecule. Its contribution is close to the value observed for the triple mutant, which exhibits maximum stability, with a shift in the thermal transition of ca. 10 degrees C. The effects of the point mutations on FAD binding and subunit association are interconnected. Because FAD binding is linked to oligomerization, the stability of the mutant apoenzyme-FAD complexes is increased. Accordingly, mutants with maximum apparent FAD binding exhibit maximum stability. Analysis of the quaternary structure of the mutant enzymes in the absence and in the presence of coenzymes gives clear evidence that both improved ligand binding and subunit interactions contribute to the observed thermal stabilization.

Alanine↗
PubMed2000

Stabilization of RNA tertiary structure by monovalent cations.

The effects of monovalent cations (Li(+), Na(+), K(+), Rb(+), Cs(+), and NH4(+)) on the thermal stability of RNA tertiary structure were investigated by UV melting. We show that with the RNA used here (nucleotides 1051-1108 of Escherichia coli 23 S rRNA with four base substitutions), monovalent cations and Mg(2+) compete in stabilizing the RNA tertiary structure, and that the competition takes place between two boundaries: one where Mg(2+) concentration is zero and the other where it is maximally stabilizing ("saturating"). The pattern of competition is the same for all monovalent cations and depends on the cation's ability to displace Mg(2+) from the RNA, its ability to stabilize tertiary structure in the absence of Mg(2+), and its ability to stabilize tertiary structure at saturating Mg(2+) concentrations. The stabilizing ability of a monovalent cation depends on its unhydrated ionic radius, and at a low monovalent cation concentration and saturating Mg(2+), there is a (calculated) net release of a single monovalent cation/RNA molecule when tertiary structure is denatured. The implications are that under these conditions there is at least one binding site for monovalent cations on the RNA, the site is specifically associated with formation of stable tertiary structure, K(+) is the most effective of the tested cations, and Mg(2+) appears ineffective at this site. At high ionic strength, and in the absence of Mg(2+), stabilization of tertiary structure is still monovalent-cation specific and ionic-radius dependent, but a larger number of cations ( approximately eight) are released upon RNA tertiary structure denaturation, and NH(4)(+) appears to be the most effective cation in stabilizing tertiary structure under these conditions. In the majority of the experiments, methanol was added as a cosolvent to the buffer. Its use allowed the examination of the behavior of monovalent ions under conditions where their effects would otherwise have been too weak to be observed. Methanol stabilizes tertiary but not secondary structure of the RNA. There was no evidence that it either causes qualitative changes in cation-binding properties of the RNA or a change in the pattern of monovalent cation/Mg(2+) competition.

Base Sequence↗
PubMed2003

Enzyme stabilization strategies based on electrolytes and polyelectrolytes for biosensor applications.

The achievements in the area of enzyme stabilization based on electrolytes, polyelectrolytes and polyols is reviewed, in the context of biosensor applications. Both the storage and operational stabilities of the biosensors can be improved using these stabilizers. The deactivation of the enzymes used for the development of biosensors from thermal shock, proteolytic degradation, and non-specific metal-catalyzed oxidation can be drastically reduced with the use of one or more of these stabilizers. It is attempted to deconvolute the effect of these additives on (a) the storage stability or shelf life, and (b) the operational stabilities of the biosensors. Even though there are a large number of techniques and reports dealing with enzyme stabilization, their application to biosensor technology is still very limited. It is thus concluded that the use of the existing enzyme stabilization techniques will have a drastic effect on the storage and operational stabilities of biosensors in the near future.

Biosensing Techniques↗
PubMed2005

Stabilization and improved in vivo performance of amorphous etoricoxib using Gelucire 50/13.

PURPOSE: Amorphous drugs have gained importance because of their advantageous biopharmaceutical properties; however, their stabilization remains a challenge. The purpose of this work was to stabilize the amorphous form of etoricoxib (ET) by using a low excipient/drug ratio to improve drug dissolution and thus bioavailability. METHODS: The effect of Gelucire and polyvinylpyrrolidone (PVP) on stabilization and bioavailability of amorphous etoricoxib (AET) was studied. X-ray powder diffractometry, differential scanning calorimetry, and scanning electron microscopy were used to study the physical state of the drug. Dissolution studies were performed for melt granules of AET with Gelucire 50/13 (MG-AET) and solid dispersion with PVP (SDP) to differentiate dissolution performance. A stability study on samples was conducted for 3 months to evaluate the physical state of the drug and its dissolution in the formulation. The in vivo performance of the optimized and stable formulation of ET was evaluated in rat. RESULTS: Dissolution of MG-AET was significantly improved as compared to AET and SDP. Both factors, amorphization of drug and melt granulation with lipid, seemed to be important for improving dissolution. Stability data revealed that MG-AET was significantly advantageous for AET stabilization, whereas PVP was not. The amount of Gelucire required for the stabilization of one part of AET was 0.5 part (by weight), whereas even 1.5 part (by weight) of PVP failed to elicit the same result. The superior in vivo performance of MG-AET has been attributed to the altered physiochemical properties of AET and the presence of lipid in the system. CONCLUSION: Gelucire can stabilize AET and improve its biopharmaceutical performance at a low excipient/drug ratio and may provide a better alternative to conventional stabilizers such as PVP.

Animals↗
PubMed2006

Thermal stability landscape for Klenow DNA polymerase as a function of pH and salt concentration.

The thermal denaturation of Klenow DNA polymerase has been characterized over a wide variety of solution conditions to obtain a relative stability landscape for the protein. Measurements were conducted utilizing a miniaturized fluorescence assay that measures Tm based on the increase in the fluorescence of 1,8-anilinonaphthalene sulfonate (ANS) when the protein denatures. The melting temperature (Tm) for Klenow increases as the salt concentration is increased and as the pH is decreased. Klenow's Tm spans a range of over 20 degrees C, from 40 to 62 degrees C, depending upon the solution conditions. The landscape reconciles and extends previously measured Tm values for Klenow. Salt effects on the stability of Klenow show strong cation dependence overlaid onto a more typical Hofmeister anion type dependence. Cationic stabilization of proteins has been far less frequently documented than anionic stabilization. The monovalent cations tested stabilize Klenow with the following hierarchy: NH4+>Na+>Li+>K+. Of the divalent cations tested: Mg+2 and Mn+2 significantly stabilize the protein, while Ni+2 dramatically destabilizes the protein. Stability measurements performed in combined Mg+2 plus Na+ salts suggest that the stabilizing effects of these monovalent and divalent cations are synergistic. The cationic stabilization of Klenow can be well explained by a model postulating dampening of repulsion within surface anionic patches on the protein.

Anilino Naphthalenesulfonates↗
PubMed2006

Stability of Beriplast P fibrin sealant: storage and reconstitution.

This study was performed to investigate the stability of Beriplast P fibrin sealant (FS) across a range of storage conditions, both pre- and post-reconstitution. Storage stability of the FS was evaluated during long-term refrigeration (24 months) with or without interim storage at elevated temperatures (40 degrees C for 1 week and 25 degrees C for 1 and 3 months). Stability of individual FS components was assessed by measuring: fibrinogen content, Factor XIII activity (FXIII), thrombin activity and aprotinin potency. The package integrity of each component was also checked (sterility testing, moisture content and pH). Storage stability was also evaluated by testing the reconstituted product for adhesion (tearing force testing after mixing the solutions) and sterility. Reconstitution stability was evaluated following 3-months' storage, for up to 50 h post-reconstitution using the same tests as for the storage stability investigations. Pre-defined specifications were met for fibrinogen content, Factor XIII activity, and thrombin activity, demonstrating storage stability. Package integrity and the functionality and sterility of the reconstituted product were confirmed throughout. Reconstitution stability was demonstrated for up to 50 h following reconstitution, in terms of both tearing force and sterility tests. In conclusion, the storage stability of Beriplast P was demonstrated over a range of 24-month storage schedules including interim exposure to elevated temperature, and the reconstituted product was stable for up to 50 h.

Aprotinin↗
PubMed2004

Do proteins always benefit from a stability increase? Relevant and residual stabilisation in a three-state protein by charge optimisation.

The vast majority of our knowledge on protein stability arises from the study of simple two-state models. However, proteins displaying equilibrium intermediates under certain conditions abound and it is unclear whether the energetics of native/intermediate equilibria is well represented in current knowledge. We consider here that the overall conformational stability of three-state proteins is made of a "relevant" term and a "residual" one, corresponding to the free energy differences of the native to intermediate (N-to-I) and intermediate to denatured (I-to-D) equilibria, respectively. The N-to-I free energy difference is considered to be the relevant stability because protein-unfolding intermediates are likely devoid of biological activity. We use surface charge optimisation to first increase the overall (N-to-D) stability of a model three-state protein (apoflavodoxin) and then investigate whether the stabilisation obtained is realised into relevant or into residual stability. Most of the mutations designed from electrostatic calculations or from simple sequence conservation analysis produce large increases in the overall stability of the protein. However, in most cases, this simply leads to similarly large increases of the residual stability. Two mutations, nevertheless, show a different trend and increase the relevant stability of the protein substantially. When all the mutations are mapped onto the structure of the apoflavodoxin thermal-unfolding intermediate (obtained independently by equilibrium phi-analysis and NMR) they cluster perfectly so that the mutations increasing the relevant stability appear in the small unstructured region of the intermediate and the others in the native-like region. This illustrates the need for specific investigation of N-to-I equilibria and the structure of protein intermediates, and indicates that it is possible to rationally stabilise a protein against partial unfolding once the structure of the intermediate conformation is known, even if at low resolution.

Anabaena↗
PubMed2001

Statistical evaluation for stability studies under stress storage conditions.

During the pharmaceutical development of a new drug, it is necessary to select as soon as possible the formulation with the best stability characteristics. The current International Commission for Harmonisation (ICH) regulations regarding stability testing requirements for a Registration Application provide the stress testing conditions with the aim of assessing the effect of severe conditions on the drug product. In practice, the well-known Arrhenius theory is still used to make a rapid stability prediction, to estimate a drug product shelf life during early stages of its pharmaceutical development. In this work, both the planning of a stress stability study to obtain a correct stability prediction from a temperature extrapolation and the suitable data treatment to discern the reliability of the stability results are discussed. The study was focused on the early formulation step of a very stable drug, Mitonafide (antineoplastic agent), formulated in a parenteral solution and in tablets. It was observed, for the solid system, that the extrapolated results using Arrhenius theory might be statistically good, but far from the real situation if the stability study is not designed in a correct way. The statistical data treatment and the stress-stability test proposed in this work are suitable to make a reliable stability prediction of different formulations with the same drug, within its pharmaceutical development.

Antineoplastic Agents↗
PubMed1997

Thermophilic proteins: stability and function in aqueous and organic solvents.

The molecular stability of thermophilic and hyperthermophilic enzymes generally reflects the growth temperatures of the parent organisms. Extracellular enzymes from the hyperthermophilic Archaea typically show very high levels of thermal stability and a number of enzymes with Tm values of greater than 100 degrees C have been reported. The mechanisms responsible for high molecular stability are typically intrinsic characteristics of the protein, as shown by the comparative stabilities of many native and recombinant proteins. However, some extrinsic stabilisation mechanisms have been demonstrated. High levels of thermal stability are positively correlated with stability in the presence of other denaturing agents, including detergents and organic solvents. This correlation suggests a common denaturation pathway where molecular mobility/flexibility is the prime determinant of susceptibility to irreversible denaturation. In single phase organic-aqueous solvents, protein destabilisation occurs via solvent-induced alteration to the protein hydration shell. However, correlations between protein stability and solvent hydrophobicity are unreliable. In two-phase organic-aqueous systems, interfacial denaturation predominates and is a function of both interfacial tension and interfacial surface area. Intracellular enzymes are protected from interfacial denaturation but are potentially susceptible to direct organic solvent effects, possibly depending on the role of the cell wall and cell membrane in the partitioning of the organic solvent into the cell cytoplasm. Immobilisation of thermophilic enzymes provides a method for enhancing both the thermal and solvent stabilities of thermophilic and mesophilic enzymes. Multi-point covalent immobilisation to glyoxal-agarose enhances thermal stability and limits protein-protein inactivation mechanisms. Miscible organic solvents have a profound influence on the specificities of enzyme reactions. The presence of high concentrations of miscible organic solvents may induce gross changes in substrate specificity and/or more subtle alterations in chiral selectivity. Correlations between the variation in enantioselectivity and both solvent hydrophobicity and solvent dielectric constant have been demonstrated although some recent studies implicate the formation of specific solvent-enzyme complexes which directly affect reaction kinetics.

Archaea↗
PubMed2001

Electrolyte- and pH-stabilities of aqueous solid lipid nanoparticle (SLN) dispersions in artificial gastrointestinal media.

The influence of artificial gastrointestinal (GI) media on the physical stability of solid lipid nanoparticle (SLN) formulations consisting of different lipids and various surfactants/stabilizers has been investigated in vitro, with respect to ionic strength and pH. Laser diffractometry and zeta potential measurements were the techniques applied. Some SLN formulations already showed aggregation/particle growth in the presence of electrolytes at neutral pH. Other lipid nanodispersions remained physically stable with respect to the influence of electrolytes, but were pH-sensitive. It was possible to produce SLN that were GIT (gastrointestinal tract) stable by an optimized stabilizer composition. There is no optimal surfactant mixture for stabilization of any lipid, e.g. SLN consisting of the lipid Cutina CP showed GIT stability in combination with the stabilizer sugar ester S1670, whereas the stabilization with the surfactant mixture Tween 80/Span 85 was not effective. Vice versa, the emulsifier Pluronic F68 stabilized the lipid Compritol ATO 888 but not the lipid Imwitor 900 sufficiently to avoid aggregation of the SLN dispersion in artificial GI media. The stabilizing properties depend obviously on the specific interactions of the lipid matrix with the emulsifier, e.g. anchoring of the stabilizer on the lipid surface and density on the surface.

Capsules↗
PubMed2001

Effect of salts on the stability and folding of staphylococcal nuclease.

The stability and folding kinetics of wild-type and a mutant staphylococcal nuclease (SNase) at neutral pH are significantly perturbed by the presence of moderate to high concentrations of salts. Very substantial increases in stability toward thermal and urea denaturation were observed; for example, 0.4 M sodium sulfate increased the free energy of wild-type SNase by more than 2 kcal/mol. For the NCA SNase mutant, the presence of the salts abolished the cold denaturation observed at neutral pH with this variant, and substantially increased its stability. Significant effects of salts on the kinetics of refolding were also observed. For NCA SNase, the presence of the salts markedly increased the folding rates (up to 5-fold). On the other hand, chloride, in particular, substantially decreased the rate of folding of the wild-type protein. Since the rates of the slow phases due to proline isomerization were increased by salt, these steps must be coupled to conformational processes. Fluorescence energy transfer between the lone tryptophan (Trp140) and an engineered fluorescent acceptor at residue 64 revealed that the addition of a high concentration of KCl led to the formation of a transient folding intermediate not observed at lower salt concentrations, and in which residues 140 and 64 were much closer than in the native state. The salt-induced effects on the kinetics of folding are attributed to the enhanced stability of the transient folding intermediates. It is likely that the combination of the high net charge, due to the high isoelectric point, and the relatively low intrinsic hydrophobicity, leads to staphylococcal nuclease having only marginal stability at neutral pH. The salt-induced effects on the structure, stability, and kinetics of staphylococcal nuclease are attributed to the binding of counterions, namely, anions, resulting in minimization of intramolecular electrostatic repulsion. This leads to increased stability, more structure, and greater compactness, as observed. Consequently, localized electrostatic repulsion is present at neutral pH in SNase, probably contributing to its marginal stability. The results suggest that, in general, marginally stable globular proteins will be significantly stabilized by salts under conditions where they have a substantial net charge.

Amino Acid Substitution↗
PubMed2005

Thermodynamic stability of carbonic anhydrase: measurements of binding affinity and stoichiometry using ThermoFluor.

ThermoFluor (a miniaturized high-throughput protein stability assay) was used to analyze the linkage between protein thermal stability and ligand binding. Equilibrium binding ligands increase protein thermal stability by an amount proportional to the concentration and affinity of the ligand. Binding constants (K(b)) were measured by examining the systematic effect of ligand concentration on protein stability. The precise ligand effects depend on the thermodynamics of protein stability: in particular, the unfolding enthalpy. An extension of current theoretical treatments was developed for tight binding inhibitors, where ligand effect on T(m) can also reveal binding stoichiometry. A thermodynamic analysis of carbonic anhydrase by differential scanning calorimetry (DSC) enabled a dissection of the Gibbs free energy of stability into enthalpic and entropic components. Under certain conditions, thermal stability increased by over 30 degrees C; the heat capacity of protein unfolding was estimated from the dependence of calorimetric enthalpy on T(m). The binding affinity of six sulfonamide inhibitors to two isozymes (human type 1 and bovine type 2) was analyzed by both ThermoFluor and isothermal titration calorimetry (ITC), resulting in a good correlation in the rank ordering of ligand affinity. This combined investigation by ThermoFluor, ITC, and DSC provides a detailed picture of the linkage between ligand binding and protein stability. The systematic effect of ligands on stability is shown to be a general tool to measure affinity.

Animals↗
PubMed1996

Unusual effects of an engineered disulfide on global and local protein stability.

The global and local stabilities of a eukaryotic ferricytochrome c variant with an engineered disulfide are examined. The disulfide connects position 20, which is usually a valine, to position 102, which is usually a threonine. The cross-linked variant is approximately 1.2 kcal mol-1 less stable than the wild-type protein at 298 K, pH 4.6, in H2O and D2O. Circular dichroism studies show that the decreased stability results from structure-induced stabilization of the denatured state [Betz, S. F., & Pielak, G. J. (1992) Biochemistry 31, 12337-12344]. Here, we use proton chemical shift, paramagnetic shift, and amide proton exchange data to obtain atomic level structural and energetic information. Chemical and paramagnetic shift data indicate only minor native state structural changes. Local stability is obtained from amide proton-deuterium exchange data, using model peptide intrinsic exchange rates. As expected, the exchange data indicate that cross-link incorporation decreases the majority of local stabilities. Near the cross-link, however, local stability seems to increase despite the overall global stability decrease. Furthermore, local stability changes for hydrophobic core residues seem to be greater than the global stability change. We interpret these observations as cross-link-induced changes in exchange competent states and relate them to changes in the denatured state.

Amino Acid Sequence↗