Thermal stabilization of antithrombin III by sugars and sugar derivatives and the effects of nonenzymatic glycosylation

A variety of neutral and acidic sugars and related compounds were evaluated in terms of their effect on the midpoint, Td, of the thermal denaturation curve of antithrombin III. The objectives were to determine which structural features of these molecules are responsible for their stabilizing properties and to identify more efficient stabilizers which combine the effects of lyotropic anions such as citrate with those of the polyols in a single molecule. The presence of one or more carboxylate groups in a sugar molecule invariably increased its stabilizing potency, whereas the number and position of hydroxyl groups appeared to have no influence on the molecules' stabilizing ability. Several compounds were shown to be effective in preserving antithrombin III activity during pasteurization for 10 h at 60 degrees C. However, the presence of reducing sugars invariably resulted in a decrease in activity following pasteurization, in spite of their ability to increase Td. In fact, when antithrombin III was pasteurized in the presence of 2 M glucose and 0.5 M citrate, it steadily lost its ability to inhibit thrombin even though Td under these conditions was 10 degrees C higher than in citrate alone where activity was preserved. This effect was shown to be coincident with the covalent incorporation of glucose into the protein molecule.     

Employment of a turbidimetric assay system to measure heat-induced protein aggregation

1. 1.|We developed a turbidimetric assay system for quantitation of heat-induced protein aggregation which is presumably caused by protein denaturation.

2. 2.|Rhodanese in 6 M guanidinium chloride was employed in the assay system, because this protein recognizes hydrophobic sites on denatured proteins and aggregates.

3. 3.|Turbidity caused by protein-rhodanase aggregation was recorded at 320 nm by using a u.v./VIS spectrophotometer.

4. 4.|When heated, alcohol dehydrogenase (ADH) aggregates with rhodanese. The increase of ADH-rhodanese aggregation was correlated with the loss of enzymatic activity.

5. 5.|These results indicated that the aggregation was proportional to the extent of ADH denaturation which assumingly caused the loss of ADH activity during heating at 45.5°C.

6. 6.|Similar results were observed when cytosolic proteins from CHO cells were heated at 45.5°C. Heated cytosolic proteins promoted aggregation by complex formation with rhodanese. The aggregation increased with increasing heat dose.

7. 7.|Therefore, the rhodanese assay system can be employed usefully to quantitate the protein aggregation after heat stress.

抗鱼肉蛋白冷冻变性机理的研究进展

简述了鱼肉蛋白冷冻变性机理的研究现状,综述了糖类、盐类、乳蛋白、不同水解物等添加物的抗冷冻变性机理,介绍了鱼肉蛋白冷冻变性的评价指标及其测定方法,展望了抗鱼肉蛋白冷冻变性的新途径及应用前景。论文内容对于深入研究抗肉类蛋白冷冻变性具有较大的参考价值。     

Effect of Sugars on Rabbit Serum Albumin Stability and Induction of Secondary Structure

The effect of sugars (sucrose, maltose, and glucose) on the thermal and chemical denaturation of rabbit serum albumin (RSA) has been examined by viscosity and far UV circular dichroism measurements. The viscosity measurements indicate a change in the reduced viscosity from 4.18 to 16.23 ml/g in the temperature range from 20 to 90°C. The T _m value for RSA obtained by viscosity measurements in the absence of sugar was found to be 63.2°C, but this value increased to 68.4, 70.3, and 73.2°C in the presence of 0.5 M sucrose, 0.5 M glucose, and 0.5 M maltose, respectively. Further, the stability of RSA in the presence of 0.5 M sugars was also investigated by measuring the mean residue ellipticity at 222 nm (MRE₂₂₂) using chemical (0-6 M guanidine hydrochloride) and thermal (20-90°C) transition processes. At the midpoint of the chemical denaturation, the increase in the MRE values at 222 nm in the presence of 0.5 M sugars were of the same order as the increase in the T _m values, i.e., maltose > glucose > sucrose. Interestingly, a mixture of 0.25 M glucose and 0.25 M fructose showed a cumulative effect on the thermal as well as chemical stability as compared to 0.5 M sucrose alone. In the case of both thermal and chemical denaturation, there was an increase in the MRE₂₂₂ values upon addition of various sugars, this indicating induction of secondary structure in the protein.     

Approaches for increasing the solution stability of proteins

Stabilization of proteins through proper formulation is an important challenge for the pharmaceutical industry. Two approaches for stabilization of proteins in solution are discussed. First, work describing the effect of additives on the thermally induced denaturation and aggregation of low molecular weight urokinase is presented. The effects of these additives can be explained by preferential exclusion of the solute from the protein, leading to increased thermal stability with respect to denaturation. Diminished denaturation leads to reduced levels of aggregation. The second approach involves stoichiometric replacement of polar counter ions (e.g., chloride, acetate, etc.) with anionic detergents, in a process termed hydrophobic ion pairing (HIP). The HIP complexes of proteins have increased solubility in organic solvents. In these organic solvents, where the water content is limited, the thermal denautration temperatures greatly exceed those observed in aqueous solution. In addition, it is possible to use HIP to selectively precipitate basic proteins from formulations that contain large amounts of stabilizers, such as human serum albumin (HSA), with a selectivity greater than 2000-fold. This has been demonstrated for various mixtures of HSA and interleukin-4. (c) 1995 John Wiley & Sons, Inc.     

Production, secretion, and stability of human secreted alkaline phosphatase in tobacco NT1 cell suspension cultures

Tobacco NT1 cell suspension cultures secreting active human secreted alkaline phosphatase (SEAP) were generated for the first time as a model system to study recombinant protein production, secretion, and stability in plant cell cultures. The SEAP gene encodes a secreted form of the human placental alkaline phosphatase (PLAP). During batch culture, the highest level of active SEAP in the culture medium (0.4 U/mL, corresponding to approximately 27 mg/L) was observed at the end of the exponential growth phase. Although the level of active SEAP decreased during the stationary phase, the activity loss did not appear to be due to SEAP degradation (based on Western blots) but due to SEAP denaturation. The protein-stabilizing agents polyvinylpirrolidone (PVP) and bacitracin were added extracellularly to test for their ability to reduce the loss of SEAP activity during the stationary phase. Bacitracin (100 mg/L) was the most effective treatment at sustaining activity levels for up to 17 days post-subculture. Commercially available human placental alkaline phosphatase (PLAP) was used to probe the mechanism of SEAP deactivation. Experiments with PLAP in sterile and conditioned medium corroborated the denaturation of SEAP by factors generated by cell growth and not due to simple proteolysis. We also show for the first time that the factors promoting activity loss are heat labile at 95 degrees C but not at 70 degrees C, and they are not inactivated after a 5 day incubation period under normal culture conditions (27 degrees C). In addition, there were no significant changes in pH or redox potential when comparing sterile and cell-free conditioned medium during PLAP incubation, indicating that these factors were unimportant.     

Probing membrane protein unfolding with pulse proteolysis

Technical challenges have greatly impeded the investigation of membrane protein folding and unfolding. To develop a new tool that facilitates the study of membrane proteins, we tested pulse proteolysis as a probe for membrane protein unfolding. Pulse proteolysis is a method to monitor protein folding and unfolding, which exploits the significant difference in proteolytic susceptibility between folded and unfolded proteins. This method requires only a small amount of protein and, in many cases, may be used with unpurified proteins in cell lysates. To evaluate the effectiveness of pulse proteolysis as a probe for membrane protein unfolding, we chose Halobacterium halobium bacteriorhodopsin (bR) as a model system. The denaturation of bR in SDS has been investigated extensively by monitoring the change in the absorbance at 560 nm (A₅₆₀). In this work, we demonstrate that denaturation of bR by SDS results in a significant increase in its susceptibility to proteolysis by subtilisin. When pulse proteolysis was applied to bR incubated in varying concentrations of SDS, the remaining intact protein determined by electrophoresis shows a cooperative transition. The midpoint of the cooperative transition (C_m) shows excellent agreement with that determined by A₅₆₀. The C_m values determined by pulse proteolysis for M56A and Y57A bRs are also consistent with the measurements made by A₅₆₀. Our results suggest that pulse proteolysis is a quantitative tool to probe membrane protein unfolding. Combining pulse proteolysis with Western blotting may allow the investigation of membrane protein unfolding in situ without overexpression or purification.     

Investigation of a side-chain-side-chain hydrogen bond by mutagenesis, thermodynamics, and NMR spectroscopy.

Anomalous NMR behavior of the hydroxyl proton resonance for Ser 31 has been reported for histidine-containing protein (HPr) from two microorganisms: Escherichia coli and Staphylococcus aureus. The unusual slow exchange and chemical shift exhibited by the resonance led to the proposal that the hydroxyl group is involved in a strong hydrogen bond. To test this hypothesis and to characterize the importance of such an interaction, a mutant in which Ser 31 is replaced by an alanine was generated in HPr from Escherichia coli. The activity, stability, and structure of the mutant HPr were assessed using a reconstituted assay system, analysis of solvent denaturation curves, and NMR, respectively. Substitution of Ser 31 yields a fully functional protein that is only slightly less stable (delta delta G(folding) = 0.46 +/- 0.15 kcal mol-1) than the wild type. The NMR results confirm the identity of the hydrogen bond acceptor as Asp 69 and reveal that it exists as the gauche- conformer in wild-type HPr in solution but exhibits conformational averaging in the mutant protein. The side chain of Asp 69 interacts with two main-chain amide proteins in addition to its interaction with the side chain of Ser 31 in the wild-type protein. These results indicate that removal of the serine has led to the loss of all three hydrogen bond interactions involving Asp 69, suggesting a cooperative network of interactions. A complete analysis of the thermodynamics was performed in which differences in side-chain hydrophobicity and conformational entropy between the two proteins are accounted for.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermal stabilization of antithrombin III by sugars and sugar derivatives and the effects on nonenzymatic glycosylation

A variety of neutral and acidic sugars and related compounds were evaluated in terms of their effect on the midpoint, T_d, of the thermal denaturation curve of antithrombin III. The objectives were to determine which structural features of these molecules are responsible for their stabilizing properties and to identify more efficient stabilizers which combine the effects of lyotropic anions such as citrate with those of the polyols in a single molecule. The presence of one or more carboxylate groups in a sugar molecule invariably increased its stabilizing potency, whereas the number and position of hydroxyl groups appeared to have no influence on the molecules' stabilizing ability. Several compounds were shown to be effective in preserving antithrombin III activity during pasteurization for 10 h at 60°C. However, the presence of reducing sugars invariably resulted in a decrease in activity following pasteurization, in spite of their ablity to increase T_d. In fact, when antithrombin III was pasteurized in the presence of 2 M glucose and 0.5 M citrate, it steadily losts its ability to inhibit thrombin even though T_d under the conditions was 10°C higher than in citrate alone where activity was preserved. This effect was shown to be coincident with the covalent incorporation of glucose into the protein molecule.     

Equilibrium phi-analysis of a molten globule: the 1-149 apoflavodoxin fragment

The apoflavodoxin fragment comprising residues 1-149 that can be obtained by chemical cleavage of the C-terminal alpha-helix of the full-length protein is known to populate a molten globule conformation that displays a cooperative behaviour and experiences two-state urea and thermal denaturation. Here, we have used a recombinant form of this fragment to investigate molten globule energetics and to derive structural information by equilibrium Phi-analysis. We have characterized 15 mutant fragments designed to probe the persistence of native interactions in the molten globule and compared their conformational stability to that of the equivalent full-length apoflavodoxin mutants. According to our data, most of the mutations analysed modify the stability of the molten globule fragment following the trend observed when the same mutations are implemented in the full-length protein. However, the changes in stability observed in the molten globule are much smaller and the Phi-values calculated are (with a single exception) below 0.4. This is consistent with an overall and significant debilitation of the native structure. Nevertheless, the fact that the molten globule fragment can be stabilised using as a guide the native structure of the full-length protein (by increasing helix propensity, optimising charge interactions and filling small cavities) suggests that the overall structure of the molten globule is still quite close to native, in spite of the lowered stability observed.     

Effect of polyols on the conformational stability and biological activity of a model protein lysozyme

The purpose of this study was to investigate the stabilizing action of polyols against various protein degradation mechanisms (eg, aggregation, deamidation, oxidation), using a model protein lysozyme. Differential scanning calorimeter (DSC) was used to measure the thermodynamic parameters, mid point transition temperature and calorimetric enthalpy, in order to evaluate conformational stability. Enzyme activity assay was used to corroborate the DSC results. Mannitol, sucrose, lactose, glycerol, and propylene glycol were used as polyols to stabilize lysozyme against aggregation, deamidation, and oxidation. Mannitol was found to stabilize lysozyme against aggregation, sucrose against deamidation both at neutral pH and at acidic pH, and lactose against oxidation. Stabilizers that provided greater conformational stability of lysozyme against various degradation mechanisms also protected specific enzyme activity to a greater extent. It was concluded that DSC and bioassay could be valuable tools for screening stabilizers in protein formulations.     

Robust and convenient analysis of protein thermal and chemical stability

We present the software CDpal that is used to analyze thermal and chemical denaturation data to obtain information on protein stability. The software uses standard assumptions and equations applied to two‐state and various types of three‐state denaturation models in order to determine thermodynamic parameters. It can analyze denaturation monitored by both circular dichroism and fluorescence spectroscopy and is extremely flexible in terms of input format. Furthermore, it is intuitive and easy to use because of the graphical user interface and extensive documentation. As illustrated by the examples herein, CDpal should be a valuable tool for analysis of protein stability.     

A microplate-based evaluation of complex denaturation pathways: structural stability of Escherichia coli transketolase

We have previously developed a rapid microplate-based approach for measuring the denaturation curves by intrinsic tryptophan fluorescence for simple monomeric and two-state unfolding proteins. Here we demonstrate that it can accurately resolve the multiple conformational transitions that occur during the denaturation of a complex multimeric and cofactor associated protein. We have also analyzed the effect of two active-site mutations, D381A and Y440A upon the denaturation pathway of transketolase using intrinsic fluorescence measurements, and we compare the results from classical and microplate-based instrumentation. This work shows that the rapid assay is able to identify changes in the denaturation pathway, due to mutations or removal of cofactors, which affect the stability of the native and intermediate states. This would be of significant benefit for the directed evolution of protein stability, optimizing enzyme stability under biocatalytic process conditions, and also for engineering specific transitions in protein unfolding pathways.     

Design, expression, and stability of a diverse protein library based on the human fibronectin type III domain

Protein libraries based on natural scaffolds enable the generation of novel molecular tools and potential therapeutics by directed evolution. Here, we report the design and construction of a high complexity library (30 x 10(13) sequences) based on the 10th fibronectin type III domain of human fibronectin (10FnIII). We examined the bacterial expression characteristics and stability of this library using a green fluorescent protein (GFP)-reporter screen, SDS-PAGE analysis, and chemical denaturation, respectively. The high throughput GFP reporter screen demonstrates that a large fraction of our library expresses significant levels of soluble protein in bacteria. However, SDS-PAGE analysis of expression cultures indicates the ratio of soluble to insoluble protein expressed varies greatly for randomly chosen library members. We also tested the stabilities of several representative variants by guanidinium chloride denaturation. All variants tested displayed cooperative unfolding transitions similar to wild-type, and two exhibited free energies of unfolding equal to wild-type 10FnIII. This work demonstrates the utility of GFP-based screening as a tool for analysis of high-complexity protein libraries. Our results indicate that a vast amount of protein sequence space surrounding the 10FnIII scaffold is accessible for the generation of novel functions by directed as well as natural evolution.     

Functional decline of sweet taste sensitivity of colobine monkeys

For many primates, sweet taste is palatable and is an indicator that the food contains carbohydrates, such as sugars and starches, as energy sources. However, we have found that Asian colobine monkeys (lutungs and langurs) have low sensitivity to various natural sugars. Sweet tastes are recognized when compounds bind to the sweet taste receptor TAS1R2/TAS1R3 in the oral cavity; accordingly, we conducted a functional assay using a heterologous expression system to evaluate the responses of Javan lutung (Trachypithecus auratus) TAS1R2/TAS1R3 to various natural sugars. We found that Javan lutung TAS1R2/TAS1R3 did not respond to natural sugars such as sucrose and maltose. We also conducted a behavioral experiment using the silvery lutung (Trachypithecus cristatus) and Hanuman langur (Semnopithecus entellus) by measuring the consumption of sugar-flavored jellies. Consistent with the functional assay results for TAS1R2/TAS1R3, these Asian colobine monkeys showed no preference for sucrose or maltose jellies. These results demonstrate that sweet taste sensitivity to natural sugars is low in Asian colobine monkeys, and this may be related to the specific feeding habits of colobine monkeys.     

Thermal fluctuation spectroscopy of DNA thermal denaturation

We have developed the technique of thermal fluctuation spectroscopy to measure the thermal fluctuations in a system. This technique is particularly useful to study the denaturation dynamics of biomolecules like DNA. Here we present a study of the thermal fluctuations during the thermal denaturation (or melting) of double-stranded DNA. We find that the thermal denaturation of heteropolymeric DNA is accompanied by large, non-Gaussian thermal fluctuations. The thermal fluctuations show a two-peak structure as a function of temperature. Calculations of enthalpy exchanged show that the first peak comes from the denaturation of AT rich regions and the second peak from denaturation of GC rich regions. The large fluctuations are almost absent in homopolymeric DNA. We suggest that bubble formation and cooperative opening and closing dynamics of basepairs causes the additional fluctuation at the first peak and a large cooperative transition from a partially molten DNA to a completely denatured state causes the additional fluctuation at the second peak.     

Water penetration in the low and high pressure native states of ubiquitin

Theoretical studies on the solvation of methane molecules in water have shown that the effect of increased pressure is to stabilize solvent separated contacts relative to direct contacts. This suggests that high pressure stabilizes waters that have penetrated into a protein's core, indicating a mechanism for the high pressure denaturation of proteins. We test this theory on a folded protein by studying the penetration of water into the native state of ubiquitin at low and high pressures, using molecular dynamics. An ensemble of conformations sampled in the folded state of ubiquitin has been determined by NMR at two pressures below the protein's denaturation pressure, 30 atm and 3000 atm. We find that 1-5 more waters penetrate the high pressure conformations than the low pressure conformations. Low volume configurations of the system are favored at high pressures, but different components of the system may experience increases or decreases in their specific volumes. We find that penetrating waters have a higher volume per water than bulk waters, but that the volume per protein residue may be lowered by solvation. Furthermore, we find that penetration of the protein by water at high pressures is driven by the difference in the pressure dependence of the probability of cavity opening in the protein and pressure dependence of the probability of cavity opening in the bulk solvent. The volume changes associated with cavity opening and closing indicate that each penetrating water reduces the volume of the system by about 12 mL/mol. The experimental volume change going from the low pressure to the high pressure native state of ubiquitin is 24 mL/mol. Our results indicate that this volume change can be explained by penetration of the protein by two water molecules.     

A cure for traffic jams: small molecule chaperones in the endoplasmic reticulum

Folding in the endoplasmic reticulum is the limiting step for the biogenesis of most secretory pathway cargo proteins; proteins which fail to fold are initially retained in the endoplasmic reticulum and subsequently often degraded. Mutations that affect secretory protein folding have profound phenotypes irrespective of their direct impact on protein function, because they prevent secretory proteins from reaching their final destination. When unicellular organisms are stressed by fluctuation of temperature or ionic strength, they synthesize high concentrations of small molecules such as trehalose or glycerol to prevent protein denaturation. These osmolytes can also stabilize mutant secretory proteins and allow them to pass secretory protein quality control in the endoplasmic reticulum. Specific ligands and cofactors such as ions, sugars, or peptides have similar effects on specific defective proteins and are beginning to be used as therapeutic agents for protein trafficking diseases.     

Calorimetric study of G-actin denaturation

The investigation of G-actin heat denaturation at various pH of the solution by scanning microcalorimetry has shown that unfolding of G-actin is not a two-state process. Since the protein structure does not behave as a single cooperative unit during heat denaturation, it is suggested that the G-actin globule consists, at least, of two interacting domains.     

Evidence that specific oligosaccharides block early events necessary for the expression of antigen-specific proliferation by human lymphocytes

We have previously shown that monosaccharides and disaccharides will block the expression of spontneous monocyte-mediated cytotoxicity. Our data were consistent with the hypothesis that human mononuclear cells express lectin-like receptors that are capable of binding to a variety sugar moieties found on target cell membranes. In this communication, we will present evidence that monosaccharides and disaccharides are also capable of blocking the expression of T cell reactivity as measured by an in vitro antigen-specific proliferative assay. The majority of sugars that blocked monocyte-mediated cytotoxicity had no effect on antigen-specific proliferation. Those sugars that did suppress antigen-induced proliferation had no effect on PHA-induced proliferation. Furthermore, some of these sugars only inhibited if they were added at the initiation of the assay; they failed to inhibit if added 24 hr after the initiation of the assay. Antigen-pulsing experiments suggested that these sugars did not block antigen uptake by human monocytes. These data suggest that a variety of cellular interactions may be mediated by receptors with specificity for simple sugars. The ability to block these naturally occurring lectins specifically both in vitro and in vivo may prove to be a powerful tool for dissecting out various forms of cellular recognition and collaboration.