Polyol osmolytes stabilize native-like cooperative intermediate state of yeast hexokinase A at low pH

Osmolytes produced under stress in animal and plant systems have been shown to increase thermal stability of the native state of a number of proteins as well as induce the formation of molten globule (MG) in acid denatured states and compact conformations in natively unfolded proteins. However, it is not clear whether these solutes stabilize native state relative to the MG state under partially denaturing conditions. Yeast hexokinase A exists as a MG state at pH 2.5 that does not show any cooperative transition upon heating. Does the presence of some of these osmolytes at pH 2.5 help in the retention of structure that is typical of native state? To answer this question, the effect of ethylene glycol (EG), glycerol, xylitol, sorbitol, trehalose and glucose at pH 2.5 on the structure and stability of yeast hexokinase A was investigated using spectroscopy and calorimetry. In presence of the above osmolytes, except EG, yeast hexokinase at pH 2.5 retains native secondary structure and hydrophobic core and unfolds with excessive heat absorption upon thermal denaturation. However, the cooperative structure binds to ANS suggesting that it is an intermediate between MG and the native state. Further, we show that at high concentration of polyols at pH 2.5, except EG, which populates a non-native ensemble, ΔH_cal/ΔH_van approaches unity indicative of two-state unfolding. The results suggest that osmolytes stabilize cooperative protein structure relative to non-cooperative ensemble. These findings have implications toward the structure formation, folding and stability of proteins produced under stress in cellular systems.     

Increased thermal stability of proteins in the presence of sugars and polyols.

Sugars and polyols stablize proteins against heat denaturation. Scanning calorimetry was used to obtain a quantitative estimate of the degree of stabilization. Solutions of ovalbumin, lysozyme, conalbumin, and alpha-chymotrypsinogen were heated at a constant rate, and the temperature of the maximum rate of denaturation was estimated (Tm). Addition of a sugar or polyol raised Tm. The magnitude of the stabilizing effect (delta Tm) depended on both the nature of the protein and the nature of the sugar or polyol, ranging from 18.5 degrees C for lysozyme at pH 3 in the presence of 50% (w/w) sorbitol to 0 degrees C for conalbumin at pH 7 in 50% glycerol solution. It is argued that this stablization is due to the effects of sugars and polyols on hydrophobic interactions. The strength of the hydrophobic interaction was measured in model systems in sucrose and glycerol solutions. Sucrose and glycerol strengthened the pairwise hydrophobic interaction between hydrophobic groups; however, they reduced the tendency for complete transfer of hydrophobic groups from an aqueous to a nonpolar environment. The extent of stabliziation by different sugars and polyols is explained by their different influences on the structure of water. The difference between the partial molar volume of the sugar or polyol and its van der Waals volume was used as a rough quantitative measure of the structure-making or structure-breaking effect. There was a linear relationship between this quantity and delta Tm.     

Effect of ethylene glycol and polyethylene glycol on the acid-unfolded state of trypsinogen

Effect of increasing concentrations of two of the polyols, ethylene glycol (EG) and polyethylene glycol (PEG), was studied by near and far circular dichroism (CD), fluorescence emission spectroscopy, and binding of hydrophobic dye, 1-anilino-8-naphthalene sulfonic acid (ANS). Far-UV CD spectra show the transition of acid-unfolded trypsinogen from an unordered state to an intermediate state having ordered secondary structure. Interestingly, near-UV CD spectra show some amounts of stabilizing effect on the tertiary structure of the protein also. Tryptophan fluorescence studies indicate the change in the environment of the tryptophan residues on addition of EG and PEG. Maximum ANS binding occurs in presence of 80% EG and 90% PEG (v/v), suggesting the presence of an intermediate or molten globule-like state at high concentrations of the two polyols.     

Specific volume and compressibility of human serum albumin-polyanion complexes

The ultrasound velocimetry, densitometry, and differential scanning calorimetry have been used to study the formation of the complexes between human serum albumin (HSA) and polyanions heparin (HEP) and/or dextran sulfate (DS). The values of the ultrasound velocity and specific volume allowed us to determine the specific adiabatic compressibility, phi(K)/beta(0), which reflects the degree of volume compressibility of the complexes. We showed that in the presence of HEP and DS the adiabatic compressibility of HSA decreases with increasing concentration of polyanions. HEP more strongly interacts with HSA than DS. pH of electrolyte in the range 4.7-8.5 weakly affects the adiabatic compressibility. Changes of compressibility of HSA can be caused by increase of the hydration due to the formation of the HSA-polyanion complexes and due to partial unfolding of HSA. The HSA-polyanion interaction resulted in decrease of phase transition temperature of the protein. This evidences about protein destabilization in the presence of polyanions.     

Cholesterol-induced variations in the volume and enthalpy fluctuations of lipid bilayers.

The sound velocity and density of suspensions of large unilamellar liposomes from dimyristoylphosphatidylcholine with admixed cholesterol have been measured as a function of temperature around the chain melting temperature of the phospholipid. The cholesterol-to-phospholipid molar ratio xc has been varied over a wide range (0 </= xc </= 0.5). The temperature dependence of the sound velocity number, of the apparent specific partial volume of the phospholipid, and of the apparent specific adiabatic compressibility have been derived from the measured data. These data are particularly discussed with respect to the volume fluctuations within the samples. A theoretical relation between the compressibility and the excess heat capacity of the bilayer system has been derived. Comparison of the compressibilities (and sound velocity numbers) with heat capacity traces display the close correlation between these quantities for bilayer systems. This correlation appears to be very useful as it allows some of the mechanical properties of membrane systems to be calculated from the specific heat capacity data and vice versa.     

Specific volume and adiabatic compressibility measurements of native and aggregated recombinant human interleukin-1 receptor antagonist: density differences enable pressure-modulated refolding

High hydrostatic pressures have been used to dissociate non-native protein aggregates and foster refolding to the native conformation. In this study, partial specific volume and adiabatic compressibility measurements were used to examine the volumetric contributions to pressure-modulated refolding. The thermodynamics of pressure-modulated refolding from non-native aggregates of recombinant human interleukin-1 receptor antagonist (IL-1ra) were determined by partial specific volume and adiabatic compressibility measurements. Aggregates of IL-1ra formed at elevated temperatures (55 degrees C) were found to be less dense than native IL-1ra and refolded at 31 degrees C under 1,500 bar pressure with a yield of 57%. Partial specific adiabatic compressibility measurements suggest that the formation of solvent-free cavities within the interior of IL-1ra aggregates cause the apparent increase in specific volume. Dense, pressure-stable aggregates could be formed at 2,000 bar which could not be refolded with additional high pressure treatment, demonstrating that aggregate formation conditions and structure dictate pressure-modulated refolding yields.     

Amyloid protofibril is highly voluminous and compressible

We report here results of the first direct measurement of partial volume and compressibility changes of a protein as it forms an amyloid protofibril. We use a high precision density meter and an ultrasonic velocity meter on a solution of intrinsically denatured, disulfide-deficient variant of hen lysozyme, and follow the time-dependent changes in volume and compressibility, as the protein spontaneously forms a protofibril. We have found a large increase in partial specific volume with time from 0.684 to 0.724 mL x g-1 (Deltanu = 0.040 mL x g-1 corresponding to 570 mL x (mol monomer)-1) and in partial specific adiabatic compressibility coefficient from -7.48 x 10(-12) to +1.35 x 10(-12) cm2 x dyn-1 (Deltabetas = 8.83 x 10(-12) x cm2 x dyn-1) as the monomer transforms into a protofibril. The results demonstrate that the protofibril is a highly voluminous and compressible entity, disclosing a cavity-rich, fluctuating nature for the amyloid protofibril. The volume and compressibility changes occur in two phases, the faster one preceding the major development of the beta-structure in the protofibril as monitored by CD.     

Compressibility changes accompanying conformational transitions of apomyoglobin

We used high-precision density and ultrasonic velocity measurements to characterize the native (N), molten globule (MG), and unfolded (U) conformations of apomyoglobin. The molten globule states that were studied in this work include the MG(pH4)(NaCl) state observed at pH 4 and 20 mM NaCl, the MG(pH4)(NaTCA) state observed at pH 4 and 20 mM sodium trichloracetate (NaTCA), the MG(pH2)(NaCl) state observed at pH 2 and 200 mM NaCl, and the MG(pH2)(NaTCA) state observed at pH 2 and 20 mM NaTCA. We used our densimetric and acoustic data to evaluate changes in adiabatic compressibility associated with the acid- or salt-induced N-to-MG, MG-to-U, MG-to-MG, and U-to-MG transitions of the protein. The N-to-MG(pH4)(NaCl) and N-to-MG(pH4)(NaTCA) transitions are accompanied by decreases in compressibility of -(3.0 +/- 0.6) x 10(-6) and -(2.0 +/- 0.6) x 10(-6) cm3 g(-1)bar(-1), respectively. The N-to-MG(pH2)(NaCl) and N-to-MG(pH2)(NaTCA) transitions are associated with compressibility changes of -(4.9 +/- 1.1) x 10(-6) and (0.7 +/- 0.9) x 10(-6) cm3 g(-1) bar(-1), respectively. We interpret these data in terms of the degree of unfolding of the various molten globule forms of apomyoglobin. In general, our compressibility data reveal significant disparities between the various equilibrium molten globule states of apomyoglobin while also quantitatively characterizing each of these states. Volumetric insights provided by our data facilitate gaining a better understanding of the folding pathways, intermediates, and kinetics of apomyoglobin folding.     

Survival of cultured cells and somatic embryos of Asparagus officinalis cryopreserved by vitrification

Cultured cells and somatic embryos derived from the mesophyll tissue of asparagus (Asparagus officinalis L.) were cryopreserved by vitrification. The vitrification solution (PVS) contains (w/v) 22% glycerol, 15% ethylene glycol, 15% propylene glycol and 7% DMSO in Murashige-Skoog medium enriched with 0.5M sorbitol. After initial cryoprotection with sorbitol supplemented MS medium containing 12% ethylene glycol, cells or embryos were exposed stepwise to 85% PVS at 0°C. They were loaded into 0.5 ml transparent straws, and were then plunged directly into liquid nitrogen. After rapid warming, PVS was removed and diluted stepwise. The highest survivals of vitrified cells and embryos were about 65 and 50%, respectively. Surviving embryos developed into plantlets.Abbreviations DMSO dimetyl sulfoxide - PVS vitrification solution - LN liquid nitrogen - DSC differential scanning calorimeter - MS Murashige-Skoog salt medium - NAA naphthalene acetic acid - BA 6-benzyladenine     

Heat-triggered conversion of protofibrils into mature amyloid fibrils of beta2-microglobulin

Heat-triggered conversion of the salt-induced thin and flexible protofibrils into well-organized thick and straight mature amyloid fibrils was achieved with beta2-microglobulin, a protein responsible for dialysis-related amyloidosis. First, protofibrils that formed spontaneously at pH 2.5 in the presence of 0.5 M NaCl were aggregated by agitating the solution. Second, the aggregated protofibrils were heated in a cell of a differential scanning calorimeter (DSC). The DSC thermogram showed an exothermic transition with sigmoidal temperature dependence, resulting in a remarkably large decrease in the heat capacity of the solution. Third, on the basis of electron microscopy together with circular dichroism spectroscopy, seeding experiments, and a thioflavin T binding assay, the sigmoidal transition was found to represent the conversion of protofibrils into mature amyloid fibrils. Furthermore, DSC thermograms obtained at various heating rates revealed that the transition curve depends on the heating rate, implying that the effects of heat associated with the conversion to the mature fibrils are kinetically controlled, precluding an interpretation in terms of equilibrium thermodynamics. Taken together, these results highlight the importance of the change in heat capacity in addressing the biological significance of interactions between solvent water and amyloid fibrils and, moreover, in detecting the formation of amyloid fibrils.     

Compressibility and specific volume of actin decrease upon G to F transformation

We measured the densities as well as the sound velocities in solutions of G-actin, F-actin and the reconstituted thin filament. Using the data obtained, we determined their partial specific volumes and partial specific adiabatic compressibilities. The objectives were to investigate the volume change of actin upon polymerization and to detect the conformational change associated with the Ca²⁺-binding to the reconstituted thin filament. The partial specific volume and the partial specific adiabatic compressibility of G-actin were 0.749 cm³/g and 9.3 · 10⁻¹² cm²/dyne, respectively. The results suggest that G-actin is a rather soft protein compared with other globular proteins. The partial specific volumes of F-actin were in a range of 0.63–0.66 cm³/g depending on the solvent conditions. The partial specific adiabatic compressibilities of F-actin were negative (−(7–13) · 10⁻¹² cm³/dyne). These data indicate that the amount of hydration may increase by several times upon polymerization assuming that the size of the cavity remains constant. We detected little difference between the partial specific adiabatic compressibility of the reconstituted thin filament in a Ca²⁺-bound state and that in a Ca²⁺-unbound state. This suggests that the Ca²⁺ binding affected not the subunit itself but the inter-subunit junction.     

Stereocontrolled Synthesis of an Octasaccharide Part of I-Active Glycolipids

The effects of glycerin and ethylene glycol on the elastic modulus and DSC thermograms of agarose and kappa-carrageenan gels were examined to clarify the relation between structure and properties. The elastic modulus of these gels as a function of the concentration of polyols increased up to a certain concentration and then decreased with increasing concentration of polyols. These polyols shifted the melting temperature of the gel to higher temperatures in kappa-carrageenan gels but to lower temperatures in agarose gels. The temperature dependence of elastic modulus was changed in opposite directions in agarose and kappa-carrageenan gels by the addition of polyols, and this is discussed on the basis of model consisting of junction zones which are connected by Langevin chains. It was suggested that the mean distance between junction zones became shorter in the presence of a small amount of polyols.     

Molecular mechanism of polyethylene glycol mediated stabilization of protein

The effect of different molar ratios of polyethylene glycol (PEG) on the conformational stability of protein, bovine serum albumin (BSA), was studied. The binding of PEG with BSA was observed by fluorescence spectroscopy by measuring the fluorescence intensity after displacement of PEG with chromophore ANS and had further been confirmed by measuring the intrinsic fluorescence of tryptophan residues of BSA. Co-lyophilization of BSA with PEG at optimum BSA:PEG molar ratio led to the formation of the stable protein particles. Circular dichroism (CD) spectroscopy study suggested that a conformational change had occurred in the protein after PEG interaction and demonstrated the highest stability of protein at the optimum BSA:PEG molar ratio of 1:0.75. Additional differential scanning calorimetry (DSC) study suggested strong binding of PEG to protein leading to thermal stability at optimum molar ratio. Molecular mechanism operating behind the polyethylene glycol (PEG) mediated stabilization of the protein suggested that strong physical adsorption of PEG on the hydrophobic core of the protein (BSA) along with surface adsorption led to the stability of protein.     

Binding of bovine pancreatic trypsin inhibitor to trypsinogen: spectroscopic and volumetric studies

We have investigated the binding of bovine pancreatic trypsin inhibitor (BPTI) to bovine trypsinogen by combining ultrasonic velocimetry, high precision densimetry, and fluorescence spectroscopy. We report the changes in volume, adiabatic compressibility, van't Hoff enthalpy, entropy, and free energy that accompany the association of the two proteins at 25 degrees C and pH 8.0. We have used the measured changes in volume and compressibility in conjunction with available structural data to characterize the binding-induced changes in the hydration properties and intrinsic packing of the two proteins. Our estimate reveals that 110 +/- 40 water molecules become released to the bulk from the hydration shells of BPTI and trypsinogen. Furthermore, we find that the intrinsic coefficient of adiabatic compressibility of the two proteins decreases by 14 +/- 2%, which is suggestive of the binding-induced rigidification of the proteins' interior. BPTI-trypsinogen association is an entropy-driven event which proceeds with an unfavorable change in enthalpy. The favorable change in entropy results from partial compensation between two predominant terms. Namely, a large favorable change in hydrational entropy slightly prevails over a close in magnitude but opposite in sign change in configurational entropy. The reduction in configurational entropy and, consequently, protein dynamics is consistent with the observed decrease in intrinsic compressibility. In general, results of this work emphasize the vital role that water plays in modulating protein recognition events.     

Ca2+ and pH dependence of hydrophobicity of alpha-lactalbumin: affinity partitioning of proteins in aqueous two-phase systems containing poly(ethylene glycol) esters of fatty acids.

Hydrophobic affinity partitioning in an aqueous two-phase system, composed of dextran and poly(ethylene glycol), has been used to study the hydrophobic binding capacity of bovine alpha-lactalbumin. The hydrophobicity of the poly(ethylene glycol)-containing phase was adjusted by including varying amounts of fatty acids bound to the polymer via an ester linkage. The change in the logarithmic partition coefficient of the protein in such systems was used as a measure of the hydrophobic binding. This value was strongly influenced by the amount of Ca2+ present as well as the pH value. The results are discussed in terms of the exposure of hydrophobic binding sites on alpha-lactalbumin and their relation to the conformational change in this protein due to Ca(2+)-binding, chelation of Ca2+ and pH dependence.     

MEARA sequence repeat of human CstF-64 polyadenylation factor is helical in solution. A spectroscopic and calorimetric study.

The primary structure of the human CstF-64 polyadenylation factor contains 12 nearly identical repeats of a consensus motif of five amino acid residues with the sequence MEAR(A/G). No known function has yet been ascribed to this motif; however, according to secondary structure prediction algorithms, it should form a helical structure in solution. To validate this theoretical prediction, we synthesized a 31 amino acid residue peptide (MEARA(6)) containing six repeats of the MEARA sequence and characterized its structure and stability by circular dichroism (CD) spectroscopy and differential scanning calorimetry (DSC). No effects of concentration on the CD or DSC properties of MEARA(6) were observed, indicating that the peptide is monomeric in solution at concentrations up to 2 mM. The far UV-CD spectra of MEARA(6) indicates that at a low temperature (1 degrees C) the MEARA(6) peptide has a relatively high helical content (76% at pH 2.0 and 65% at pH 7.0). The effects of pH and ionic strength on the CD spectrum of MEARA(6) suggest that a number of electrostatic interactions (e.g., i, i + 3 Arg/Glu ion pair, charge-dipole interactions) contribute to the stability of the helical structure in this peptide. DSC profiles show that the melting of MEARA(6) helix is accompanied by positive change in the enthalpy. To determine thermodynamic parameters of helix-coil transition from DSC profiles for this peptide, we developed a new, semiempirical procedure based on the calculated function for the heat capacity of the coiled state for a broad temperature range. The application of this approach to the partial molar heat capacity function for MEARA(6) provides the enthalpy change for helix formation calculated per amino acid residue as 3.5 kJ/mol.     

Preparation of self-assembled silk sericin nanoparticles

Silk sericin (SS) possessing moisture-retaining property was reacted with activated poly(ethylene glycol) (PEG) to obtain self-assembled SS nanoparticles. The aliphatic and aromatic hydroxyl groups of serine and tyrosine residues as the reaction sites in SS were clarified by amino acid analysis and 1H NMR spectroscopy, respectively. From IR and circular dichroism (CD) measurements, introduction of PEG into SS induced the conformational change from random coil to beta-sheet. DSC thermogram of sericin-PEG conjugate suggests that mutual miscibility between PEG and SS chains was poor. Nanoparticles of sericin-PEG conjugate with sizes measured by dynamic light scattering ranging about 200-400 nm in diameter, were prepared by the diafiltration method. Shape of sericin-PEG conjugate nanoparticles observed by scanning and transmission electron microscopes was spherical. The results suggest that sericin-PEG conjugates are self-associated to form spherical nanoparticles through hydrophobic interaction.     

Effects of solute methoxylation on glass-forming ability and stability of vitrification solutions

The effects of replacing hydroxyl groups with methoxyl (OCH(3)) groups in the polyols ethylene glycol (EG), propylene glycol (PG), glycerol, and threitol were studied by differential scanning calorimetry (DSC) during cooling of aqueous solutions to -150 degrees C and subsequent rewarming. For 35% (w/w) PG, 40% EG, and 45% glycerol, a single substitution of a terminal hydroxyl group with a methoxyl group reduced the critical cooling rate necessary to avoid ice on cooling (vitrify) from approximately 500 to 50 degrees C/min. This reduction was approximately equivalent to increasing the parent polyol concentration by 5% (w/w). The critical warming rate calculated to avoid formation of ice on rewarming (devitrification) was also reduced by methoxyl substitution, typically by a factor of 10(4) for dilute solutions. Double methoxylation (replacement of both terminal hydroxyls) tended to result in hydrate formation, making these compounds less interesting. An exception was threitol, for which substituting both terminal hydroxyls by methoxyls reduced the critical rewarming rate of a 50% solution by a factor of 10(7) without any hydrate formation. These glass-forming and stability properties of methoxylated compounds, combined with their low viscosity, enhanced permeability, and high glass transition temperatures, make them interesting candidate cryoprotective agents for cryopreservation by vitrification or freezing. Copyright 1999 Academic Press.     

Protein stability and dynamics in the pressure-temperature plane

The pressure-temperature stability diagram of proteins and the underlying assumptions of the elliptical shape of the diagram are discussed. Possible extensions, such as aggregation and fibril formation, are considered. An important experimental observation is the extreme pressure stability of the mature fibrils. Molecular origins of the diagram in terms of models of the partial molar volume of a protein focus on cavities and hydration. Changes in thermal expansivity, compressibility and heat capacity in terms of fluctuations of the enthalpy and volume change of the unfolding should also focus on these parameters. It is argued that the study of water-soluble polymers might further our understanding of the stability diagram. Whereas the role of water in protein behaviour is unquestioned, the role of cavities is less clear.     

Interaction of the antimicrobial peptide gramicidin S with dimyristoyl–phosphatidylcholine bilayer membranes: a densitometry and sound velocimetry study

We determined changes in the volume and adiabatic compressibility of large multi- and unilamellar vesicles composed of dimyristoylphosphatidylcholine containing various concentrations of the antimicrobial peptide gramicidin S (GS) by applying densitometry and sound velocimetry. Gramicidin S incorporation was found to progressively decrease the phase transition temperature of DMPC vesicles as well as to decrease the degree of cooperativity of the main phase transition and to increase the volume compressibility of the vesicles. GS probably enhanced thermal fluctuations at the region of main phase transition and provide more freedom of rotational movement for the phospholipid hydrocarbon chains. The ability of GS to increase the membrane compressibility and to decrease the phase transition temperature is evidence for regions of distorted membrane structure around incorporated gramicidin S molecules. At relatively high GS concentration (10 mol%), more significant changes of specific volume and compressibility appear. This might suggest changes in the integrity of the lipid bilayer upon interaction with high concentrations of GS.