Carrageenan from Sarconema scinaioides (Gigartinales, Rhodophyta) of Indian waters

Carrageenan was extracted from the red seaweed Sarconema scinaioides of Indian waters and was characterized. The crude carrageenan as well as its alkali modified derivative was composed of 3,6-anhydro galactose, 6-O-methyl galactose as well as galactose moieties in various proportions. Linkage analysis exhibited that these two carrageenan samples consisted of 4-linked 3,6-anhydrogalactose residue sulphated at position 2, and 3-linked galactose residue sulphated at position 4. The physicochemical and rheological data along with molecular weight data, FT-IR, 1D and 2D NMR (¹H, ¹³C, COSY and HSQC) spectrometry suggested that the polysaccharide was composed predominantly of iota- along with a small amount of its precursor nu (ν)-carrageenan, unlike the hybrid carrageenans (iota-, pyruvated- and kappa-carrageenans) from this seaweed reported in the literature. This Indian seaweed species would be a potentially important source of iota-carrageenan.     

The interaction between Beta-lactoglobulin and sodium N-dodecyl sulphate.

1. The binding of sodium n-dodecyl sulphate to beta-lactoglobulin was studied in the pH range 3.5-7.0 by equilibrium dialysis, ultracentrifugation and microcalorimetry. 2. At low binding concentrations (less than 30 bound surfactants anions per protein molecule) the complexes formed aggregates in solution. 3. At higher binding concentrations aggregation does not occur at low ionic strength (0.01 mol/litre), but continues at high ionic strength (0.1 mol/litre). 4. At 25 degrees C the enthalpy of interaction of sodium n-dodecyl sulphate with beta-lactoglobulin can be interpreted as the sum of the enthalpies of formation of a complex with 2 bound surfactant anions, with an enthalpy change of -9.5 kJ-mol-1 of bound surfactant, and complexes containing at least 22 bound surfactant anions, with limiting enthalpies per bound surfactant anion of -12.4 kJ-mol-1 at pH 3.5 and -3.25 kJ-mol-1 at pH 5.5. 5. The binding of surfactant and the enthalpy of interaction at pH 3.5 ARE NOT SIGNIFICANTLY AFFECTED BY THE ADDITION Of 8 M-urea. 6. The data indicate that at low binding concentrations the interaction is of an ionic nature, and is accompanied by a conformational change in the protein.     

A fluorescence study of the temperature-dependent polymerization of bovine beta-casein A1.

The intrinsic fluorescence properties of bovine beta-casein A1 solutions have been studied under a variety of conditions. The protein shows both tyrosine and tryptophan fluorescence emission, the former being more pronounced at low temperature (5 degrees C) and in the presence of urea. Approximate relative quantum yields for the tyrosine and tryptophan residues were determined using free tyrosine and free tryptophan respectively as standards. The tryptophan emission intensity of beta-casein shows an increase with temperature indicating a temperature-dependent transition of the protein. Two-state analysis of the emission-intensity--temperature data yielded positive enthalpy and entropy values for the transition over a range of protein concentrations (0.018--0.18%). A marked protein concentration effect was apparent which indicated that the transition was a consequence of polymerization rather than a prerequisite for polymerization. The size of the polymers appeared to be concentration-dependent. Decreased ionic strength and increased pH both caused a reduction in the increase in emission intensity when the temperature was increased. At higher pH both the enthalpy and the entropy for the transition were reduced. Experimental precision was insufficient to allow the effect of decreased ionic strength on these parameters to be determined. Many of the properties of the temperature-dependent transition can be explained if the transition is considered to be one of micelle formation.     

Energetics of solvent and ligand-induced conformational changes in alpha-lactalbumin

The energetics of structural changes in the holo and apo forms of a-lactalbumin and the transition between their native and denatured states induced by binding Ca2+ and Na+ have been studied by differential scanning and isothermal titration microcalorimetry and circular dichroism spectroscopy under various solvent conditions. Removal of Ca2+ from the protein enhances its sensitivity to pH and ionic conditions due to noncompensated negative charge-charge interactions at the cation binding site, which significantly reduces its overall stability. At neutral pH and low ionic strength, the native structure of apo-alpha-lactalbumin is stable below 14 C and undergoes a conformational change to a native-like molten globule intermediate at temperatures above 25 degrees C. The denaturation of either holo- or apo-alpha-lactalbumin is a highly cooperative process that is characterized by an enthalpy of similar magnitude when calculated at the same temperature. Measured by direct calorimetric titration, the enthalpy of Ca2+-binding to apo-LA at pH 7.5 is -7.1 kJ mol(-1) at 5.0 degrees C. which is essentially invariant to protonation effects. This small enthalpy effect infers that stabilization of alpha-lactalbumin by Ca2+ is primarily an entropy driven process, presumably arising from electrostatic interactions and the hydration effect. In contrast to the binding of calcium, the interaction of sodium with apo-LA does not produce a noticeable heat effect and is characterized by its ionic nature rather than specific binding to the metal-binding site. Characterization of the conformational stability and ligand binding energetics of alpha-lactalbumin as a function of solvent conditions furnishes significant insight regarding the molecular flexibility and regulatory mechanism mediated by this protein.     

Study of the interaction between the antitumour protein alpha-sarcin and phospholipid vesicles.

alpha-Sarcin is a single polypeptide chain protein which exhibits antitumour activity by degrading the larger ribosomal RNA of tumour cells. We describe the interaction of a alpha-sarcin with lipid model systems. The protein specifically interacts with negatively-charged phospholipid vesicles, resulting in protein-lipid complexes which can be isolated by ultracentrifugation in a sucrose gradient. alpha-Sarcin causes aggregation of such vesicles. The extent of this interaction progressively decreases when the molar ratio of phosphatidylcholine increases in acidic vesicles. The kinetics of the vesicle aggregation induced by the protein have been measured. This process is dependent on the ratio of alpha-sarcin present in the protein-lipid system. A saturation plot is observed from phospholipid vesicles-protein titrations. The saturating protein/lipid molar ratio is 1:50. The effect produced by the antitumour protein on the lipid vesicles is dependent on neither the length nor the degree of unsaturation of the phospholipid acyl chain. However, the aggregation is dependent on temperature, being many times higher above the phase transition temperature of the corresponding phospholipid than below it. The effects of pH and ionic strength have also been considered. An increase in the ionic strength does not abolish the protein-lipid interaction. The effect of pH may be related to conformational changes of the protein. Binding experiments reveal a strong interaction between alpha-sarcin and acidic vesicles, with Kd = 0.06 microM. The peptide bonds of the protein are protected against trypsin hydrolysis upon binding to acidic vesicles. The interaction of the protein with phosphatidylglycerol vesicles does not modify the phase transition temperature of the lipid, although it decreases the amplitude of the change of fluorescence anisotropy associated to the co-operative melting of 1,6-diphenyl-1,3,5-hexatriene (DPH)-labelled vesicles. The results are interpreted in terms of the existence of both electrostatic and hydrophobic components for the interaction between phospholipid vesicles and the antitumour protein.     

Complexation of β-lactoglobulin fibrils and sulfated polysaccharides

Fibrils of β-lactoglobulin, formed by heating at pH 2, were titrated with a sulfated polysaccharide (κ-carrageenan) to determine the morphology and mechanism of complex formation at low pH. Structural information on the resultant complexes was gathered using transmission electron microscopy, atomic force microscopy, Doppler electrophoresis, and small-angle neutron scattering. Electrophoresis demonstrated that the carrageenan complexed with protein fibrils until reaching a maximum complexation efficiency at a protein/polysaccharide (r) weight ratio of 5:3. Neutron scattering and microscopy indicated an increasing formation of spherical aggregates attached along the protein fibrils with increases in the carrageenan concentration. These globular particles had an average diameter of 30 nm. Small-angle neutron scattering of these complexes could be accurately described by a form factor corresponding to multistranded twisted ribbons with spherical aggregates along their contour length, arranged in a necklace configuration.     

pH and cation-induced thermodynamic stability of human hyaluronan binding protein 1 regulates its hyaluronan affinity

Hyaluronan-binding protein 1 (HABP1) is a trimeric protein with high negative charges distributed asymmetrically along the faces of the molecule. Recently, we have reported that HABP1 exhibits a high degree of structural flexibility, which can be perturbed by ions under in vitro conditions near physiological pH (Jha, B. K., Salunke, D. M., and Datta, K. (2003) J. Biol. Chem. 278, 27464-27472). Here, we report the effect of ionic strength and pH on thermodynamic stability of HABP1. Trimeric HABP1 was shown to unfold reversibly upon dissociation ruling out the possibility of existence of folded monomer. An increase in ionic concentration (0.05-1 M) or decrease in pH (pH 8.0-pH 5.0) induced an unusually high thermodynamic stability of HABP1 as reflected in the gradual increase in transition midpoint temperature, enthalpy of transition, and conformational entropy. Our studies suggest that the presence of counter ions in the molecular environment of HABP1 leads to dramatic reduction of the intramolecular electrostatic repulsion either by de-ionizing the charged amino acid residues or by direct binding leading to a more stable conformation. A regulation on cellular HA-HABP1 interaction by changes in pH and ionic strength may exist, because the more stable conformation attained at higher ionic strength or at acidic pH showed maximum affinity toward HA as probed either in solid phase binding assay on HA-immobilized plates or an in-solution binding assay using intrinsic fluorescence of HABP1.     

Thermal and Alkaline Denaturation of Bovine β-Casein

The secondary structure of bovine beta-casein was characterized using circular dichroism (CD) and FTIR spectroscopies under physiologically relevant conditions. Analytical ultracentrifugation technique was used to follow the highly temperature, pH and concentration dependent self-association behavior. CD measurements provide convincing evidence for short segments of polyproline II-like structures in beta-casein in addition to a wide range of secondary structure elements, such as 10-20% alpha-helix, approximately 30% turns, 32-35% extended sheet. Results obtained at extreme pH (10.5) revealed structural destabilization in the monomeric form of the protein. At least four distinct structural transitions at 10, 33, 40 and 78 degrees C were observed at pH 6.75 by CD analysis, compared to only two transitions, 26 and 40 degrees C, at pH 10.5. Calculations from analytical ultracentrifugation suggest that the transitions at lower temperature (< or = 30 degrees C) occur primarily in the monomer. It is hypothesized that the transition at 10 degrees C and neutral pH may represent a general conformational change or cold denaturation. Those middle ranged transitions, i.e. 33 and 40 degrees C are more likely the reflection of hydrophobic changes in the core of beta-casein. As beta-casein undergoes self-association and increases in size, the transition at higher temperature (78 degrees C) is perhaps caused by the apparent conformational change within the micelle-like polymers. It has been shown that beta-casein binds the hydrophobic fluorescent probe ANS with high affinity in much similar fashion to molten globular proteins. The effect of urea denaturation on the bound complex effectively supports this observation.     

Exploring the energy profile of the Q(A)(-) to Q(B) electron transfer reaction in bacterial photosynthetic reaction centers: pH dependence of the conformational gating step

Both large- and small-scale conformational changes are needed as proteins carry out reactions. However, little is known about the identity, energy of, and barriers between functional substates on protein reaction coordinates. In isolated bacterial photosynthetic reaction centers, the electron transfer from the reduced primary quinone, Q(A)(-), to the secondary quinone, Q(B), is rate limited by conformational changes at low pH and by proton binding at high pH. The kinetics and thermodynamics of this reaction were determined between 200 and 300 K from pH 6 to pH 10.5. A model with two substates of the reactant, P(+)Q(A)(-)Q(B), one protonated (state A) and one unprotonated (alpha), and one state of the product, P(+)Q(A)Q(B)(-) (B), was able to simulate the dependence of the rate on temperature and pH fairly well. The equilibrium between the three states were measured in situ at each temperature. Proton binding (alpha to A transition) has a favorable DeltaH and unfavorable DeltaS as does the conformational changes required for electron transfer at low pH (A to B). The pK for the A to alpha transition is 9.7 at room temperature, consistent with previous measurements, and equivalent to 13.5 at 200 K. The activation barriers were determined for each transition. Both the alpha to A and the A to B transitions are limited primarily by the activation enthalpy with modest DeltaS.     

Conformational transition and polyelectrolyte behaviour of a succinoglycan polysaccharide

We report the chemical characterization and the relationship between the physicochemical properties and conformational change of a succinoglycan polysaccharide produced by Pseudomonas sp, NCIB 11592. The expected chemical structure is confirmed, with a ratio of D-glucose: D-galactose: pyruvate: succinate of 7:1:1:1. The molecular weight of the native form is 4.2 x 10(6) but after a single heating cycle through the disordered state the molecular weight is reduced to 3.0 x 10(6). The polymer has a polymolecularity index of 1.3 in both cases. The conformational change was studied by different methods which enabled us to define the exact nature of the ordered and disordered states. The conformational transition depends on the temperature, the ionic strength and the nature of the counterion. The polyelectrolyte behaviour is in favour of a single chain conformation with an intramolecular helix-coil transition. The enthalpy change during this transition is greater than that expected solely on the basis of the polyelectrolyte contribution. It may be associated with changes in solvation or a rearrangement of water molecules in close association with the polymer.     

Differential scanning calorimetry of alpha 2-macroglobulin and alpha 2-macroglobulin-proteinase complexes.

Differential scanning calorimetry is shown to detect substantial structural alterations occurring on the association of proteinases with the serum glycoprotein alpha 2-macroglobulin. At pH 7.5, the thermally induced unfolding of the macroglobulin occurs at approx. 60 degrees C with a transition enthalpy of 17 J/g. Association of active thermolysin, trypsin and papain shifts the transition temperature to 77 degrees C (transition enthalpy 5 J/g), indicating that a substantial conformational change accompanies the binding event. The stoicheiometry of the thermolysin--alpha 2-macroglobulin association producing this change appears to be unity, implying the presence of co-operative subunit interactions in the mechanism of association. The calorimetric method provides a novel approach for the evaluation of conformational variants induced on protein-protein association or pre-existing in the purified macroglobulin.     

Thermodynamic stability of porcine beta-lactoglobulin. A structural relevance.

The proposed biological function of beta-lactoglobulins as transporting proteins assumes a binding ability for ligands and high stability under the acidic conditions of the stomach. This work shows that the conformational stability of nonruminant porcine beta-lactoglobulin (BLG) is not consistent with this hypothesis. Thermal denaturation of porcine BLG was studied by high-sensitivity differential scanning calorimetry within the pH range 2.0-10.0. Dependences of the denaturation temperature and enthalpy on pH were obtained, which reveal a substantial decrease in both parameters in acidic and basic media. The denaturation enthalpy follows a linear dependence on the denaturation temperature. The slope of this line is 9.4 +/- 0.6 kJ.mol-1. K-1,which is close to the denaturation heat capacity increment DeltadCp = 9.6 +/- 0.5 kJ.mol-1.K-1, determined directly from the thermograms. At pH 6.25 the denaturation temperatures of porcine and bovine BLG coincide, at 83.2 degrees C. At this pH the denaturation enthalpy of porcine BLG is 300 kJ.mol-1. The denaturation transition of porcine BLG was shown to be reversible at pH 3.0 and pH 9.0. The transition profile at both pH values follows the two-state model of denaturation. Based on the pH-dependence of the transition temperature and the linear temperature dependence of the transition enthalpy, the excess free energy of denaturation, DeltadGE, of porcine BLG was calculated as a function of pH and compared with that of bovine BLG derived from previously reported data. The pH-dependence of DeltadGE is analysed in terms of the contributions of side-chain H-bonds to the protein stability. Interactions stabilizing native folds of porcine and bovine BLG are discussed.     

Perturbation of the equilibrium between open and closed conformations of the periplasmic C4-dicarboxylate binding protein from Rhodobacter capsulatus.

A kinetic and thermodynamic analysis has been carried out on the conformational transitions of the periplasmic C4-dicarboxylate binding protein (DctP) from the photosynthetic bacterium Rhodobacter capsulatus. This protein is distinct from other periplasmic binding proteins characterized to date in that the transition between the putative closed-unliganded (BP1) and open-unliganded (BP2) conformations is slow compared to the rate of ligand binding [Walmsley, A. R., Shaw, J. G., & Kelly, D. J. (1992) J. Biol. Chem. 276, 8064-8072]. Using stopped-flow fluorescence techniques, we have probed the conformational dynamics of the closed to open transition of DctP in the absence and presence of ligand. Both the forward rate constant for the BP1 to BP2 interconversion (k1) and the fumarate dissociation rate constant (k-3) were found to increase in a biphasic manner between pH 5 and pH 11. The data were fitted to a two-pKa function which gave pKa values of 10.3 and 5.4 for the BP1 to BP2 interconversion and 8.9 and 4.5 for the closed-liganded (BP3L) to open-liganded (BP2L) transition. An increase in ionic strength at constant pH resulted in a hyperbolic increase in both k1 and k-3 to maximal rates that were similar in each case to the values obtained in pH variation experiments. Measurement of the temperature dependencies of k1 and k-3 also gave similar activation energies. Gibbs free energy, enthalpy, and entropy changes were determined for the open to closed transitions of DctP in both the presence and absence of ligand.(ABSTRACT TRUNCATED AT 250 WORDS)     

On the link between conformational changes,ligand binding and heat capacity

BackgroundConformational changes coupled to ligand binding constitute the structural and energetics basis underlying cooperativity, allostery and, in general, protein regulation. These conformational rearrangements are associated with heat capacity changes. ITC is a unique technique for studying binding interactions because of the simultaneous determination of the binding affinity and enthalpy, and for providing the best estimates of binding heat capacity changes.Scope of reviewStill controversial issues in ligand binding are the discrimination between the “conformational selection model” and the “induced fit model”, and whether or not conformational changes lead to temperature dependent apparent binding heat capacities. The assessment of conformational changes associated with ligand binding by ITC is discussed. In addition, the “conformational selection” and “induced fit” models are reconciled, and discussed within the context of intrinsically (partially) unstructured proteins.Major conclusionsConformational equilibrium is a major contribution to binding heat capacity changes. A simple model may explain both conformational selection and induced fit scenarios. A temperature-independent binding heat capacity does not necessarily indicate absence of conformational changes upon ligand binding. ITC provides information on the energetics of conformational changes associated with ligand binding (and other possible additional coupled equilibria).General significancePreferential ligand binding to certain protein states leads to an equilibrium shift that is reflected in the coupling between ligand binding and additional equilibria. This represents the structural/energetic basis of the widespread dependence of ligand binding parameters on temperature, as well as pH, ionic strength and the concentration of other chemical species. This article is part of a Special Issue entitled Microcalorimetry in the BioSciences — Principles and Applications, edited by Fadi Bou-Abdallah.     

Adsorption of RNase A on cationic polyelectrolyte brushes: a study by isothermal titration calorimetry

We present a study of the adsorption of a positively charged protein to a positively charged spherical polyelectrolyte brush (SPB) by isothermal titration calorimetry (ITC). ITC is used to determine the adsorption isotherm as a function of temperature and of salt concentration (at physiological pH 7.2). At low ionic strength, RNase A is strongly adsorbed by the SPB particles despite the fact that both the SPB particles and the protein are positively charged. Virtually no adsorption takes place when the ionic strength is raised through added salt. This is strong evidence for counterion release as the primary driving force for protein adsorption. We calculated that ~2 counterions were released upon RNase A binding. The adsorption of RNase A into like-charged SPB particles is entropy-driven, and protein protonation was not significant. Temperature-dependent measurements showed a disagreement between the enthalpy derived via the van't Hoff equation and the calorimetric enthalpy. Further analysis shows that van't Hoff analysis leads to the correct enthalpy of adsorption. The additional contributions to the measured enthalpy are potentially sourced from unlinked equilibria such as conformational changes that do not contribute to the binding equilibrium.     

Thermodynamics of lipid protein associations. Thermodynamics of helix formation in the association of high density apolipoprotein A-I (apoA-I) to dimyristoyl phosphatidylcholine.

The structure and phospholipid-binding properties of human plasma high density apolipoprotein A-I (apoA-I) has been studied at pH 7.4 and 3.1 by microcalorimetry, circular dichroism and density gradient ultracentrifugation. At pH values of 7.4 and 3.1, apoA-I binds to dimyristoyl phosphatidylcholine (DMPC) to form complexes of similar composition (molar ratio of DMPC/apoA-I of 100) and helical content (67%). At pH 7.4, the lipid-protein association is accompanied by an increase in helical content from 58 to 67% and an exothermic enthalpy of binding (deltaHB) of -90 kcal/mol apoA-I. At pH 3.1, the helical content of apoA-I is increased from 48 to 67% on binding to DMPC and the enthalpy of binding was -170 kcal/mol. We suggest that the difference in the enthalpies of binding (-80 kcal/mol) at pH 3.1 compared to 7.4 is due to the greater coil leads to helix transition at the lower pH.     

Phospholipid membranes affect tertiary structure of the soluble cytochrome b5 heme-binding domain

The influence of charged phospholipid membranes on the conformational state of the water-soluble fragment of cytochrome b5 has been investigated by a variety of techniques at neutral pH. The results of this work provide the first evidence that aqueous solutions with high phospholipid/protein molar ratios (pH 7.2) induce the cytochrome to undergo a structural transition from the native conformation to an intermediate state with molten-globule like properties that occur in the presence of an artificial membrane surface and that leads to binding of the protein to the membrane. At other phospholipid/protein ratios, equilibrium was observed between cytochrome free in solution and cytochrome bound to the surface of vesicles. Inhibition of protein binding to the vesicles with increasing ionic strength indicated for the most part an electrostatic contribution to the stability of cytochrome b5-vesicle interactions at pH 7.2. The possible physiological role of membrane-induced conformational change in the structure of cytochrome b5 upon the interaction with its redox partners is discussed.     

Dynamic properties of monomeric insect erythrocruorin III from Chironomus thummi-thummi: relationships between structural flexibility and functional complexity.

We have investigated the kinetics of geminate carbon monoxide binding to the monomeric component III of Chironomus thummi-thummi erythrocruorin, a protein that undergoes pH-induced conformational changes linked to a pronounced Bohr effect. Measurements were performed from cryogenic temperatures to room temperature in 75% glycerol and either 0.1 M potassium phosphate (pH 7) or 0.1 potassium borate (pH 9) after nanosecond laser photolysis. The distributions of the low temperature activation enthalpy g(H) for geminate ligand binding derived from the kinetic traces are quite narrow and are influenced by temperature both below and above approximately 170 K, the glass transition temperature. The thermal evolution of the CO binding kinetics between approximately 50 K and approximately 170 K indicates the presence of some degree of structural relaxation, even in this temperature range. Above approximately 220 K the width of the g(H) progressively decreases, and at 280 K geminate CO binding becomes exponential in time. Based on a comparison with analogous investigations of the homodimeric hemoglobin from Scapharca inaequivalvis, we propose a link between dynamic properties and functional complexity.     

Differential scanning calorimetric studies on bovine serum albumin: II. Effects of neutral salts and urea

Using defatted and SH-blocked bovine serum albumin (BSA), measurements of differential scanning calorimetry (d.s.c.) have been made mainly in NaSCN solution. BSA undergoes a heat-induced conformational transition in a particular range of pH and ionic strength and is separated into two thermally independent units, each of which has different thermostability in acidic and alkaline pH regions. Comparisons were made of the pH dependencies of the enthalpy of thermal denaturation (ΔH) and the temperature of thermal denaturation (T_d) in 0.01 NaSCN with those in 0.01 NaCl. It has been found that the stabilizing effect of NaSCN on BSA is larger than that of NaCl at pH 3.5–8, and that the heat-induced transition occurs by the electrostatic repulsive forces among the positively charged amino acid residues in a segment Arg 184–Arg 216 containing Trp 212 and the primary binding sites of anions. At ionic strength 0.01, the relative effectiveness of anions in suppressing the heat-induced transition and increasing the thermostability of BSA follows the order ClO₄⁻ − SCN⁻ > I⁻ > SO₄²⁻ > Br⁻ > Cl⁻. At ionic strength 0.1, the heat-induced transition is suppressed in all the salt solutions, and a T_d increase follows the order ClO₄⁻ SCN⁻ > I⁻ > Br⁻ > Cl⁻ SO₄²⁻. Thus, the highly chaotropic ions thermostabilize BSA more markedly than kosmotropic ions in the low and moderate salt concentrations. In contrast, chaotropic ions destabilize BSA and kosmotropic ions stabilize BSA at the higher concentrations. An adequate amount of NaCl or NaSCN prevents the destruction of the environment of the binding site in the segment containing Trp 212 in 4 urea solution at pH 7.0.