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• W2149173814 abstract "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. 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. The diverse biological roles of hyaluronan, a complex polysaccharide in vertebrates, are now established beyond any doubt. These include acting as a vital structural component of connective tissues, the formation of loose hydrated matrices that allow cells to divide and migrate during development, and in intracellular signaling (1Lee J.Y. Spicer A.P. Curr. Opin. Cell Biol. 2000; 12: 581-586Crossref PubMed Scopus (453) Google Scholar, 2Day A.J. Prestwich G.D. J. Biol. Chem. 2002; 277: 4585-4588Abstract Full Text Full Text PDF PubMed Scopus (476) Google Scholar, 3Turley E.A. Bourguignon L. Noble P.W. J. Biol. Chem. 2002; 277: 4589-4592Abstract Full Text Full Text PDF PubMed Scopus (882) Google Scholar, 4Toole B.P. Wright T.N. Tammi M.I. J. Biol. Chem. 2002; 277: 4593-4596Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar, 5Fraser J.R.E. Laurent T.C. Comper W.D. Extracellular Matrix, Molecular Components and Interactions. 2. Harwood Academic Publishers, Amsterdam1996: 141-199Google Scholar). Such variable activities may result from its interaction with a family of proteins known as hyaladherin (2Day A.J. Prestwich G.D. J. Biol. Chem. 2002; 277: 4585-4588Abstract Full Text Full Text PDF PubMed Scopus (476) Google Scholar). Hyaluronan-binding protein 1 (HABP1), 1The abbreviations used are: HABP1, Hyaluronan-binding protein 1; MES, 4-morpholineethanesulfonic acid; ABTS, 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid); HRP, horseradish peroxidase; ASA, accessible surface areas; DSC, differential scanning calorimetric; HA, hyaluronan. one of the members of hyaladherin family was identified (6D'Souza M. Datta K. Biochem. Int. 1985; 10: 43-51PubMed Google Scholar) and its role in different cellular processes like cell adhesion and tumor invasion, sperm maturation, and motility (7Deb T.B. Datta K. J. Biol. Chem. 1996; 271: 2206-2212Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 8Gupta S. Babu B.R. Datta K. Eur. J. Cell Biol. 1991; 56: 58-67PubMed Google Scholar, 9Gupta S. Datta K. Exp. Cell Res. 1991; 195: 386-394Crossref PubMed Scopus (39) Google Scholar, 10Ranganathan S. Ganguly A.K. Datta K. Mol. Reprod. Dev. 1994; 38: 69-76Crossref PubMed Scopus (80) Google Scholar, 11Ranganathan S. Bharadwaj A. Datta K. Cell. Mol. Biol. Res. 1995; 41: 467-476PubMed Google Scholar) are under intensive investigation in our laboratories. Molecular cloning of human HABP1 revealed its multifunctional nature as its sequence was found to be identical with p32, a protein copurified with splicing factor SF2 and with the receptor of the globular head of complement factor C1q (gC1qR). It is represented as synonyms of p32/C1QBP (accession number NP_001203) in human chromosome 17. This molecule has generated a considerable interest in the last few years largely because of its multifarious functions as well as its localization in various subcellular compartments including cell surface in different cell types (8Gupta S. Babu B.R. Datta K. Eur. J. Cell Biol. 1991; 56: 58-67PubMed Google Scholar, 9Gupta S. Datta K. Exp. Cell Res. 1991; 195: 386-394Crossref PubMed Scopus (39) Google Scholar, 10Ranganathan S. Ganguly A.K. Datta K. Mol. Reprod. Dev. 1994; 38: 69-76Crossref PubMed Scopus (80) Google Scholar, 11Ranganathan S. Bharadwaj A. Datta K. Cell. Mol. Biol. Res. 1995; 41: 467-476PubMed Google Scholar, 12Krainer A.R. Mayeda A. Kozak D. Binns G. Cell. 1991; 66: 383-393Abstract Full Text PDF PubMed Scopus (413) Google Scholar, 13Ghebrehiwet B. Lim B.L. Peerschke E.I. Willis A.C. Reid K.B. J. Exp. Med. 1994; 179: 1809-1821Crossref PubMed Scopus (317) Google Scholar, 14Ghebrehiwet B. Peerschke E.I. Immunobiology. 1998; 199: 225-238Crossref PubMed Scopus (75) Google Scholar, 15Ghebrehiwet B. Jesty J. Peerschke E.I. Immunobiology. 2002; 205: 421-432Crossref PubMed Scopus (51) Google Scholar, 16Ghebrehiwet B. Lim B.L. Kumar R. Feng X. Peerschke E.I. Immunol. Rev. 2001; 180: 65-77Crossref PubMed Scopus (156) Google Scholar, 17Soltys B.J. Kang D. Gupta R.S. Histochem. Cell Biol. 2000; 114: 245-255Crossref PubMed Scopus (116) Google Scholar). However, only one transcript of HABP1 was detected from different types of tissues suggesting that no other isoform(s) of HABP1 exist in different cell types (16Ghebrehiwet B. Lim B.L. Kumar R. Feng X. Peerschke E.I. Immunol. Rev. 2001; 180: 65-77Crossref PubMed Scopus (156) Google Scholar). Studies on the crystal structure of p32/HABP1 revealed that it exists as a homotrimer, and each protomer consists of seven consecutive twisted anti-parallel β-sheets flanked by one NH2-terminal and two COOH-terminal α-helices and that the terminal α-helices have extensive intra- as well as intermolecular contacts. It has been postulated that these terminal helices are critical for maintaining the trimeric assembly and proteinprotein interactions (18Jiang J. Zhang Y. Krainer A.R. Xu R.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3572-3577Crossref PubMed Scopus (224) Google Scholar). In a previous report, we have shown that HABP1 exists predominantly as a non-covalently linked trimer near physiological conditions and as a hexamer (dimer of trimers) in the oxidative environment (19Jha B.K. Salunke D.M. Datta K. Eur. J. Biochem. 2002; 269: 298-306Crossref PubMed Scopus (21) Google Scholar). Being acidic in nature and having highly polar amino acid residues distributed asymmetrically on the surface, the electrostatic interactions in HABP1 are expected to have a role in dictating its folding topology and tertiary interactions. However, the relative role of the contribution of electrostatic interactions to protein stability, compared with that of hydrophobic interactions, has been the subject of long standing query (20Pace C.N. Methods Enzymol. 1995; 259: 538-544Crossref PubMed Scopus (95) Google Scholar). Part of the difficulty, in discriminating the contributions from electrostatic effect, stems from the fact that there are several different ways in which they contribute to the net stability of the native conformation. In addition, both attractive and repulsive electrostatic interactions are possible. In general, models to account for co-solute effects on protein stability may be classified in a number of ways; two major classifications are those in which the co-solute directly interacts with the protein, i.e. binding, or those involving effects on the solvent, e.g. excluded volume effects (21Saunders A.J. Davis-Searles P.R. Allen D.L. Pielak G.J. Erie D.A. Biopolymers. 2000; 53: 293-307Crossref PubMed Scopus (152) Google Scholar). Previously, we have shown that HABP1 exists in an expanded molten globule-like state at low ionic strength around alkaline pH because of charge-charge repulsion emanating from the presence of multiple negatively charged carboxyl groups, and that the addition of low concentrations of cations leads to a substantial compaction of HABP1 (22Jha B.K. Salunke D.M. Datta K. J. Biol. Chem. 2003; 278: 27464-27472Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). The effect of cation is attributed to their minimizing the charge repulsion by binding to the negatively charged groups, which in turn diminish the repulsive interaction (23Goto Y. Calciano L.J. Fink A.L. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 573-577Crossref PubMed Scopus (583) Google Scholar). Similar effects, in cases involving the presence of anions, appear to result in the refolding of intrinsically disordered protein that have a net positive charge at neutral pH (24Uversky V.N. Gillespie J.R. Fink A.L. Proteins. 2000; 41: 415-427Crossref PubMed Scopus (1783) Google Scholar). In terms of the overall spectrum of protein stability ranging from intrinsically unstructured protein at one extreme to very stable, globular proteins at the other, HABP1 is in the middle. In other words, there are many other proteins that may have marginal or even lower stability under physiological conditions. Thus, the effects of salts and pH on HABP1 are likely to be applicable to a number of other eukaryotic proteins, and to further our understanding of the broad range of interactions of ions with proteins. Recently, we have reported the structural flexibility of HABP1 under a wide range of ionic environments. At low ionic strength HABP1 exists in highly expanded and loosely held trimeric structures, whereas, the presence of salt induces compact trimeric structure (22Jha B.K. Salunke D.M. Datta K. J. Biol. Chem. 2003; 278: 27464-27472Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Thus the possibility of minimizing the intramolecular repulsion by counter ions leading to stable conformation needs to be examined. Also being an oligomeric protein, the studies on HABP1 can provide important insights into the relative contributions of the various forces under different conditions of pH, ion concentration, and temperature that stabilize the oligomeric structures of proteins in various stable or meta-stable conformations. EAH-Sepharose 4B, all empty XK, pre-packed Resource-Q, and Superose-6 HR10/30 columns and the gel filtration calibration kit were from Amersham Biosciences. All other chemicals (unless otherwise mentioned) were of the highest purity grade available and obtained from Sigma and highly purified HA-octasaccharide was a generous gift from Dr. Akira Asari of Seikagaku Corp., Japan. Milli-Q™ grade water was used for preparing all the solutions required for the study. Urea was purchased from Sigma and further purified by re-crystallization before use. Purification and Concentration Determination of HABP1—Recombinant HABP1 was produced in Escherichia coli as described earlier (7Deb T.B. Datta K. J. Biol. Chem. 1996; 271: 2206-2212Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). The protein was purified by a 65–90% ammonium sulfate fractionation, followed by ion exchange chromatography on a Resource-Q (6 ml) column (Amersham Biosciences), interfaced with a Pharmacia FPLC™ system (Amersham Biosciences) using a linear gradient of 0–1 m NaCl in 20 mm HEPES, 1 mm EDTA, 1 mm EGTA, 5% glycerol, and 0.2% 2-mercaptoehanol, pH 7.5, followed by hyaluronan-Sepharose affinity column chromatography as reported earlier (19Jha B.K. Salunke D.M. Datta K. Eur. J. Biochem. 2002; 269: 298-306Crossref PubMed Scopus (21) Google Scholar, 22Jha B.K. Salunke D.M. Datta K. J. Biol. Chem. 2003; 278: 27464-27472Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Affinity purified HABP1 was further purified by size exclusion chromatography as described earlier (19Jha B.K. Salunke D.M. Datta K. Eur. J. Biochem. 2002; 269: 298-306Crossref PubMed Scopus (21) Google Scholar). For all practical purposes the concentration of a known aliquot was determined in 20 mm phosphate buffer, pH 6.5, containing 6 m guanidine-HCl by measuring the absorbance at 280 nm at 25 °C on a Cary 100 Bio UV visible double beam spectrophotometer (Varian Inc., Mulgrave, Australia) interfaced with a Peltier thermal controller. The molar extinction coefficient of denatured HABP1 was calculated and found to be 22,190 at 280 nm, which corresponds to A0.1%280nm = 0.932 (22Jha B.K. Salunke D.M. Datta K. J. Biol. Chem. 2003; 278: 27464-27472Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Gel Permeation Chromatography—Gel permeation chromatography of HABP1 was carried out on a Pharmacia Superose-6™ HR10 × 30 analytical column interfaced with Pharmacia FPLC™ system at a constant flow rate of 0.3 ml/min. The buffer concentration used was 10 mm: phosphate, pH 6.5–8; MES, pH 5.5–6.3; acetate, pH 4.0–5.4; and citrate phosphate, pH 3.0–4.4, keeping the ionic concentration constant at 150 mm using NaCl at all pH values. The standard molecular mass markers of known molecular mass and Stokes radii: alcohol dehydrogenase (150 kDa, 46 Å): bovine serum albumin (67 kDa, 35.5 Å), ovalbumin (43 kDa, 30.5 Å), chymotrypsinogen (25 kDa, 20.9 Å), and ribonuclease A (13.7 kDa, 16.4 Å), were independently run to calibrate the column prior to sample run. Stokes radii were determined from a plot of Stokes radius versus Kav, where Kav is defined as (Ve - Vo)/(Vt - Vo), where Ve = elution volume; Vo = void volume; Vt = total bed volume of column (25Siegel L.M. Monty K.J. Biochim. Biophys. Acta. 1966; 112: 346-362Crossref PubMed Scopus (1547) Google Scholar). Circular Dichroism Spectral Studies—All of the far UV CD spectra of HABP1 were recorded on a JASCO 715 spectropolarimeter in the buffer of different pH values. The scans were taken between 250 and 200 nm at 10 °C. The samples were prepared 4–24 h before recording the spectra in the desired buffer and filtered through 0.22-μ Millipore membranes. A rectangular cuvette of 1-mm path length was used throughout the experiment. The concentration of protein was kept constant at 10 μm. Data were recorded at a scan speed of 50 nm/min with a response time of 1 s at a bandwidth of 1 nm and accumulated at 0.1-nm step intervals. Typically, 3–5 scans were taken for each sample. The buffer baseline was subtracted in each case. Thermal denaturation of HABP1 (5 μm) was monitored by change in molar ellipticity at 222 nm with increasing temperature on a JASCO 715 spectropolarimeter, equipped with a Peltier temperature controller (JASCO PTC-348 WI). The wavelength was decided on the basis of the minima of the difference spectra obtained by subtracting the native protein scan from the thermally denatured protein scan. The samples and buffers were prepared as described above. Samples were heated at a constant rate of 60 K h-1 and data were accumulated at 0.1-K intervals. Analyses of the data were carried out with Origin version 5.0 using a two-state transition model. Differential Scanning Calorimetric Studies—All calorimetric scans were performed on the ultrasensitive differential scanning calorimeter, VP-DSC (Microcal Inc.). The protein samples on which DSC scans were performed as a function of salt concentration were prepared in 10 mm phosphate buffer, pH 7.2. The ionic concentration was varied using NaCl. Samples for the pH-dependent scans were prepared in different buffers (phosphate, pH 6.5–8; MES, pH 5.5–6.3; acetate, pH 4.0–5.4) at a fixed ionic concentration of 200 mm. The protein samples were extensively dialyzed against the desired buffer, typically dialysis was done in 1:1000 samples to buffer ratio with 5 changes (1:1000 × 5) at 4 °C. The sample and the last dialysis buffer were filtered and degassed before being scanned in the calorimeter. The 0.5-ml sample was introduced into the sample cell and a similar amount of the last dialysate was introduced into the reference cell and the calorimeter was up-scanned at a constant rate. Protein concentration was determined by measuring A at 280 nm as described earlier. The calorimetric unit was interfaced to a microcomputer for automatic data collection and analysis. The best least squares fit of the two-state transition model, A3 ⇔ 3B, where A is the folded state and B is the unfolded state, to the data were determined using Origin version 5.0 software supplied with the instrument. Analyses of normalized data utilizing the progress base line connection of pre- and post-transition base line of the DSC thermogram yielded the van't Hoff enthalpy (ΔHv), transition midpoint temperature (Tm, the temperature at half the peak area), and the transition peak area, which when divided by the number of moles of protein in the cell, yields the calorimetric enthalpy (ΔHc). The ratio of ΔHc/ΔHv yields the cooperativity of transition (26Swaminathan C.P. Surolia N. Surolia A. J. Am. Chem. Soc. 1998; 120: 5153-5159Crossref Scopus (87) Google Scholar). All the scans at ionic concentrations of 100 mm NaCl or above were found to be reversible. Hyaluronan Binding Assays—Microtiter plate binding assays were carried out to investigate the effect of pH on the interactions of HA with HABP1. The assays, which are based on those described previously (22Jha B.K. Salunke D.M. Datta K. J. Biol. Chem. 2003; 278: 27464-27472Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 27Parkar A. Kahmanna J.D. Howatb S.L. Baylissb M.T. Day A.J. FEBS Lett. 1998; 428: 171-176Crossref PubMed Scopus (64) Google Scholar), determine colorimetrically the level of binding of HABP1 to wells coated with HA. All washes were performed in standard assay buffer (SAB: 10 mm phosphate buffer, pH 7.2, containing 150 mm NaCl and 0.05% (v/v) Tween 20). Plastic Costar flat-bottomed high binding microtiter plates (EIA/RIA) were coated (in triplicates) overnight with 100 μl/well of 10 μg/ml HA in 20 mm carbonate buffer (Na2CO3/NaHCO3), pH 9.6, at 4 °C. Control wells were treated with buffer alone. The coating solution was removed and the plates were washed three times and nonspecific binding sites were blocked by incubation with 1.5% (w/v) bovine serum albumin for 90 min at 37 °C, followed by three washes. Finally, the respective wells were rinsed three times with buffers of different pH values (10 mm sodium acetate, pH 4.0–6.5, and sodium phosphate, pH 6.5–8.5). 100 μl of HABP1 or HABP1-biotin from a 50 μg/ml solution in 10 mm sodium phosphate buffer, pH 6.5–8.5, or acetate buffer, pH 4.0–6.5, containing 150 mm NaCl was added to each well and incubated for 4 h at room temperature or overnight at 4 °C. Plates were washed three times and 100 μl of a 1:10,000 dilution of polyclonal anti-HABP1 antibody (in SAB containing 1% bovine serum albumin) was added and incubated for 90 min, followed by five washes. A 100 μl/well of 1:10,000 dilution of horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG in SAB containing 1% bovine serum albumin was added to each well and incubated for 1 h at room temperature. For competitive inhibition assays biotinylated HABP1 at different pH values were preincubated with varying concentrations of unlabeled HABP1 and added to HA-coated plates and the bound HABP1-biotin was probed with HRP-conjugated extravidin (1:30,000). The unbound secondary antibodies/extravidin-HRP were sipped out of the wells after 1 h incubation at 37 °C and washed five times and finally rinsed with substrate buffer (100 mm phosphate citrate buffer, pH 5.0). 100 μl of 0.6 mg/ml solution of ABTS in substrate buffer containing 3 μl/ml H2O2 was added to each well and incubated for 10 min at 37 °C for color to develop. Absorbance at 405 nm was determined on a microtiter plate reader (Bio-Rad model 550 microplate reader). All absorbance readings were corrected against blank wells. Solution Binding Assay for HA-HABP1 Interaction—All fluorescence measurements were performed on a LS-55 luminescence spectrometer (PerkinElmer Life Sciences) interfaced with a Multi Temp III (Amersham Biosciences) water circulator to keep the temperature constant. Binding of HA to HABP1 was monitored by adding small aliquots of HA-octa- or polysaccharide to 0.8 μm HABP1 dissolved in a similar buffer at constant temperature. In every case the total volume of aliquots added was always ≤1% of the total reaction volume. A parallel control containing similar disaccharide equivalents of chondritin sulfate A of similar aliquots was run and used as F0. The fluorescence emission intensity was measured by using an excitation wavelength of 295 nm. For titration experiments, emission was monitored at 350 nm. In every case excitation and emission slit-width of 2.5 and 5 nm, respectively, were used. Fluorescence data were corrected for excitation intensity. Five successive readings with an integration time of 10 s were recorded and averaged value was subtracted from F0 to get ΔF and plotted against ligand concentration. ΔFmax calculated from the plot was further used for the estimation of dissociation constant as reported earlier (28Bagshaw C.R. Harris D.A. Harris D.A. Bashford C.L. Spectrophotometry and Spectrofluorimetry, A Practical Approach. IRL Press Oxford, United Kingdom1988: 91-113Google Scholar). Calculation of the Accessible Surface Area Changes upon Trimer Dissociation and Monomer Unfolding—The polar, apolar, and total accessible surface areas (ASA) were calculated with the program Insight, using the methods of Lee and Richards (29Lee B. Richards F.M. J. Mol. Biol. 1971; 55: 379-400Crossref PubMed Scopus (5360) Google Scholar). For the folded trimeric HABP1 the coordinates were downloaded (Protein Data Bank code 1ID p32) and analyzed for accessible surface area. The program calculates the atomic accessible surface defined by rolling a spherical probe of a given size around the van der Waals surface. A probe of 1.4-Å radius and a slice thickness of 0.05 Å were used. The output file contained summed atomic ASA over each residue. The ASA of the unfolded protein was calculated as the sum of the accessibility of the residues in an extended Ala-X-Ala tripeptide because of the primary structure of the protein. We took the Ala-X-Ala surface values from the tripeptide designed in extended conformations for each residue using Insight software. Hydrodynamic Behavior of Trimeric HABP1 as a Function of pH—To examine if electrostatic repulsion in HABP1, an acidic protein, is indeed responsible for the expanded structure around neutral and basic pH, size exclusion chromatography was carried out as a function of pH keeping the ionic concentration constant. HABP1 showed a gradual decrease in elution volume suggesting a gradual increase in the hydrodynamic volume (Vh) of the molecule with increase in pH (Fig. 1A). Therefore, size exclusion chromatographic data were further analyzed for calculating the hydrodynamic size of the molecule by comparing with the standard molecular weight markers of known Stokes radii. The pH-induced structural compaction seems to be the result of reduction in total charges because of de-ionization of various polar amino acid residues, which are asymmetrically distributed on trimeric HABP1. The total charge present on HABP1 at different pH values was calculated using ANTHEPROT and plotted against pH along with the Stokes radius at the corresponding pH (Fig. 1B). The total charge on trimeric HABP1 at pH 7.2 is -83 as compared with that of +8.0 at pH 4.0. The important observations of our study are concerned with strikingly different states of compaction of HABP1, induced by pH. For example, HABP1 trimer under alkaline pH is so expanded that it elutes much earlier than the compact trimer. There is a gradual increase in the compactness of trimer with decreasing pH. The decrease is such that the compact form of the trimer has a Stokes radius of 30.7 ± 1.4 Å (n = 3) at pH 4.5 compared with the most expanded trimer, which has a Stokes radius of 40.5 ± 1.5 Å at pH 9.0 (n = 3). All these experiments were performed at a fixed ionic concentration. Model for Fitting the Differential Scanning Calorimetric Data of HABP1 Unfolding—We have shown previously that the absence of counter ion drastically affects the structure as well as conformation of trimeric HABP1 in solution, which to a great extent seems to be responsible for the generation of binding sites for different ligands under different conditions of ionic strength (22Jha B.K. Salunke D.M. Datta K. J. Biol. Chem. 2003; 278: 27464-27472Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Thus, the presence of counter ions in its molecular environment is an absolute requirement for HABP1 to exist in any sort of functionally relevant structure/conformation, especially so for its hyaluronan binding capabilities. Consequently, we proposed that the combination of the high net charge asymmetrically distributed along the faces of the molecule and relatively low intrinsic hydrophobicity of HABP1 resulted in its expanded structure around neutral pH in low ionic environment. Addition of counter ions in the molecular environment minimizes the intra-molecular electrostatic repulsion in HABP1 leading to its stable and compact conformations, which is also reflected in its differential affinity toward different types of ligands. In this study we have, therefore, scrutinized in detail, the conformational stability and folding of HABP1 in a wide range of salt and pH values using ultrasensitive differential scanning calorimeter. A typical endotherm of HABP1 with the details of the mode of curve fitting procedure, notation, and the definition of various thermodynamic parameters determined are illustrated in Fig. 2. HABP1 (14 μm) in 10 mm sodium phosphate-buffered saline, pH 7.2, was filled in the sample cell and the reference cell, containing buffer alone, was up-scanned. The respective baseline-subtracted data (where the baseline was obtained with buffer in both the sample and the reference cells of the calorimeter) were used in each case for analyses (Fig. 2, a and b). HABP1 showed a perfect thermodynamic as well as biochemical reversibility from 100 mm salt onwards, as shown in Fig. 3, a and b, respectively. Therefore the data obtained from the DSC measurements were analyzed by a two-state reversible unfolding, i.e. unfolding upon dissociation, fitting model.Fig. 3Thermodynamic and biochemical reversibility. HABP1 exhibits perfect thermodynamic as well as biochemical reversibility. A, raw data obtained from DSC scans of HABP1 (open circle) and rescanning the same protein sample (closed diamond). 14 μm HABP1 in 10 mm phosphate-buffered saline (100 mm NaCl) was up-scanned at a heating rate of 20 K h-1 beyond its transition midpoint temperature. The same sample was then cooled to 10 °C and up-scanned again. The graph shows the plot of excess Cp versus temperature. B, microtiter plate-based HA binding assay of native (solid circle) and renatured (open circle) HABP1 shows perfect biochemical reversibility. For this binding assay 100 μl/well of native and thermally denatured HABP1, renatured by slow cooling, were coated in a 96-well enzyme-linked immunosorbent assay plate in triplicates as described under “Materials and Methods” and incubated with biotin-labeled HA. The bound HA was detected with 1:10,000 dilution of HRP-conjugated extravidin using ABTS as substrate. Bound HA was plotted against the increasing concentration of HABP1. The S.D. (n = 3) is shown as error bars on each data point.View Large Image Figure ViewerDownload (PPT) Effect of Protein Concentration, Scan Rate, Salt, and pH on Thermodynamic Stability of HABP1—It has been demonstrated by using a number of independent techniques that HABP1 is predominantly trimeric under the experimental conditions in this study (22Jha B.K. Salunke D.M. Datta K. J. Biol. Chem. 2003; 278: 27464-27472Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). For oligomeric proteins, the measured (effective) heat effect of unfolding and melting temperature depends on protein concentration. To see such an effect in the case of HABP1, concentration-dependent c" @default.
• W2149173814 created "2016-06-24" @default.
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• W2149173814 date "2004-05-01" @default.
• W2149173814 modified "2023-09-28" @default.
• W2149173814 title "pH and Cation-induced Thermodynamic Stability of Human Hyaluronan Binding Protein 1 Regulates Its Hyaluronan Affinity" @default.
• W2149173814 cites W1494638726 @default.
• W2149173814 cites W1508745139 @default.
• W2149173814 cites W1533569014 @default.
• W2149173814 cites W1536613246 @default.
• W2149173814 cites W1567493631 @default.
• W2149173814 cites W1720678970 @default.
• W2149173814 cites W1967493968 @default.
• W2149173814 cites W1982629607 @default.
• W2149173814 cites W1985645723 @default.
• W2149173814 cites W1986699235 @default.
• W2149173814 cites W1987433783 @default.
• W2149173814 cites W1987893498 @default.
• W2149173814 cites W1989054182 @default.
• W2149173814 cites W1991871934 @default.
• W2149173814 cites W1998882239 @default.
• W2149173814 cites W2001458612 @default.
• W2149173814 cites W2002442479 @default.
• W2149173814 cites W2004360551 @default.
• W2149173814 cites W2006605358 @default.
• W2149173814 cites W2029794817 @default.
• W2149173814 cites W2033371740 @default.
• W2149173814 cites W2035485234 @default.
• W2149173814 cites W2035536358 @default.
• W2149173814 cites W2039610628 @default.
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