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

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Introduction
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 signalling (1)(2)(3)(4)(5). Such variable activities may result from its interaction with a family of protein known as hyaladherin (2) Hyaluronan binding protein 1 (HABP1), one of the members of hyaladherin family was identified (6) and its role in different cellular processes like cell adhesion and tumor invasion, sperm maturation and motility (7)(8)(9)(10)(11) 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 co-purified with splicing factor SF2 and with the receptor of 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 due to its multifarious functions as well as its localization in various sub-cellular compartments including cell surface in different cell types (8)(9)(10)(11)(12)(13)(14)(15)(16)(17). However, only one transcript of HABP1 was detected from different type of tissues suggesting that no other isoform(s) of HABP1 exist in different cell types (16).
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 NH2terminal and two COOH-terminal α-helices and that the terminal α-helices have extensive intraas well as intermolecular contacts. It has been postulated that these terminal helices are critical for maintaining the trimeric assembly and protein-protein interactions (18). 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 (19). 4 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, relative role of the contribution of electrostatic interactions to protein stability, compared to that of hydrophobic interactions, has been the subject of long-standing query (20). 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 (21). Previously, we have shown that HABP1 exists in an expanded molten-globule like state at low ionic strength around alkaline pH due to 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 (22). 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 (23). 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 (24).
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 protein, and to further our understanding of the broad range of interactions of ions with proteins. Recently, we have reported the structural 5 flexibility of HABP1 under a wide range of ionic environments. At low ionic strength HABP1 exists in highly expanded and loosely held trimeric structure, whereas, the presence of salt induces compact trimeric structure (22). Thus 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.

Materials
EAH-Sepharose 4B, all empty XK, pre-packed Resource-Q, and Superose-6 HR10/30 column and the gel filtration calibration kit were from Amersham Bioscience. All other chemicals (unless otherwise mentioned) were of the highest purity grade available and obtained from Sigma Chemicals Co. (St. Louis, MO, USA) and highly purified HA-octasaccharide was a generous gift from Dr. Akira Asari of Seikagaku corporation Japan. MilliQ™ grade water was used for preparing all the solutions required for the study. Urea was purchased from Sigma and further purified by re-crystallisation before use.
Affinity purified HABP1 was further purified by size exclusion chromatography as described earlier (19). For all practical purposes the concentration of a known aliquot was determined in 20mM phosphate buffer pH 6.5 containing 6M Gdn-HCl by measuring the absorbance at 280 nm at 25 0 C on a cary100 Bio UV-Visible double beam spectrophotometer (Varian Inc. Australia) interfaced with peltier thermal controller. The molar extinction coefficient of denatured HABP1 was calculated and found to be 22190 at 280 nm, which corresponds to (Abs 0.1% ) 280nm = 0.932 where V e = elution volume; V o = void volume; V t = total bed volume of column (25).

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. The scans were taken between 250 nm to 200 nm at 10 0 C. The samples were prepared 4-24 hours before recording the spectra in the desired buffer and filtered through 0.22µ Millipore membranes. A rectangular cuvette of 1mm path length was used throughout the experiment. The concentration of protein was kept constant at 10µM. Data were recorded at a scan speed of 50 nm/min with a response time of one second 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 (5µM) was monitored by change in molar ellipticity at 222 nm with increasing temperature on JASCO 715 spectropolarimeter, equipped with a Peltier temperature controller (JASCO PTC-348 WI). The wavelength was decided on the basis of the by guest on March 24, 2020 http://www.jbc.org/ Downloaded from 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 Kh -1 and data were accumulated at 0.1K 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. USA). The protein samples on which DSC scans were performed as a function of salt concentration were prepared in 10mM phosphate buffer, pH 7.2.
The ionic concentration was varied using NaCl. Samples prepared 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 200mM). 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 X 5) at 4 0 C. The sample and the last dialysis buffer were filtered and degassed before being scanned in the calorimeter. 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 OD 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, A 3 ⇔ 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 (∆H v ), transition mid point temprature (T m , the temprature at half the peak area) and the transition peak area, which when divided by number of moles of protein in the cell, yields the calorimetric enthalpy (∆H c ). The ratio of ∆H c /∆H v yields the cooperativity of transition (26). All the scans at ionic concentrations above 100mM NaCl were found to be reversible

Hyaluronan binding assays
Microtitre 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 (22, 27), determine colorimetrically the level of binding of HABP1 to wells coated with HA. All washes were performed in standard assay buffer (SAB: 10mM phosphate buffer, pH 7.2 containing 150mM NaCl and 0.05% (v/v) Tween-20). Plastic Costar flat bottom high binding microtitre plates (EIA/RIA) were coated (in triplicates) overnight with 100 µl/well of 10 µg/ml of HA in 20mM carbonate buffer (Na 2 CO 3 -NaHCO 3 ) pH 9.6 at 4 0 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 0 C, followed by three washes. Finally, the respective wells were rinsed three times with buffers of different pH (10mM Na-acetate, pH 4.0-6.5 and Na-phosphate pH 6.5-8.5). 100 µl HABP1 or HABP1-biotin from a 50 µg/ml solution in 10mM sodium phosphate buffer (pH 6.5-8.5) or acetate buffer pH 4.0-6.5 containing 150mM of NaCl was added to each well and incubated for 4 h at room temperature or overnight at 4 0 C. Plates were washed three times and 100µl of a 1:10,000 dilution of polyclonal anti-HABP1 antibody (in SAB containing 1% BSA) was added and incubated for 90 min, followed by five washes. A 100 µl/well of 1:10,000 dilution of Horse Radish Peroxidase (HRP) conjugated goat anti rabbit IgG in SAB containing 1% BSA was added to each well and incubated for 1 hour at room temperature. For competitive inhibition assays biotinylated HABP1 at different pH was pre-incubated with varying concentration of unlabelled 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 hour incubation at 37 0 C and washed five times and finally rinsed with substrate buffer (100mM phosphate-citrate buffer pH 5.0). 100µl of 0.6 mg/ml solution of 2,2'-Azino-bis(3-ethylbenzthiazoline-6-Sulfonic acid), ABTS, in substrate buffer containing 3µl/ml of H 2 O 2 was added to each well and incubated for 10 minutes at 37 0 C for colour to develop. Absorbance at 405 nm was determined on a microtitre 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 LS-55 Luminescence Spectrometer (Perkin-Elmer Instruments) interfaced with Multi Temp III (Pharmacia Biotech) water circulator to keep the temperature constant. Binding of HA to HABP1 was monitored by adding small aliquots of HA-octa-or polysaccharide to 0.8µM HABP1 dissolved in 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 equivalent of condritin sulfate A of similar aliquots was run and used as F 0 . The fluorescence emission intensity was measured by using excitation wavelength of 295nm. For titration experiments, emission was monitored at 350nm. In every case excitation and emission slit-width of 2.5nm and 5nm respectively were used. Fluorescence data were corrected for excitation intensity. Five successive readings with an integration time of 10 second were recorded and averaged value was subtracted from F 0 to get ∆F and plotted against ligand concentration. ∆F max calculated from the plot was further used for the estimation of dissociation constant as reported earlier (28).

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 (29). For the folded trimeric HABP1 the coordinates were downloaded (PDB 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 due to 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 expanded structure around neutral and basic pH, size exclusion chromatography was carried out as a function of pH keeping ionic concentration constant. HABP1 showed a gradual decrease in elution volume suggesting a gradual increase in the hydrodynamic volume (V h ) 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 Stoke's radii. The pH induced structural compaction seems to be the result of reduction in total charges due to de-ionization of various polar amino acid residues, which are asymmetrically distributed on trimeric HABP1. The total charge present on HABP1 at different pH was calculated using ANTHERPROT and plotted against pH along with the Stoke's radius at corresponding pH (Fig. 1B). The total charge on trimeric HABP1 at pH 7.2 is -83 as compared to 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 Stoke's radius of 30.7±1.4 Å (n = 3) at pH 4.5 compared to the most expanded trimer, which has a Stoke's 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 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 (22). 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 towards different types of ligands.
In this study we have, therefore, scrutinized in detail, the conformational stability and folding of HABP1 in wide range of salt and pH using ultra-sensitive 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 Figure, Table-1  kcal-mol -1 at 1M of salt concentration. Since Tp is determined from the raw data and not from the fits using the two-state transition model, the agreement of ∆Hv(S) with ∆Hv (which is determined from application of the two-state model to the data) constitutes an independent confirmation that not only does the denaturation of HABP1 results in dissociation, but the application of the two-state transition model to the data is justified.
To test any kinetic effect on the thermal unfolding of HABP1, DSC melts were performed at scans rates from 10 Kh -1 to 90 Kh -1 in 10mM phosphate buffer containing 200mM of NaCl, pH 7.2. The scan rate dependence of the transition mid point temperature, Tm, of HABP1 is given in Table-2. The data suggest that the difference in Tm as a function of scan rate is insignificant, and fall well within the instrumental response hence, asserting to the fact that the thermodynamic model used for calculation is correct and scan rate independent.
It was observed that HABP1 DSC scans did not give any enthalpically relevant transition below 50mM salt concentration. It suggested that any enthalpically relevant structural formation in HABP1 starts at 50mM salt, below which it remains in a molten globule like state. The enthalpically relevant transition could not be observed in such cases although the molecule may have some degree of intact structure (34). The transition becomes apparent as soon as the ionic strength of the buffer is increased, suggesting a disorder to ordered transition of HABP1 with increasing salt concentration as observed in the case of some other proteins which exist as natively disordered structures (24, 34-38).
The thermal stability of HABP1 increases gradually with increasing ionic strength of the buffer. An appreciable transition is observed at around 50mM salt with a transition mid point temperature (T m ) of 304.52 ±1.65 K (n = 3), which increases linearly till 1M of salt (Table-3 (Fig. 5b).
The ratio ∆Hc/∆Hv ≈ 0.32 (±0.03) is constant at 100mM and higher salt concentrations (Table 3 and 4). This is indicative of the fact that three independent unfolding units exist in the HABP1, which may be identified as the three subunits of the molecule. Thus, the completely denatured HABP1 exists as a monomer, while in native state it exists as a trimer. The raw data were processed as described earlier and the representative curves are shown in Figure 6a along with the fit. The results of the fits of the data are given in Table-4. It is evident from the data that HABP1 is more stable under acidic pH conditions. The transition mid point temperature at pH 8.0 is 40.18±0.96 0 C with a very small value of ∆Hc of 6.38±0.68 kcal mol -1 compared to values of 60.57±0.65 0 C with a ∆Hc of 59.5 ± 0.983 kcal mol -1 at pH 5.0. This large shift in the transition mid point temperature is expected because of the fact that electrostatic repulsive force in HABP1 seems to be a major source of its destabilization. At lower pH the negatively charged amino acid residues are protonated allowing the protein to fold in a more compact fashion.

Thermodynamic Parameters using CD thermal melt
HABP1 exhibited a CD spectrum typical of α+β class of proteins with double minima at 220 nm and 210 nm in the pH range 4.5 -8.5 (Fig. 7a). HABP1, 7.0µM in trimer equivalents under different conditions of pH and ionic strength were heated at 60 Kh -1 and the molar ellipticity was monitored at 220 nm (a minimum of the difference spectra of native and thermally denatured HABP1) and plotted against temperature (Fig. 7b). Analysis of CD thermal melt data were made as described for another trimeric protein considering the unfolding upon dissociation (39). Thus the unfolding equilibrium can be expressed as Where, F 3 is the folded trimer and U is unfolded monomer. The equilibrium constant for above reaction can be written as: the raw data is presented in Figure 7 and the thermodynamic parameter obtained from the above fitting is presented in Table-

Change in accessible surface area (∆ASA) upon folding and trimer formation
For the accessible surface area (ASA) calculation the crystal structure of HABP1 (PDB ID 1P32) was used. Based on this structure, the Lee & Richards method is used for accessibility calculation (29) and the surface area of HABP1 trimer is presented in Table-6.
In order to obtain the accessible surface area change upon folding a monomeric unit of HABP1: ∆ASA monomer; folding = ASA free monomer; unfolded -ASA free monomer; folded -------(7) We calculated first the accessible surface of the unfolded protein ASA free monomer, unfolded . For this purpose, the accessible surface areas of the residues in a disordered/extended Ala-X-Ala tri-peptide were multiplied by the number of their occurrences in the polypeptide chain and then totaled (39). In order to calculate the accessible surface area change per monomer upon trimerization, the following equation was used: ∆ASA monomer; association = ASA free monomer; folded -ASA trimerized monomer -------- (8) We also intended to obtain the accessible surface area of the monomer that is associated with two other monomers in a trimer, ASA trimerized monomer . The results reveal that 88% of the surface is

Functional implication of pH induced structural changes in HABP1
HABP1 binds hyaluronan specifically with high affinity (7-9, 19, 22 there is either no binding or very little binding of HABP1 with HA below pH 4 or above pH 8.5 ( Fig. 8A). To see whether these interactions are specific at different pH, HA-coated plates were incubated with biotinylated HABP1 in the presence of unlabelled HABP1 at different pH and the bond HABP1-biotin was plotted with increasing concentration of unlabelled HABP1 (Fig. 8B).
The data suggested that these interactions are specific as unlabelled HABP1 compete biotin labeled HABP1 at different pH. There is an increase in HA binding with decreasing pH with maximum binding around pH 5.0. A significant increase in binding at pH 5.0 was observed presumably due to the more compact structure of HABP1. In addition to the compact structure of HABP1 at this pH, it also has very little negative charges. The electrostatic repulsion may be one of the factors limiting the HABP1 binding to HA in alkaline pH range.
In addition, the effect of pH on HA-HABP1 interactions was also analyzed by in-solution binding assay using fluorometric titration as described in methods section. Since each trimeric assembly of HABP1 has nine tryptophans spread all over the molecule therefore, change in fluorescence emission spectra was used as probe fort HA-HABP1 interactions. The fluorescence emission spectra of HABP1 show a maximum at 353nm when excited at 295nm, which upon binding to HA shift to 350nm with reduced emission intensity (Fig. 8C). An added advantage of this kind of binding assay was the fact that HA neither absorbs nor emits light in the wavelength region of 295-360 nm (curve 4, Fig. 8C). The relative fluorescence was plotted with increasing concentration of HA in disaccharide equivalents and presented in figure 8D. The data obtained from fluorimetric titration were analyzed using following standard equation derived elsewhere reports (42)(43). However, at acidic pH, de-ionization of these amino acids abolishes the electrostatic repulsion allowing the protein to fold in a more compact fashion. A similar effect was observed by the surface charge screening with counter ion (22). Thus our data suggest that, in general, both proton and counter ions affect the charge interactions at the surface of HABP1 in a similar fashion. These interpretations are supported by observations on several other proteins that have a high degree of repulsive surface charge density (38,(44)(45)(46).
We examined if the ionic strength and pH induced structural compaction has any effect on thermodynamic stability of HABP1. We observed that the HABP1 oligomer unfolds reversibly owing to a two-state unfolding behavior. In other words, the dissociation of oligomer to monomer and the unfolding of the protein occurs simultaneously. It is true with a number of oligomeric proteins where existence of folded monomer as they exist in the respective oligomer was not observed (39,42,47). From our studies on transition mid point temperature (Tm) as well as enthalpy of transition of HABP1, it is apparent that either increasing the ionic strength or decreasing the pH results in enhanced thermal stability. Such increased thermodynamic stability may be attributed to the screening of negative charges at higher ionic strength by counter ions or the de-ionization of polar amino acid residues under acidic pH. Consistency of thermodynamic parameters obtained (such as ∆Hc and Tm) using either the two-state or the model independent method also supports the fact that HABP1 unfolds via a two-state mechanism. The intrinsic stability of HABP1 as calculated from calorimetric scans was found to be 25kcal-mol -1 under standard conditions at pH 7.2. These observations suggest that the ordering effects either due to charge screening or by de-ionization of acidic amino acid residues results in a compact structure of HABP1 under these conditions. This observation is consistent with the fact that selective ion binding during protein folding causes the loss of entropy due to selective displacement of water.
Under such conditions contribution of enthalpy take predominance in protein folding process (43).
We have recently reported using ANS binding studies that HABP1 exists in a molten globule like state at low salt concentrations (0 and 20mM NaCl), the CD spectra in absence of salt shows a minima at 205nm suggesting the existence of random coil structure of HABP1 (22).
However addition of salt (25-300mM) facilitates structure formation in HABP1, which is reflected in the gradual increase in its molar ellipticity at 220nm and the capacity to bind HA (22). In the present study an enthalpically significant structure was detected only at 50mM and higher salt concentrations, although as shown previously, HA binding occurred even at lower salt concentrations such as 25mM where no peak was obtained in a DSC experiment. Therefore as proposed earlier, absence of enthalpically relevant transition may not necessarily be interpreted as a complete loss of structure or the existence of unfolded/denatured protein (34). We should also mention here that the DSC data collected at 50mM salt was not completely reversible which prevented us from thermodynamically evaluating the DSC profile.
HABP1 has large number uncompensated charges (48 negative and 25 positive) and lack of hydrophobic residues (hydropathicity of -0.7) making it a protein of low intrinsic hydrophobicity and high net charge around neutral pH. This protein has specific features such as anomalous migration in SDS-PAGE, high vulnerability to proteolysis and lack of secondary structure at low ion concentration (22). All these characteristics are reminiscent of "Intrinsically Unstructured Proteins" (24, 36, 48-51). Therefore, we analyzed the amino acid frequencies in HABP1 and compared them with that in ordered (Swis-PROT and globular proteins) and intrinsically unstructured proteins (24, 36, 48-51). According to reports, P, E/D, K, S and Q are termed as disorder promoting and W, Y, F, C, I, L and N are order promoting amino acid residues, whose relative frequencies in a polypeptide chains determine their extent of flexibility. In summary, the asymmetric charge distribution along the faces of HABP1 in association with cations and pH in the molecular environment play critical roles in maintaining its physical stability and three-dimensional structure at secondary, tertiary and quaternary levels in solution.
These factors govern its structural transitions from disordered to ordered and regulate its affinity towards HA. Thus, it seems logical to conclude that the electrostatic repulsion, together with an accessible hydrophobic core of HABP1 and the consequent dynamics due to its intrinsic flexibility imparts a repertoire of ligand recognition specificity and biological functions to this protein.