βB1-Crystallin: Thermodynamic Profiles of Molecular Interactions

Background β-Crystallins are structural proteins maintaining eye lens transparency and opacification. Previous work demonstrated that dimerization of both βA3 and βB2 crystallins (βA3 and βB2) involves endothermic enthalpy of association (∼8 kcal/mol) mediated by hydrophobic interactions. Methodology/Principal Findings Thermodynamic profiles of the associations of dimeric βA3 and βB1 and tetrameric βB1/βA3 were measured using sedimentation equilibrium. The homo- and heteromolecular associations of βB1 crystallin are dominated by exothermic enthalpy (−13.3 and −24.5 kcal/mol, respectively). Conclusions/Significance Global thermodynamics of βB1 interactions suggest a role in the formation of stable protein complexes in the lens via specific van der Waals contacts, hydrogen bonds and salt bridges whereas those β-crystallins which associate by predominately hydrophobic forces participate in a weaker protein associations.


Introduction
The transparency and refraction of the mammalian lens are dependent on the molecular associations of the crystalline proteins. These proteins form a bc-crystallin superfamily those members are similar in structure and contain Greek key motifs. The b-crystallins constitute the major proportion of the lens proteins and seven subtypes have been identified, four of which are acidic (bA1, bA2, bA3, and bA4), and three basic (bB1, bB2, and bB3). Most bcrystallins are monomer-dimer systems [1][2][3] but some, for example, bA2 and bA4, have low intrinsic solubility and exhibit only weak self-associations [4,5]. The in vivo heteromolecular interactions of acidic with basic b-crystallins can circumvent solubility issues [4][5][6]. Under physiological conditions only bcrystallins are known to associate into dimers, tetramers, and higher-order oligomers [3,[7][8][9]. Although the interactions of the bcrystallins have been well studied [4,10], the detailed molecular mechanisms of most associations remain obscure. We previously demonstrated that the self-associations of both bA3 and bB2 are mediated by hydrophobic interactions [11]. Here we describe the energetics controlling bB1 dimerization and tetramer formation with bA3. bB1 and bA3 crystallins are major component in the human lens [12,13] and both recently were found in non-lens tissues including the retina [14][15][16]. We show that the molecular associations of bB1 crystallin are dominated by exothermic enthalpy. This indicates that bB1 plays an important role in the formation of stable protein complexes mediated by specific (stronger) interactions stabilized by van der Waals forces, hydrogen bonds, and salt bridges.

Results
Protein molecular weights and associative behavior bB1 (monomer, 28 kDa) and bA3 (monomer, 25 kDa) elute during size-exclusion chromatography (SEC) with apparent molecular weights of 35 and 42 kDa, respectively, intermediate between that of monomers and dimers (Fig. S1). Sedimentation equilibrium analysis indicated the proteins are reversible monomer-dimer systems [2]. When equimolar amounts of bB1 and bA3 were mixed and incubated for 24 hrs at 20uC, a single symmetrical peak with apparent molecular weight of ,70 kDa was observed (Fig. S1). Sedimentation equilibrium analysis of the mixture indicated a weight-average molecular weight of 95 kDa close to that predicted for a weak heterotetramer (M r 106 kDa). The bestfit model for the equilibrium data was a heterodimerheterotetramer system with a K d of 8.78 mM (Table S1). This result is very similar to that measured for the analogous interaction with murine b-crystallins [17].

bB1 dimerization energetics
To gain information on the energetics of the bB1 interactions, the temperature dependence of association was determined by sedimentation equilibrium over the range 5-30uC. The equilibrium profiles are shown in Fig. S2 Panel A. bB1 is a reversible monomer-dimer system with the equilibrium position shifting towards monomeric protein at higher temperatures as indicated by increased K d values (Table S1). For example, the dimer fraction of bB1 (total 18 mM) decreased from 81% at 10uC to 50% at 30uC. The calculated free energy DG a values for dimerization also decrease with increase in temperature. This is due to the negative contributions from both enthalpy DH a and entropy DS a ( Table 1) which were derived from plots (non-linear) of In K d and C o versus 1/T (Fig. 1A, B).

bA3 dimerization energetics
To be consistent with our previous work with murine bA3 crystallin [11], we analyzed the association behavior of the human protein (sequence identity with murine protein 95%). As expected, human bA3 formed tighter dimers (lower K d 's) at higher temperatures (Table S1) [11]. Thus, in contrast to bB1, the dimer fraction of bA3 increases with temperature (58% at 5uC and 80% at 30uC). There was a linear dependence of the lnK 0 /C 0 on 1000/T using the change in heat capacity DC p zero or non zero values. (Table S1, and Fig. 1A, B). Hence, the self-association of human bA3 is characterized by positive enthalpy DH a and entropy DS a at zero and nonzero DC p ( Table 1), which is similar to our previously published analysis of the murine protein [2].

bB1/bA3 tetramerization energetics
The bB1/bA3 complex is best modeled as a reversible heterodimer-heterotetramer system over the temperature range studied (5-30uC) with a tendency to form weaker tetramers at is constrained to be 0, resulting in a linear function; Panel B: DC p is not constrained and has a nonzero value. Panel C: temperature dependence of Gibbs free energy gained in formation of bB1/bA3. DDG d (bB1/bA3) is defined as a difference between Gibbs free energy changes of tetrameric bB1/ bA3 and that of individual components (bB1 and bA3). Concentrations for bB1, bA3, and bB1/bA3 crystallins were each 0.5 mg/ml. doi:10.1371/journal.pone.0029227.g001 higher temperature (Fig. S1, Fig. S2 Panel B, and Table S1).
The relationship between the logarithm of the K d and the reciprocal of the absolute temperature demonstrated by the van't Hoff plot is not linear (Fig. 1A, B). When DDG a is plotted against the reciprocal of the absolute temperature (Fig. 1C) DDG a values negatively increase with increasing temperature. Tetramer formation is, therefore, associated with exothermic enthalpy DH a and entropy DS a ( Table 1) and, analogous to bB1 dimerization, DG a decreases with increasing temperature (Fig. 1C).

Discussion
We have previously shown that both murine bA3 and bB2 are monomer -dimer systems with a tendency to form tighter dimers at higher temperatures [11]. Moreover, the self-association of these crystallins, characterized by positive enthalpy and entropy changes, is entropically driven and mediated by hydrophobic interactions. These endothermic associations (DH.0) are dominated by hydrophobic effects entropically driven by water. Here we have shown a similar energetic profile for human bA3 (Table S1) indicating that nonpolar regions of the protein, previously accessible to solvent in the isolated subunits, become buried upon dimer formation [18].
The self-association of bB1 energetically differs from that of bA3 and bB2 in that its dimers are destabilized at higher temperatures. The thermodynamic profile ( Table 1) indicates that both the dimerization of bB1 and formation of the bB1/bA3 complex are exothermic processes (DH,0). With tetrameric bB1/ bA3 formation, decreasing negative values of DG confirm that the complex is less stable at higher temperatures. Large exothermic enthalpy change DH a = 229.668.1 kcal/mol and negative entropy DS a = 297.9627.4 e.u. are accompanied with a negative heat capacity change DC p = 21.661 cal/deg mol. Thus, the profile suggests that tetramer formation is controlled by enthalpy and interactions between the subunits are mediated by van der Waals interactions, hydrogen bonds, and salt bridges [18].
A summary overview of the homo-and hetero-associations of bcrystallins is presented in Fig. 2. bB1 mediates protein interactions using van der Waals contacts and hydrogen bonds which suggest that contact involve complementary shapes of protein surfaces with a higher biological specificity [19]. In contrast the formation of dimeric bA3 and bB2, are driven by hydrophobic forces which are usually less specific. In these associations, hydrophobic residues at the surface interfaces become excluded from direct contact with surrounding water molecules.
Currently we cannot completely rule out the possibility that heterotetramers are formed by the association of homodimers rather than heterodimers but in either case it does not affect our analyses and conclusions. However; the precise mechanism of bB1/bA3 association appears to involve the association of heterodimers [2]. The kinetics, equilibrium position and balance of so-called ''close'' and ''open'' conformational isomers could be affected by interactions of core domains or with the N-terminal extensions of bA3 and bB1 [1,17]. The dimerization of bB1 may 'induce' a conformational shift which favors interaction with bA3. Such a model would explain why both, homo-and heteroassociation of bB1 are driven by enthalpy.
Previous dynamic light scattering analysis demonstrated that in fetal calf lenses soluble crystallins form a broad distribution of protein complexes with sizes of 8-14 nm with band c-crystallins at the leading edge of this distribution [20]. Proteomic analysis has demonstrated that large molecular complexes are often built around a stable core of proteins, which are expanded thorough the attachment of weakly bound exchangeable peripheral proteins often stabilized by dynamic transient interactions [21,22]. These exchangeable components could be for example, so-called 'weak' dimers which have relatively high K d 's [23]. The protein interfaces in 'weak' dimers are loosely packed and more hydrophobic than in average protein transient complexes.
bB2 and bA3 crystallins could have a propensity to be components of a peripheral protein network. The less specific and more transient nature of their interactions would give these crystallins more flexibility for binding. On the other hand, the more specific and stronger exothermic interactions involving bB1 make this crystallin more suitable for formation of the stable core of the lens proteins.
Although we have described the interactions of the crystallins as being mediated by either the weaker hydrophobic or the more specific van der Waals interactions, both may occur but on average one dominates energetically. It is known, for example, from antibody -antigen interactions, that initial contacts may involve hydrophobic interactions via interface aromatic residues followed by more specific and tighter H-bonding and salt bridges [18,24].
In conclusion, the global thermodynamics of bB1 interactions indicate that they contribute in more stable protein complexes in the lens via specific van der Waals contacts, hydrogen bonds and salt bridges whereas those b-crystallins which associate by predominately hydrophobic forces are more likely to participate in a weaker protein associations.

Materials and Methods
Expression, purification and association of bB1and bA3crystallins Wild type recombinant murine bB1 and human bA3 were expressed as soluble proteins in E.coli and purified as previously described by ion-exchange and size-exclusion chromatographies [2,17]. Murine bB1 was used which has a 95% sequence similarity (.80% sequence identity) with the human protein and is, therefore, a reasonable surrogate. The purified proteins were dialyzed overnight against Buffer A (50 mM Tris-HCl, 1 mM EDTA, 0.15 M NaCl, 1 mM TCEP, at pH 7.5) at 4uC. Protein concentrations were estimated from A 280/260 (Beckman Coulter DU650, CA) and adjusted to 0.5 mg/ml. For the formation of complexes between bB1 and bA3, an equimolar mixture (,20 mM each) was incubated at room temperature for 24 h. Aliquots (250 ml) were loaded on an analytical grade Superdex 75 HR10/ 30 column, precalibrated with standards (bovine serum albumin, 67 kDa, ovalbumin, 43 kDa, chymotrypsinogen, 25 kDa, and ribonuclease A, 13.7 kDa; Sigma, MO). Samples were eluted at a flow rate of 0.5 ml/min and 0.5 ml fractions were collected.

Analytical Ultracentrifugation
A Beckman Optima XL-I analytical ultracentrifuge with absorption optics, an An-60 Ti rotor, and standard double-sector centerpiece cells were used for sedimentation equilibrium experiments. All analyses were performed using duplicate protein samples. Data were collected after 16 hours at 18,500 rpm at 20uC. The baselines were established by overspeeding at 45,000 rpm for another 4 hours. Equilibrium profiles were analyzed by standard Optima XL-I Origin-based data analysis software. Solvent density was estimated as previously described [25]. Monomeric molecular weights M r and molar extinction coefficients were used for calculation of dissociation constants K d . The M r and K d were measured in duplicate and averaged. Equilibrium data was collected with 5uC temperature increments for the ranges: 5-25uC and 15-30uC.

Energetics of monomer-dimer and dimer-tetramer equilibrium
The temperature dependence of association was examined for homodimer and hetero-tetramer associations between 5-30uC, using a previously described [11] equation: where K d is the dissociation constant, measured by AUC; C o is the molar concentration of protein (mM); R is universal gas constant; T is temperature (K); and DC p , DHu, and DSu are changes in protein heat capacity, enthalpy and entropy, respectively. The experimental data were fitted in two ways: first; where DC p was constrained to be zero and second; where DC p was nonzero. The effect of protein concentration was excluded from the analysis by normalization to the protein molar concentration C o (See formula 1). Figure S1 Size-exclusion chromatography profiles obtained for individual proteins and the bB1/bA3 complex. Top panel: bA3, bB1, and bB2 self-associate in a reversible manner to form dimers. The homo-associations of bA3 and bB2 exhibit endothermic enthalpy and are driven by entropy as a result of hydrophobic interactions between protein molecules. In contrast, the self-association of bB1 is driven by exothermic enthalpy due to van der Waals interactions and hydrogen bonds at the dimer interface. Bottom panel: The bB1/bA3 complex is likely formed by the association of hetero-dimers but we cannot rule out that it is formed from homodimers. Similar to that of bB1 alone, the formation of the tetramer is driven by exothermic enthalpy. Structures of bB1 and bB2 were obtained from the protein database RCSB (files: 1 oki and 1 blb, respectively). Closed and open structures of bA3 were modeled as described earlier [1]. From our results we cannot say which monomer conformation exists within the hetero-tetramer. However, the majority of known crystal structures of b-crystallins (3 of 4) are of the closed monomer type suggesting this is the most stable conformation. Therefore, the structure of the hypothetical tetrameric bB1/bA3 complex was generated using the crystal packing of bB1 crystallin as a template (PDB file: 1 oki). doi:10.1371/journal.pone.0029227.g002

Supporting Information
The chromatographic profile obtained immediately after mixing of equimolar amounts of bB1 and bA3 is shown in green and following 24 hours of incubation, by the red line.  Author Contributions