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β-Crystallin association
Experimental Eye Research 79 (2004) 377–383 www.elsevier.com/locate/yexer
b-Crystallin association J.F. Hejtmancika,*, P.T. Wingfieldb, Y.V. Sergeeva a
National Eye Institute NIH, Ophthalmic Genetics and Visual Function Branch (OGVFB), Building 10, Room 10B10, 10 CENTER DRIVE MSC 1860, Bethesda, MD 20892, USA b National Institute of Arthritis and Musculoskeletal and Skin Diseases, NIH, Bethesda, MD, 20892, USA Received 2 March 2004; accepted 9 June 2004 Available online 29 July 2004
Abstract b-Crystallins are major protein constituents of the mammalian lens, where their stability and association into higher order complexes are critical for lens clarity and refraction. Dimerization is an initial step in formation of b-crystallin complexes. b-crystallin association into dimers is energetically highly favoured, but rapidly reversible under physiological conditions. b-Crystallin dimers can exchange monomers, probably through a transient and energetically unfavoured monomer intermediate state. As predicted by molecular modelling, the fraction of b-Crystallin present as dimers increases with increasing temperature, implying that b-crystallin association is entropically driven. q 2004 Elsevier Ltd. All rights reserved. Keywords: crystallin; lens; association; b-crystallin
1. Introduction The stability of crystallins and their ability to associate in appropriate intermolecular interactions are critical for transparency and refraction of the eye lens, where they make up over 90% of the water-soluble protein. In the vertebrate eye lens, three major classes of ubiquitous crystallins are found: a-, b- and g-crystallins (Wistow and Piatigorsky, 1988; Bloemendal and de Jong, 1991). The b- and g-crystallins share a common polypeptide chain fold, have conserved sequences, and together form a super-family of bg-crystallins (Lubsen et al., 1988). In contrast, the a-crystallins form a separate family of proteins related to the small heat-shock proteins (Caspers et al., 1995). Mutations in bg-crystallin genes can lead to nonspecific aggregation of crystallins resulting in cataract formation (Chambers and Russell, 1991). In addition, (Cartier et al., 1992; Litt et al., 1997; Kannabiran et al., 1998; Klopp et al., 1998; Graw, 1999; Heon et al., 1999; Ren et al., 2000) mutations which alter g-crystallin association without changing the polypeptide chain fold can also cause cataracts * Corresponding author. Dr J. F. Hejtmancik, National Eye Institute NIH, Ophthalmic Genetics and Visual Function Branch (OGVFB), Building 10, Room 10B10, 10 CENTER DRIVE MSC 1860, Bethesda, MD 20892, USA. E-mail address: [email protected] (J.F. Hejtmancik). 0014-4835/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. DOI:10.1016/j.exer.2004.06.011
(Stephan et al., 1999; Kmoch et al., 2000; Pande et al., 2000; Pande et al., 2001). bg-Crystallins comprise two domains connected by an 8 –10 amino acid inter-domain connecting peptide (Fig. 1). Each domain has an identical polypeptide chain fold, namely a b-sandwich of two anti-parallel b-sheets, known as a ‘Greek key’ motif (Wistow et al., 1983; Sergeev et al., 1988; White et al., 1989; Bax et al., 1990; Lapatto et al., 1991). The relative position of the two domains in gB- and bB2crystallins (gB and bB2) differ in crystallographic structures, having either ‘closed’ or ‘opened’ conformations, respectively (Mayr et al., 1994). The surface location of the inter-domain interfaces in both bovine gB and bB2 are very similar (Bax and Slingsby, 1989; Lapatto et al., 1991). However, in monomeric gB, intra-molecular domains form the interface, whereas, in homodimeric bB2, the interface consists of residues from the N-terminal domain of one monomer and residues from the C-terminal domain of the second monomer in a switched domain fashion. The physical properties of b-sheet residues forming the inter-domain interface in bA3 crystallin (bA3) are similar to those in gB and bB2, especially those residues showing significant accessibility changes upon formation of the interface (Sergeev and Hejtmancik, 1997). This suggests that the conserved layer structure of the interface may be important for dimer formation. The similarity of the domain docking sites may also explain subunit exchange between homo- and
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Fig. 1. Structure of gB-crystallin (left), showing intra-domain binding resulting in monomeric proteins and bB2-crystallin showing inter-domain binding resulting in dimeric protein.
heterodimers of bA3 and bB2 (Sergeev and Hejtmancik, 1997). The study of crystallin mutants in human cataracts and animal models provides insight into those regions of the bg-crystallin molecules important for protein association.
2. bA3- and bB2-Crystallin expression and size exclusion chromatography Recombinant bA3- and bB2-crystallins (rbA3 and rbB2) are faithfully expressed in a baculovirus system and appear to associate as homodimers or as heterodimers in
a biologically appropriate fashion (Hope et al., 1994a,b; Hejtmancik et al., 1997). The carboxy terminal sequence of murine bB2-crystallin is identical with those of human and cow and shares five of six amino acids with the chicken (Sergeev and Hejtmancik, 1997). The amino termini are N-acetylated, consistent with sequencing results of most b-crystallins isolated from bovine lenses (Berbers et al., 1984). The overall conformations of the rbA3 and rbB2 core domain structures as assessed by comparing their far-UV circular dichroism spectrum to mixed b-crystallins isolated from the mouse lens extremely similar. All show high b-sheet content, similar to
Fig. 2. Size exclusion chromatography of bA3- and bB2-crystallins on a Superdex 75 10/30 HR column showing migration consistent with molecular mass intermediate between monomer and dimer size. Molecular weight (filled circles) and Stokes radii (unfilled circles) calibration curves are shown. Molecular standards are bovine serum albumin, ovalbumin, carbonic anhydrase, chymotrypsinogen A and ribonuclease A.
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Table 1 Summary of molecular mass estimates from gel-filtration chromatography and sedimentation equilibrium centrifugation and Kd estimated from sedimentation equilibrium Protein
rbA3 rbB2
Calculated parameters, (Mr , (kDa))
25·1 23·3
Gel-filtration, (Mr , (kDa))
40 37
Sedimentation equilibrium
Mr (kDa)
Kd M
42·6 (^0·7) 37·6 (^0·2)
5·1 (^0·7) 4·6 (^1·1)
Data are taken from Sergeev et al. (2004).
previous results and expectations from the crystal structures of bg-crystallins. Size exclusion chromatography shows that rbA3-crystallin and rbB2-crystallin associate into dimers (Fig. 2, Table 1), consistent with results from other groups (Kroone et al., 1994; Trinkl et al., 1994; Werten et al., 1996). Molecular mass estimates for bB2- and bA3-crystallin from size exclusion chromatography (37 and 40 kDa, respectively) agree with the weight average estimates from centrifugation (37·6 and 42·6 kDa, respectively), but are smaller than the calculated dimer molecular masses (46·6 and 50·2 kDa respectively, see later). These intermediate molecular weight estimates, i.e. larger than a monomer and less than a dimer, are best explained by a reversible monomer – dimer association. Lower association strengths for mouse b-crystallins might contribute not only to the lower size estimates relative to calculated masses estimated by sieve chromatography, but also to the slight asymmetry of the chromatographic peaks seen in Fig. 2. The presence of a sharp leading edge followed by a broader trailing edge also suggests that these proteins are best described by a reversible monomer – dimer association (Winzor and Scheraga, 1963).
3. Subunit exchange Chromatography of equal amounts of rbA3- and rbB2crystallin under physiological conditions immediately after mixing results in two peaks corresponding to the elution positions of the individual purified proteins alone (Fig. 3). When the rbA3- and rbB2-crystallin fractions are allowed to stand at room temperature for 6 hr before chromatography, a single broad peak eluting at an intermediate position between those of pure bB2- and bA3-crystallin results. These results are similar to those obtained upon chromatography of equal amounts of rbA3 and rbB2 which had been disassociated by mixing in the presence of 6 M urea followed by re-association through removal of the urea by dilution. All the peaks, but especially the combined peak, show a slight asymmetry, with a sharp frontal region and a trailing back region typical of self-associating systems (Winzor and Scheraga, 1963). The observation that
Fig. 3. Size exclusion chromatography of bA3-and bB2-crystallins on a Superdex 75 column immediately after mixing (solid line) and after being allowed to equilibrate at room temperature for 6 hr (dotted line). Arrows show the elution points of purified rbA3- (solid) and rbB2 (open) crystallins. Data are taken from (Hejtmancik et al., 1997), where experimental details are provided.
an intermediate peak is formed upon re-association of the bB2- and bA3-crystallin suggests that heterodimers are present, and this is supported by the presence of both bA3and bB2-crystallin in the intermediate peak demonstrated by SDS-PAGE and Western blot analysis with antibodies to bA3- and bB2-crystallins. The formation of heterodimers between rbA3- and rbB2crystallin was confirmed by isoelectric focusing (Fig. 4). Isoelectric focusing of rbB2-crystallin alone gives a single band with a pI of 6·4, while isoelectric focusing of rbA3 gives a major band at pI 6·1 as well as several bands which presumably represent different oxidation states of this crystallin as has been reported previously for b-crystallins (Zigler, 1994). When rbA3- and rbB2-crystallin are mixed at room temperature and a physiological pH without denaturants, rbA3/rbB2 heterodimers are observed within
Fig. 4. Isoelectric focusing of bA3- and bB2-crystallin on a native Pharmacia PhastGel (pH 3–9), at various times after mixing equimolar amounts. Samples labeled A3 and B2 were allowed to stand at room temperature for 18 h before isoelectric focusing. Data are taken from (Hejtmancik et al., 1997), where experimental details are provided.
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Fig. 5. Equilibrium sedimentation of rbA3 and rbB2. Absorbance is shown in the lower panels, and residuals between the theoretical values predicted for a monomer–dimer equilibrium and the actual data points are shown in the upper panels. Data are taken from (Sergeev et al., 2004), where experimental details are provided.
10 min (Fig. 4). Isoelectric focusing of re-associated rbA3/ rbB2-crystallin heterodimers demonstrates that the new species has a pI of 6·25, intermediate between rbB2 (pI ¼ 6·4) and rbA3 (pI ¼ 6·1). The intermediate band increases in intensity as the rbB2- and rbA3-crystallin bands decrease. Equilibrium between rbA3 and rbB2 and the heterodimer is reached by 4– 6 hr, at which time approximately 50% of the rbA3- and rbB2-crystallin is in the heterodimer form. Berbers et al. had previously shown that it is possible to dissociate b-crystallin aggregates reversibly without denaturing their core domains by placing them in 6 M urea (Berbers et al., 1982). However, mouse bB2-crystallin and bA3-crystallin homodimers will exchange monomers to form heterodimers at room temperature in the absence of denaturing agents. This exchange is apparent within minutes and reaches equilibrium within 4 –6 hr. The distribution of the species at equilibrium indicates equivalent association or possibly a slight preferential association of acidic and basic monomers into heterodimers as previously reported by Slingsby and Bateman (Slingsby and Bateman, 1990). Precise quantitative analysis of the proportion of heterodimers was difficult due to the multiple bands resulting from isoelectric focusing of rbA3-crystallin.
4. Analytical ultracentrifugation studies Analytical ultracentrifugation provides a more precise characterization of b-crystallin association. To prevent aggregation artifacts TCEP (Pierce) was used in these studies. TCEP is a better reductant than DTT because it is resistant to oxidation by air, and it does not contribute to protein absorbance at 280 or 290 nm (as does oxidized DTT), which can interfere with baseline calculations. The molecular weights estimated by analytical ultracentrifugation, 42·6 KDa for rbA3 and 37·6 KDa for rbB2, are in good agreement with those estimated by gel filtration (Table 1). However, because the apparent
molecular weights estimated by both techniques were less than expected for a stable or tightly associated dimer, existence of a monomer– dimer equilibrium was suspected. With the centrifugation data, diagnostic graphs of the average molecular weight as a function of protein concentration were plotted. The estimated average molecular weight systematically increases with increasing protein concentration, approaching the calculated dimer molecular weight at the bottom of the analytical cell (data not shown). Hence, a monomer – dimer model data was applied to the data (Fig. 5). The goodness of the fit is shown by the residuals (differences between the data points and predictions of the model, top panels of Fig. 5) from the model scattering closely and randomly around the zero value. This is the case for both rbA3 and rbB2. Attempts to obtain better fits using other models, including monomer –dimer – trimer and monomer – dimer – tetramer models, all resulted in significantly worse x2 statistics. The equilibrium constant ðKd Þ for rbA3 was calculated to be 5·1 ^ 0·7 £ 1026 M , predicting that, for example, at 1 mg/ml, slightly over three quarters of the protein is dimeric. Similarly, the Kd for rbB2 was calculated to be 4·6 ^ 1·1, so that about 80% of the protein is predicted to be dimeric at 1 mg ml21. Table 2 summarizes the equilibrium constants determined for rbA3- and rbB2-crystallins as a function of Table 2 Summary of dissociation constants and DGa for rbA3- and rbB2-crystallins rbA3
rbB2
T8C
Kd (mM)
DGa (kcal/mol)
Kd (mM)
DGa (kcal/mol)
5 10 15 20 25 30 35
3·0 1·5 1·8 3·0 2·0 0·8 0·6
20·7 (^0·1) 21·1 (^0·1) 21·0 (^0·2) 20·7 (^0·2) 21·0 (^0·4) 21·5 (^0·7) 21·8 (^0·9)
6·9 2·9 1·9 2·5 3·3 2·6 2·2
20·3 20·8 21·0 20·9 20·8 20·9 21·0
(^0·6) (^0·4) (^0·7) (^1·2) (^1·2) (^0·9) (^0·8)
(^1·9) (^1·0) (^0·6) (^0·8) (^0·9) (^0·8) (^1·8)
(^0·2) (^0·2) (^0·2) (^0·2) (^0·2) (^0·2) (^0·6)
Data are taken from Sergeev et al. (2004), where experimental details are given.
J.F. Hejtmancik et al. / Experimental Eye Research 79 (2004) 377–383 Table 3 Summary of thermodynamic values for dimerization of rbA3- and rbB2crystallins Protein
DHa (kcal/mol)
2TDSa ; (kcal/mol)
DSa ; (e.u.)
DGa ; (kcal/mol)
rbA3 rbB2
7·9 (^2·8) 3·4 (^2·1)
28·9 (^2·8) 24·3 (^2·0)
30·4 (^9·4) 14·8 (^7·1)
21·0 (^4·0) 20·9 (^3·0)
Data are taken from Sergeev et al. (2004), where details are given.
temperature and also show the corresponding calculated Gibbs free energy changes. For both b-crystallins there is a reciprocal relationship between Kd and temperature; dimerization tends to increase (shown by a lowering of the Kd ) as the temperature increases. This is also reflected by the increasingly negative free energy of association, DGa ; at higher temperatures. While the values for 208 are slightly displaced from those seen in earlier studies (Hejtmancik et al., 1997; Sergeev et al., 2000, 2004), the trend between rbA3 and rbB2 and for each with increasing temperature is consistent. Measurement of dimer formation at different temperatures allows separation of the free energy of association into components resulting from changes in enthalpy and entropy (Table 3). For both rbA3 and rbB2, the negative values of DGa are derived from positive values of both enthalpy, DHa ; and entropy, DSa ; indicating that protein association is entropically driven ð2TDSa , 0 while DHa . 0) (Ross, 1981; Sackett, 1991). Experimentally, this is seen as dimerization increasing with temperature (Table 2).
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5. Implications for the lens The association of crystallins into higher order complexes is thought to be of critical importance for maintaining lens transparency (Hejtmancik and Piatigorsky, 2000). Both bA3- and bB2-crystallins appear to associate reversibly into homo- or hetero-dimers, which might then either dissociate into monomers or further associate into multimeric complexes. Exchange of crystallins between multimeric complexes seems likely to occur through monomeric intermediates, including a short-lived energetically unfavorable ‘open’ monomeric form (Fig. 6). For both bA3and bB2-crystallins the driving force for dimerization appears to be entropic, probably derived from differences in the hydration shells of the monomer and dimer (Sergeev et al., 2004). In the human lens the ratio of bB2- to bA3/A1-crystallin, as judged from mRNA levels assessed by microarray analysis, is about 2:1 in the epithelial cells and falls to about 4:3 in the fiber cells (M. Kantorow and J. Hawse, unpublished data). Thus, the molar ratio of bB2- and bA3-crystallin in the lens fiber cells is roughly similar to the equimolar ratio used in most of our association studies. However, many additional proteins, including both b- and a-crystallins, are present in the lens, each of which could interact with these proteins in ways that are difficult to predict. In addition, the environment within different parts of the lens changes substantially with lens development. Thus, the experimental system described here is best viewed as a highly simplified tool for observing specific aspects of
Fig. 6. Model of b-crystallin monomer and dimer structures co-existing in equilibrium in solution. Monomer and dimer forms coexist in rapid equilibrium in solution in near-physiological conditions. Dimers could theoretically further associate into higher order complexes. The linker connecting the N- and Cterminal domains adopts two possible conformations: either folded for the ‘closed’ form of the monomer or extended for the ‘opened’ form of the monomer and dimer. Below the structures is a diagram representing the calculated Gibbs free energy changes for the monomer–dimer states. In this model, transition from a ‘closed’ monomer to a dimer state would occur through an energetically unfavorable ‘open’ monomer state, and subunit exchange between multimeric complexes would occur through a monomeric intermediate.
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b-crystallin association rather than a working model of the lens, although it could certainly be expanded to address some of these questions in the future. These studies on the energetics of dimerization cannot provide information on the rates of subunit exchange of the various b-crystallin species. We have only rough estimates for this rate, derived from formation of bA3- and bB2heterodimers from their respective homodimers (Hejtmancik et al., 1997). At the high protein concentrations found in the lens (over 400 mg ml21), essentially all the b-crystallin would exist as higher order aggregates, consistent with studies of light scattering at high crystallin concentrations (Delaye and Gromiec, 1983). However, as protein concentration increases b-crystallin dimers should continue to dissociate as a first order reaction at the same rate, while the second order association rate should increase as the square of the concentration, so that even at high concentrations crystallin complexes should still undergo significant levels of monomer exchange rather than existing as static structures. This is particularly true since subunit exchange would be much more rapid at physiological temperatures than at room temperature, at which the exchange experiments were performed (Joss and Ralston, 1996).
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b-Crystallin association J.F. Hejtmancika,*, P.T. Wingfieldb, Y.V. Sergeeva a
National Eye Institute NIH, Ophthalmic Genetics and Visual Function Branch (OGVFB), Building 10, Room 10B10, 10 CENTER DRIVE MSC 1860, Bethesda, MD 20892, USA b National Institute of Arthritis and Musculoskeletal and Skin Diseases, NIH, Bethesda, MD, 20892, USA Received 2 March 2004; accepted 9 June 2004 Available online 29 July 2004
Abstract b-Crystallins are major protein constituents of the mammalian lens, where their stability and association into higher order complexes are critical for lens clarity and refraction. Dimerization is an initial step in formation of b-crystallin complexes. b-crystallin association into dimers is energetically highly favoured, but rapidly reversible under physiological conditions. b-Crystallin dimers can exchange monomers, probably through a transient and energetically unfavoured monomer intermediate state. As predicted by molecular modelling, the fraction of b-Crystallin present as dimers increases with increasing temperature, implying that b-crystallin association is entropically driven. q 2004 Elsevier Ltd. All rights reserved. Keywords: crystallin; lens; association; b-crystallin
1. Introduction The stability of crystallins and their ability to associate in appropriate intermolecular interactions are critical for transparency and refraction of the eye lens, where they make up over 90% of the water-soluble protein. In the vertebrate eye lens, three major classes of ubiquitous crystallins are found: a-, b- and g-crystallins (Wistow and Piatigorsky, 1988; Bloemendal and de Jong, 1991). The b- and g-crystallins share a common polypeptide chain fold, have conserved sequences, and together form a super-family of bg-crystallins (Lubsen et al., 1988). In contrast, the a-crystallins form a separate family of proteins related to the small heat-shock proteins (Caspers et al., 1995). Mutations in bg-crystallin genes can lead to nonspecific aggregation of crystallins resulting in cataract formation (Chambers and Russell, 1991). In addition, (Cartier et al., 1992; Litt et al., 1997; Kannabiran et al., 1998; Klopp et al., 1998; Graw, 1999; Heon et al., 1999; Ren et al., 2000) mutations which alter g-crystallin association without changing the polypeptide chain fold can also cause cataracts * Corresponding author. Dr J. F. Hejtmancik, National Eye Institute NIH, Ophthalmic Genetics and Visual Function Branch (OGVFB), Building 10, Room 10B10, 10 CENTER DRIVE MSC 1860, Bethesda, MD 20892, USA. E-mail address: [email protected] (J.F. Hejtmancik). 0014-4835/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. DOI:10.1016/j.exer.2004.06.011
(Stephan et al., 1999; Kmoch et al., 2000; Pande et al., 2000; Pande et al., 2001). bg-Crystallins comprise two domains connected by an 8 –10 amino acid inter-domain connecting peptide (Fig. 1). Each domain has an identical polypeptide chain fold, namely a b-sandwich of two anti-parallel b-sheets, known as a ‘Greek key’ motif (Wistow et al., 1983; Sergeev et al., 1988; White et al., 1989; Bax et al., 1990; Lapatto et al., 1991). The relative position of the two domains in gB- and bB2crystallins (gB and bB2) differ in crystallographic structures, having either ‘closed’ or ‘opened’ conformations, respectively (Mayr et al., 1994). The surface location of the inter-domain interfaces in both bovine gB and bB2 are very similar (Bax and Slingsby, 1989; Lapatto et al., 1991). However, in monomeric gB, intra-molecular domains form the interface, whereas, in homodimeric bB2, the interface consists of residues from the N-terminal domain of one monomer and residues from the C-terminal domain of the second monomer in a switched domain fashion. The physical properties of b-sheet residues forming the inter-domain interface in bA3 crystallin (bA3) are similar to those in gB and bB2, especially those residues showing significant accessibility changes upon formation of the interface (Sergeev and Hejtmancik, 1997). This suggests that the conserved layer structure of the interface may be important for dimer formation. The similarity of the domain docking sites may also explain subunit exchange between homo- and
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Fig. 1. Structure of gB-crystallin (left), showing intra-domain binding resulting in monomeric proteins and bB2-crystallin showing inter-domain binding resulting in dimeric protein.
heterodimers of bA3 and bB2 (Sergeev and Hejtmancik, 1997). The study of crystallin mutants in human cataracts and animal models provides insight into those regions of the bg-crystallin molecules important for protein association.
2. bA3- and bB2-Crystallin expression and size exclusion chromatography Recombinant bA3- and bB2-crystallins (rbA3 and rbB2) are faithfully expressed in a baculovirus system and appear to associate as homodimers or as heterodimers in
a biologically appropriate fashion (Hope et al., 1994a,b; Hejtmancik et al., 1997). The carboxy terminal sequence of murine bB2-crystallin is identical with those of human and cow and shares five of six amino acids with the chicken (Sergeev and Hejtmancik, 1997). The amino termini are N-acetylated, consistent with sequencing results of most b-crystallins isolated from bovine lenses (Berbers et al., 1984). The overall conformations of the rbA3 and rbB2 core domain structures as assessed by comparing their far-UV circular dichroism spectrum to mixed b-crystallins isolated from the mouse lens extremely similar. All show high b-sheet content, similar to
Fig. 2. Size exclusion chromatography of bA3- and bB2-crystallins on a Superdex 75 10/30 HR column showing migration consistent with molecular mass intermediate between monomer and dimer size. Molecular weight (filled circles) and Stokes radii (unfilled circles) calibration curves are shown. Molecular standards are bovine serum albumin, ovalbumin, carbonic anhydrase, chymotrypsinogen A and ribonuclease A.
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Table 1 Summary of molecular mass estimates from gel-filtration chromatography and sedimentation equilibrium centrifugation and Kd estimated from sedimentation equilibrium Protein
rbA3 rbB2
Calculated parameters, (Mr , (kDa))
25·1 23·3
Gel-filtration, (Mr , (kDa))
40 37
Sedimentation equilibrium
Mr (kDa)
Kd M
42·6 (^0·7) 37·6 (^0·2)
5·1 (^0·7) 4·6 (^1·1)
Data are taken from Sergeev et al. (2004).
previous results and expectations from the crystal structures of bg-crystallins. Size exclusion chromatography shows that rbA3-crystallin and rbB2-crystallin associate into dimers (Fig. 2, Table 1), consistent with results from other groups (Kroone et al., 1994; Trinkl et al., 1994; Werten et al., 1996). Molecular mass estimates for bB2- and bA3-crystallin from size exclusion chromatography (37 and 40 kDa, respectively) agree with the weight average estimates from centrifugation (37·6 and 42·6 kDa, respectively), but are smaller than the calculated dimer molecular masses (46·6 and 50·2 kDa respectively, see later). These intermediate molecular weight estimates, i.e. larger than a monomer and less than a dimer, are best explained by a reversible monomer – dimer association. Lower association strengths for mouse b-crystallins might contribute not only to the lower size estimates relative to calculated masses estimated by sieve chromatography, but also to the slight asymmetry of the chromatographic peaks seen in Fig. 2. The presence of a sharp leading edge followed by a broader trailing edge also suggests that these proteins are best described by a reversible monomer – dimer association (Winzor and Scheraga, 1963).
3. Subunit exchange Chromatography of equal amounts of rbA3- and rbB2crystallin under physiological conditions immediately after mixing results in two peaks corresponding to the elution positions of the individual purified proteins alone (Fig. 3). When the rbA3- and rbB2-crystallin fractions are allowed to stand at room temperature for 6 hr before chromatography, a single broad peak eluting at an intermediate position between those of pure bB2- and bA3-crystallin results. These results are similar to those obtained upon chromatography of equal amounts of rbA3 and rbB2 which had been disassociated by mixing in the presence of 6 M urea followed by re-association through removal of the urea by dilution. All the peaks, but especially the combined peak, show a slight asymmetry, with a sharp frontal region and a trailing back region typical of self-associating systems (Winzor and Scheraga, 1963). The observation that
Fig. 3. Size exclusion chromatography of bA3-and bB2-crystallins on a Superdex 75 column immediately after mixing (solid line) and after being allowed to equilibrate at room temperature for 6 hr (dotted line). Arrows show the elution points of purified rbA3- (solid) and rbB2 (open) crystallins. Data are taken from (Hejtmancik et al., 1997), where experimental details are provided.
an intermediate peak is formed upon re-association of the bB2- and bA3-crystallin suggests that heterodimers are present, and this is supported by the presence of both bA3and bB2-crystallin in the intermediate peak demonstrated by SDS-PAGE and Western blot analysis with antibodies to bA3- and bB2-crystallins. The formation of heterodimers between rbA3- and rbB2crystallin was confirmed by isoelectric focusing (Fig. 4). Isoelectric focusing of rbB2-crystallin alone gives a single band with a pI of 6·4, while isoelectric focusing of rbA3 gives a major band at pI 6·1 as well as several bands which presumably represent different oxidation states of this crystallin as has been reported previously for b-crystallins (Zigler, 1994). When rbA3- and rbB2-crystallin are mixed at room temperature and a physiological pH without denaturants, rbA3/rbB2 heterodimers are observed within
Fig. 4. Isoelectric focusing of bA3- and bB2-crystallin on a native Pharmacia PhastGel (pH 3–9), at various times after mixing equimolar amounts. Samples labeled A3 and B2 were allowed to stand at room temperature for 18 h before isoelectric focusing. Data are taken from (Hejtmancik et al., 1997), where experimental details are provided.
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Fig. 5. Equilibrium sedimentation of rbA3 and rbB2. Absorbance is shown in the lower panels, and residuals between the theoretical values predicted for a monomer–dimer equilibrium and the actual data points are shown in the upper panels. Data are taken from (Sergeev et al., 2004), where experimental details are provided.
10 min (Fig. 4). Isoelectric focusing of re-associated rbA3/ rbB2-crystallin heterodimers demonstrates that the new species has a pI of 6·25, intermediate between rbB2 (pI ¼ 6·4) and rbA3 (pI ¼ 6·1). The intermediate band increases in intensity as the rbB2- and rbA3-crystallin bands decrease. Equilibrium between rbA3 and rbB2 and the heterodimer is reached by 4– 6 hr, at which time approximately 50% of the rbA3- and rbB2-crystallin is in the heterodimer form. Berbers et al. had previously shown that it is possible to dissociate b-crystallin aggregates reversibly without denaturing their core domains by placing them in 6 M urea (Berbers et al., 1982). However, mouse bB2-crystallin and bA3-crystallin homodimers will exchange monomers to form heterodimers at room temperature in the absence of denaturing agents. This exchange is apparent within minutes and reaches equilibrium within 4 –6 hr. The distribution of the species at equilibrium indicates equivalent association or possibly a slight preferential association of acidic and basic monomers into heterodimers as previously reported by Slingsby and Bateman (Slingsby and Bateman, 1990). Precise quantitative analysis of the proportion of heterodimers was difficult due to the multiple bands resulting from isoelectric focusing of rbA3-crystallin.
4. Analytical ultracentrifugation studies Analytical ultracentrifugation provides a more precise characterization of b-crystallin association. To prevent aggregation artifacts TCEP (Pierce) was used in these studies. TCEP is a better reductant than DTT because it is resistant to oxidation by air, and it does not contribute to protein absorbance at 280 or 290 nm (as does oxidized DTT), which can interfere with baseline calculations. The molecular weights estimated by analytical ultracentrifugation, 42·6 KDa for rbA3 and 37·6 KDa for rbB2, are in good agreement with those estimated by gel filtration (Table 1). However, because the apparent
molecular weights estimated by both techniques were less than expected for a stable or tightly associated dimer, existence of a monomer– dimer equilibrium was suspected. With the centrifugation data, diagnostic graphs of the average molecular weight as a function of protein concentration were plotted. The estimated average molecular weight systematically increases with increasing protein concentration, approaching the calculated dimer molecular weight at the bottom of the analytical cell (data not shown). Hence, a monomer – dimer model data was applied to the data (Fig. 5). The goodness of the fit is shown by the residuals (differences between the data points and predictions of the model, top panels of Fig. 5) from the model scattering closely and randomly around the zero value. This is the case for both rbA3 and rbB2. Attempts to obtain better fits using other models, including monomer –dimer – trimer and monomer – dimer – tetramer models, all resulted in significantly worse x2 statistics. The equilibrium constant ðKd Þ for rbA3 was calculated to be 5·1 ^ 0·7 £ 1026 M , predicting that, for example, at 1 mg/ml, slightly over three quarters of the protein is dimeric. Similarly, the Kd for rbB2 was calculated to be 4·6 ^ 1·1, so that about 80% of the protein is predicted to be dimeric at 1 mg ml21. Table 2 summarizes the equilibrium constants determined for rbA3- and rbB2-crystallins as a function of Table 2 Summary of dissociation constants and DGa for rbA3- and rbB2-crystallins rbA3
rbB2
T8C
Kd (mM)
DGa (kcal/mol)
Kd (mM)
DGa (kcal/mol)
5 10 15 20 25 30 35
3·0 1·5 1·8 3·0 2·0 0·8 0·6
20·7 (^0·1) 21·1 (^0·1) 21·0 (^0·2) 20·7 (^0·2) 21·0 (^0·4) 21·5 (^0·7) 21·8 (^0·9)
6·9 2·9 1·9 2·5 3·3 2·6 2·2
20·3 20·8 21·0 20·9 20·8 20·9 21·0
(^0·6) (^0·4) (^0·7) (^1·2) (^1·2) (^0·9) (^0·8)
(^1·9) (^1·0) (^0·6) (^0·8) (^0·9) (^0·8) (^1·8)
(^0·2) (^0·2) (^0·2) (^0·2) (^0·2) (^0·2) (^0·6)
Data are taken from Sergeev et al. (2004), where experimental details are given.
J.F. Hejtmancik et al. / Experimental Eye Research 79 (2004) 377–383 Table 3 Summary of thermodynamic values for dimerization of rbA3- and rbB2crystallins Protein
DHa (kcal/mol)
2TDSa ; (kcal/mol)
DSa ; (e.u.)
DGa ; (kcal/mol)
rbA3 rbB2
7·9 (^2·8) 3·4 (^2·1)
28·9 (^2·8) 24·3 (^2·0)
30·4 (^9·4) 14·8 (^7·1)
21·0 (^4·0) 20·9 (^3·0)
Data are taken from Sergeev et al. (2004), where details are given.
temperature and also show the corresponding calculated Gibbs free energy changes. For both b-crystallins there is a reciprocal relationship between Kd and temperature; dimerization tends to increase (shown by a lowering of the Kd ) as the temperature increases. This is also reflected by the increasingly negative free energy of association, DGa ; at higher temperatures. While the values for 208 are slightly displaced from those seen in earlier studies (Hejtmancik et al., 1997; Sergeev et al., 2000, 2004), the trend between rbA3 and rbB2 and for each with increasing temperature is consistent. Measurement of dimer formation at different temperatures allows separation of the free energy of association into components resulting from changes in enthalpy and entropy (Table 3). For both rbA3 and rbB2, the negative values of DGa are derived from positive values of both enthalpy, DHa ; and entropy, DSa ; indicating that protein association is entropically driven ð2TDSa , 0 while DHa . 0) (Ross, 1981; Sackett, 1991). Experimentally, this is seen as dimerization increasing with temperature (Table 2).
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5. Implications for the lens The association of crystallins into higher order complexes is thought to be of critical importance for maintaining lens transparency (Hejtmancik and Piatigorsky, 2000). Both bA3- and bB2-crystallins appear to associate reversibly into homo- or hetero-dimers, which might then either dissociate into monomers or further associate into multimeric complexes. Exchange of crystallins between multimeric complexes seems likely to occur through monomeric intermediates, including a short-lived energetically unfavorable ‘open’ monomeric form (Fig. 6). For both bA3and bB2-crystallins the driving force for dimerization appears to be entropic, probably derived from differences in the hydration shells of the monomer and dimer (Sergeev et al., 2004). In the human lens the ratio of bB2- to bA3/A1-crystallin, as judged from mRNA levels assessed by microarray analysis, is about 2:1 in the epithelial cells and falls to about 4:3 in the fiber cells (M. Kantorow and J. Hawse, unpublished data). Thus, the molar ratio of bB2- and bA3-crystallin in the lens fiber cells is roughly similar to the equimolar ratio used in most of our association studies. However, many additional proteins, including both b- and a-crystallins, are present in the lens, each of which could interact with these proteins in ways that are difficult to predict. In addition, the environment within different parts of the lens changes substantially with lens development. Thus, the experimental system described here is best viewed as a highly simplified tool for observing specific aspects of
Fig. 6. Model of b-crystallin monomer and dimer structures co-existing in equilibrium in solution. Monomer and dimer forms coexist in rapid equilibrium in solution in near-physiological conditions. Dimers could theoretically further associate into higher order complexes. The linker connecting the N- and Cterminal domains adopts two possible conformations: either folded for the ‘closed’ form of the monomer or extended for the ‘opened’ form of the monomer and dimer. Below the structures is a diagram representing the calculated Gibbs free energy changes for the monomer–dimer states. In this model, transition from a ‘closed’ monomer to a dimer state would occur through an energetically unfavorable ‘open’ monomer state, and subunit exchange between multimeric complexes would occur through a monomeric intermediate.
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b-crystallin association rather than a working model of the lens, although it could certainly be expanded to address some of these questions in the future. These studies on the energetics of dimerization cannot provide information on the rates of subunit exchange of the various b-crystallin species. We have only rough estimates for this rate, derived from formation of bA3- and bB2heterodimers from their respective homodimers (Hejtmancik et al., 1997). At the high protein concentrations found in the lens (over 400 mg ml21), essentially all the b-crystallin would exist as higher order aggregates, consistent with studies of light scattering at high crystallin concentrations (Delaye and Gromiec, 1983). However, as protein concentration increases b-crystallin dimers should continue to dissociate as a first order reaction at the same rate, while the second order association rate should increase as the square of the concentration, so that even at high concentrations crystallin complexes should still undergo significant levels of monomer exchange rather than existing as static structures. This is particularly true since subunit exchange would be much more rapid at physiological temperatures than at room temperature, at which the exchange experiments were performed (Joss and Ralston, 1996).
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