Molecular Vision 2001; 7:228-233 <>
Received 1 August 2001 | Accepted 25 September 2001 | Published 3 October 2001

Heat-induced quaternary transitions in hetero- and homo-polymers of a-crystallin

M. R. Burgio,1,2 P. M. Bennett,3 J. F. Koretz1,2

1Center for Biophysics and 2Department of Biology, Science Center, Rensselaer Polytechnic Institute, Troy, NY; 3Randall Centre for Molecular Mechanisms of Cell Function, Guy's, King's and St. Thomas' School of Biomolecular Sciences, New Hunt's House, Guy's Campus, London

Correspondence to: M. R. Burgio, Center for Biophysics and Department of Biology, Science Center, Rensselaer Polytechnic Institute, 110 8th Street, Troy, NY, 12180; Phone: (518) 276-6492; FAX: (518) 276-2344; email:


Purpose: To compare the effects of heat incubation on the structure and function of native a-crystallin, urea denatured/renatured a-crystallin, and aA and aB-crystallin homo-polymers purified from bovine lenses.

Methods: Each of the a-crystallin samples were incubated for 1 h at temperatures ranging from 35 °C to 70 °C. After heat incubation structural perturbations in each of the samples were studied using non-denaturing gel electrophoresis, transmission electron microscopy (TEM) and far-UV circular dichroism. The chaperone-like activity of each of the heat-treated samples was measured using the DTT induced insulin aggregation assay.

Results: The native a-crystallin samples showed secondary structure perturbations, an increase in aggregate size and asymmetry, and an increase in chaperone-like activity after heat incubation above 50 °C. The other three sample types showed secondary structure perturbations beginning at lower incubation temperatures, and a progressive decrease in chaperone-like activity with exposure to increasing temperatures. TEM showed all samples formed large asymmetric high molecular weight aggregates after incubation at 65 °C.

Conclusions: The urea denaturation/renaturation of a-crystallin has been shown to result in the loss of a small amount of a-helix, but to have no effect on chaperone-like activity under standard test conditions. The present results indicate this lost a-helix may be responsible for the differential effects of heat incubation on the different forms of a-crystallin.


a-crystallin is the major protein component of the mammalian lens, where its solubility and transparency in the visible wavelength range, even at very high concentrations, provide the additional refractive power for image focus on the retina [1]. There are two a-crystallin isoforms, aA and aB, which are independent gene products [2]. Each is found individually and characteristically in specific tissues of the body, with the aB-crystallin being the more widely distributed of the two [3,4]. Only in the lens are both isoforms found together, where they form hetero-oligomers of about 12-14 nm diameter.

The sequences of the two isoforms are highly conserved, and have been shown to be homologous with small heat shock proteins from a variety of sources [5-7]. As a member of the small heat shock protein superfamily, a-crystallin would be expected to exhibit long-term stability and resistance to denaturation; this is a property of major functional importance in the lens. It has also been shown that a-crystallin can exhibit chaperone-like activity, preventing the super-aggregation and/or precipitation of partially denatured proteins, initially at 66 °C with g-crystallin [8], and later at 20 °C or 37 °C using several different model systems. It has been suggested that this is a major function in the lens, which would aid in maintenance of transparency through scavenging damaged and denatured material [9,10]. In addition, there is evidence that, at concentrations higher than those usually used in the laboratory, a-crystallin can act to chaperone itself [11].

Native a-crystallin has been shown to undergo a heat induced conformational change at elevated temperatures. Quaternary structure changes have been observed using FPLC [12], quasi-elastic light scattering [13], transmission electron microscopy [14], and non-denaturing gel electrophoresis [15]. Heat induced secondary structure perturbations have been observed using far-UV CD [12,16,17] and Fourier transform infrared spectroscopy [18]. These changes in structure have also been correlated with changes in the chaperone-like activity by a number of groups [12,15,17-19].

In this study we investigated the effects of heat incubation followed by cooling back to room temperature on bovine lens a-crystallins. The structural and functional changes of native a-, renatured a-, aA-, and aB-crystallin following heat incubation were compared and contrasted. Samples that had been urea denatured then renatured, including the aA- and aB-crystallin homo-aggregates, showed significant structural and functional differences from native a-crystallin samples after heat incubation under the same conditions. These differences appear to be correlated with the loss of a small amount of a-helix that occurs in the denaturation/renaturation process.


Bovine calf lenses from animals less than three months old were obtained from the Greenville Packing Company (Greenville, NY) and stored at -80 °C until use. a-Crystallin was prepared using a modification of the method of Thomson and Augusteyn [20]. Thawed lenses were hand homogenized in standard buffer (0.1 M NaCl, 50 mM Imidazole, 0.02% NaN3, pH 7.5), and centrifuged to clarify the suspension. The supernatant was then placed on a buffer-equilibrated Sepharose CL-6B column (Sigma), and eluted fractions corresponding to the a-crystallin peak were pooled and stored at 4 °C until use.

Homopolymers of aA- and aB-crystallin were purified from native a-crystallin using the method of Stevens and Augusteyn [21]. Gel filtration using Sephadex G-75 SF in 0.1 M glycine buffer (pH 2.5) was use to isolate aA- and aB-crystallin. Homopolymers of each isoform were denatured by dialysis against 9 M urea and subsequently renatured by dialysis against standard buffer. Renatured a-crystallin was prepared by denaturing and renaturing purified a-crystallin using the same method.

All samples were diluted with standard buffer to a concentration of 1.0-1.5 mg/ml. Protein concentrations were determined using absorbance spectrophotometry by subtracting the absorbance at 360 nm from the absorbance at 280 nm and dividing by the extinction coefficient (0.83). They were incubated for 1 h in a Neslab RTE-111 circulating water bath at the appropriate temperature and subsequently cooled back to room temperature before all measurements and assays were performed.

Non-denaturing composite gels were prepared using a modification of the method of Moulin, et al. [22]. The resolving gel was prepared to a final concentration of 3% acrylamide and 0.7% agarose (Fisher) in 375 mM Tris-HCl buffer, pH 8.8, in a Mini Protean II Gel Electrophoresis Cell (Bio-Rad) with frosted inner glass plates. The stacking gel was prepared to a final concentration of 2% acrylamide and 0.7% agarose in a 125 mM Tris-HCl buffer, pH 6.8. Each sample (4 mg) was added to 5X sample buffer (312.5 mM Tris-HCl, 50% glycerol, 0.05% bromophenol blue, pH 6.8) and run at 200 mV for 30 min at 4 °C, with 2 mg of Apoferritin (MW: 450,000) run as a standard. The gels were fixed and stained with a Coomassie brilliant blue R-250, citric acid, and methanol solution and destained in a citric acid-methanol solution.

Electron micrographs were taken of all the samples incubated at room temperature (RT), 50 °C, and 65 °C. Specimens were diluted 1:4 in PBS. A drop of the specimen was applied to carbon coated grids, washed with PBS, and negatively stained with 2% uranyl acetate. The specimens were observed in a JEOL 200CX transmission electron microscope at 100 kV. Micrographs were taken at a magnification of 50,000x.

Far UV-CD spectra of a-crystallin samples were taken at room temperature using a Jasco J-710 spectropolarimeter. Spectra were collected from 260 to 178 nm using a standard slit width of 1.0 nm and a scan speed of 200 nm/min. The reported spectra are the average of 32 scans, smoothed by Fast Fourier Transform noise reduction and normalized to 1 mg/ml. Samples of a-crystallin were dialyzed in 200 mM Phosphate buffer, pH 7.5, using Spectra/Por 3 molecular-porous membrane tubing (MWCO: 3500; Spectrum Medical Industries), subsequently adjusted to 1-1.5 mg/ml and placed into a 0.01 cm path length cylindrical cell before measurements were taken.

The chaperone-like activity of each sample was checked by measuring the DTT induced aggregation of insulin in the presence of the sample at room temperature. Sample, insulin, and DTT were mixed in 1 ml cuvettes to final concentrations of 0.375 mg/ml, 0.5 mg/ml, and 20 mM respectively with the DTT being added immediately before measurements were taken. The absorbance of each sample was then measured at 350 nm every 30 s over the course of 1 h with a Beckman DU-640 spectraphotometer.


Heat induced quaternary structure changes in the native a-, renatured native a-, and aA-crystallins were observed using non-denaturing gel electrophoresis, with a decrease in the mobility of a protein sample interpreted as an increase in aggregate size. Figure 1 show non-denaturing composite gels of a-crystallin samples heated to temperature for 1 h and then cooled back to room temperature. The native samples show an increase in molecular weight beginning at 50 °C, and becoming progressively more pronounced with increasing temperature to 70 °C. The renatured a- and aA-crystallin samples show an increase in molecular weight beginning at 45 °C and 50 °C respectively, with the molecular weight increases becoming more pronounced with increasing incubation temperature. The size changes in the renatured a- and aA-crystallin samples appeared to be much greater than those observed for the native a-crystallin samples at the same incubation temperatures. We were unable to resolve non-denaturing gels of aB-crystallin aggregates because aB-crystallin's high isoelectric point results in poor mobility through the gel matrix in the pH range used.

In order to visualize the quaternary structure change in all the a-crystallin samples, we used transmission electron microscopy. Figure 2 show electron micrographs of a-crystallin samples incubated at RT, 50 °C, and 65 °C, then allowed to cool to room temperature. The samples incubated at RT reveal all the a-crystallins to be similar in size and shape distribution. At 50 °C the native a-, renatured a-, and aA-crystallins show readily observable increases in size and heterogeneity over RT samples. The aB-crystallin particles incubated at 50 °C appear to maintain similar size and shape characteristics to those observed in the sample incubated at room temperature. The micrographs of samples incubated at 65 °C revealed that all of the a-crystallins formed large, asymmetric particles. In the native a-, aA-, and aB-crystallin samples, many elongated structures can be observed. Some of these elongated structures resemble beads on a string while others are much smoother.

Figure 3 shows heat induced secondary structure changes in a-crystallin samples as monitored using far UV-CD spectroscopy. Spectra were taken at room temperature for all crystallins following incubation at RT, 35, 40, 45, 50, 55, 60, 65, and 70 °C. Spectra were collected from 178 to 260 nm but are only presented from 190 to 260 nm. Noise in the signal below 190 nm made this area of the spectra unreliable. Native a-crystallin shows a progressive increase in negative ellipticity between 210 and 215 nm with increasing incubation temperature above 55 °C. Renatured a-, aA-, and aB-crystallin samples show an increase in negative ellipticity as well as an increase and slight blue shift in the positive ellipticity peak around 198 nm. These changes are first observed at 35, 40, and 55 °C in the renatured a-, aA-, and aB-crystallin samples respectively, and are progressive with increasing incubation temperature above the initial transition temperature. The magnitude of these changes are much less pronounced in the native samples than in the other a-crystallin samples. It should be noted that there is a significant difference in the magnitude of the positive peak at about 198 nm between the native and the renatured a-crystallin samples incubated at room temperature. Difference spectra between the native and renatured a-crystallin indicate there is a loss of a-helix in the protein during the denaturation/renaturation process. This result has been reported previously by this laboratory [11] and by others [23].

Figure 4 shows the inhibition of DTT induced insulin super aggregation by a-crystallin samples incubated at RT, 35, 45, 55, and 65 °C. These data show an increase in chaperone-like activity of native samples after incubation at temperatures 55 °C and above, with a clear division in effectiveness above and below the transition temperature. Conversely the renatured a-, aA-, and aB-crystallin samples all show a progressive decrease in chaperone-like activity with increasing incubation temperatures.


The data presented here indicate that heat incubation produces structural and functional changes in renatured a-, aA-, and aB-crystallins as well as in the native a-crystallin. The chain like structures observed in the 65 °C transmission electron micrographs may indicate the particles formed after heat incubation have a greater propensity to interact with each other than the native particles. These chains could also be artifacts of the negative staining process, however.

There are major differences in the heat-induced secondary structural transitions that separate the native a-crystallin from the other a-crystallins. Renatured a-crystallin exhibits more severe structural changes that are observable at lower incubation temperatures than the native a-crystallin. The effects of heat incubation on chaperone-like activity are also completely opposite, with the native a-crystallin samples showing an improvement and renatured a-crystallin samples showing a progressive decline in activity with increasing incubation temperature. Further the aA- and aB-crystallin samples, which have to be denatured/renatured after their purification, show results more similar to the renatured a-crystallin than the native.

Being able to compare the properties of aA- and aB-crystallin homo-aggregates with the properties of native a-crystallin is important in understanding why both isoforms exist together only in the mammalian lens. One study using recombinant aA- and aB-crystallin indicates they may be co-expressed in the lens to enhance the thermal stability of a-crystallin [24]. Our results indicate that the denaturation/renaturation process produces structural changes in the aA- and aB-crystallin that may alter their response to heat incubation. It is difficult to determine whether the observed response to heat incubation is the "native" response of the homo-aggregates or the result of their denaturation/renaturation.

Whether aA- andaB-crystallins expressed in and purified from E. coli exhibit a heat response more similar to native or renatured a-crystallin has yet to be determined. However, heat induced structural transitions have been studied in human recombinant aA- and aB-crystallins. These crystallins were shown to have perturbed secondary and tertiary structures by circular dichroism after incubation at 62 °C for 2 h. These changes in secondary structure were similar to the changes we observed in the purified bovine lens aA- and aB-crystallin. The effects of heat incubation on the chaperone-like activity and quaternary structure of the recombinant species were not investigated [25].

The major structural difference between the native and renatured a-crystallin appears to be the loss of a small amount of a-helix, as observed by far-UV CD. The homologous small heat shock protein HSP16.5 from Methanococcus jannaschii has a solved crystal structure that contains two short a-helices, one in the a-crystallin domain between b7 and b8 and another at the border between the c-terminal extension and the a-core region [26]. Sequence homology between aA-crystallin, aB-crystallin and HSP16.5 indicates these two a-helices may be present in the a-crystallins as well. The loss of one or both of these a-helices may be responsible for the differences in structure and activity between the native and renatured samples of a-crystallin.

Mutation studies have demonstrated the c-terminal end to be important for chaperone-like function [27,28]. It has also been shown that at 60-66 °C native a-, renatured a-, aA-, and aB-crystallins inhibit the super-aggregation of a-crystallin with about the same level of effectiveness [29,30], indicating that the loss of chaperone-like activity in renatured a-crystallins occurs during the cooling process. The a-helical segment in the c-terminal end could be important for controlling the orientation of the c-terminal extension. At high temperatures the c-terminal ends of all the a-crystallins are probably flexible and pointing out into solution. Without the a-helix to control its orientation, however, the c-terminal ends of the renatured samples could fall into one or more conformations during cooling to room temperature that are detrimental to the protein's chaperone-like activity.

Additionally, the c-terminal end of HSP 16.5 is involved in inter-subunit interactions. Two hydrophobic residues at the end of the c-terminal extension of HSP 16.5 appear to interact with a hydrophobic patch on the a-core region of an adjacent subunit. This promotes dimer-dimer interaction of HSP 16.5 and is important in the formation of its overall quaternary structure. The key residues involved in this interactions are well conserved among the small heat shock proteins, including in aA- and aB-crystallin. A loss of structure on the c-terminal of a-crystallin could destabilize this interaction and account for the greater propensity for structural changes among the renatured samples. It is possible there are other a-helices present in a-crystallin that are involved in intersubunit interactions and/or chaperone activity and are lost upon renaturation. An a-helix in the c-terminal extension, however, seems a likely candidate to explain the observed differences between the native and renatured a-crystallin samples.


The support of NIH Grant EY10011 for this work is gratefully acknowledged. The authors would also like to thank Professor John C. Salerno for constructive discussions.


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