Tsinghua Science and Technology Tsinghua University, China ISSN: 1007-0212 Vol. 6, Num. 5, 2001, pp. 406-409

Tsinghua Science and Technology, December 2001, 6(5), pp. 406-409

Heat Treatment of Small Heat Shock Proteins a-Crystallin and  Hsp16.3:  Structural Changes vs. Chaperone-like Activity*

MAO Qilong  , KE Danxia  , CHANG Zengyi

Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China

* Supported by the National Natural Science Foundation of China (No.39700025) and the National Science Foundation for Outstanding Young Scientists in China (No.39725008)
 ** To whom correspondence should be addressed

Received: 2000-09-12

Code Number: ts01088

Abstract:

Both a-crystallin from bovine eye lens and Hsp16.3 from  Mycobacterium tuberculosis  are members of the small heat shock protein family.  They were preincubated at  100°C for 15 min and then cooled on ice immediately.  The chaperone-like activities of preheated proteins were measured at  37° using DTT-treated insulin B chains as substrates. Both preheated proteins exhibited greatly enhanced chaperone-like activities, accompanied with almost unchanged secondary structures and surface hydrophobicity but with a minor change in tertiary structures.  The dramatically enhanced chaperone-like activities of preheated a-crystallin and  Hsp16.3  may have resulted from the irreversible change in the tertiary structure as detected by near-UV CD spectra.

Key words: chaperone activity; heat treatment; small heat shock protein

Small  heat shock proteins are a conserved, abundant and ubiquitious protein family[1].  They have been detected in all types of organisms, including archaea, bacteria, and eukarya.  In spite of a relatively low overall homology between different members, they are grouped together based on (a) containing a conserved a-crystallin domain that is preceded by a variable N-terminal region and followed by an unconserved C-terminal sequence; (b) consisting of mainly b-sheets in the secondary structure; and (c) forming large multimeric complexes of 9 - 40 subunits (12 - 43 kDa in size)[1,2].

a-Crystallin, originally recognized as the major protein in the lens of vertebrate eyes, is believed to play an important role in maintaining the transparency and refractive properties of the eye lens[3]. It consists of two related 20-kDa subunits, aA- and aB-crystallin. aA- and aB-crystallins are also expressed in non-lenticular tissues including the heart, muscle, lung and kidney. aB-crystallin was found to be involved in a series of diseases, including cataracts, cardio- myopathy, desmin-related myopathy (DRM), Alexander's disease, Creutzfeldt-Jacob disease, Parkinson's disease, and Alzheimer's disease[4].

Hsp16.3  from Mycobacterium tuberculosis (namely 14- or 16-kDa antigen, 16-kDa  a-crystallin  homolog or Acr protein) was originally identified as an immunodominant antigen and a major membrane protein[5]. It is mainly synthesized at the stationary phase and strongly associated with the cell wall thickening under low oxygen conditions[6]. A previous study has shown that purified recombinant Hsp16.3 forms a trimer-of trimers structure[5].

Both  Hsp16.3  and a-crystallin have been found to share low homologous sequences with small heat shock proteins and to prevent thermal or chemical-induced proteins from aggregation in vitro, thus being molecular chaperones[5,7] . They are both able to remain clear even after preincubation at high temperatures over  85 °C . This study investigated the effect of preheat treatment (at  10 °C for 15 min) on the chaperone-like activities and the structural changes of Hsp16.3 and a-crystallin. The results clearly demonstrate that the minor differences in the tertiary structure between untreated and preheated proteins might result in greatly enhanced chaperone-like activities of Hsp16.3/a-crystallin.

Materials and Methods

1.1 Materials

Dithiothreitol (DTT), a-crystallin (from bovine eye lens) and insulin (from bovine pancreas) were all obtained from Sigma.   Hsp16.3  was overexpressed from pET-Hsp16.3 in  E. coli (DE3) host cells and purified to almost homogeneity according to methods previously described[5]. All other chemical reagents were local products of analytical grade.

1.2 Chaperone-like activity assay

The chaperone-like activities of Hsp16.3 and a-crystallin were measured by monitoring the turbidity of DTT-treated insulin B chains at  37°C. The aggregation of insulin B chains (0.4 mg/mL) were initiated by the addition of DTT (20 mmol·L^-1 ) and monitored at 360 nm by a UV-8500 spectrophotometer.  The reaction buffer was a 50 mmol·L^-1  sodium phosphate buffer (pH 7.0).

1.3 Circular dichroism measurements

Near-UV and far-UV circular dichroism (CD) spectra of a-crystallin and Hsp16.3 were measured on a Jasco J-715 spectropolarimeter at  37  °C . The spectra were recorded using 1 mm path length cells.  Protein concentrations of 4 mg/mL and 0.4 mg/mL were used for near-UV and far-UV CD spectra measurements, respectively.  All the spectra were the cumulative average of 15 repeat scans.

1.4 Fluorescence measurements

Fluorescence spectra were measured with a Hitachi F-4000 fluorescence spectrophotometer at  37  °C .  The excitation and emission bandpasses were both set at 5 nm. For ANS-binding fluorescence spectra, the excitation monochromator was set at 390 nm and the emission monochromator was scanned from 400 nm to 660 nm. The concentrations of proteins at  0.1 mg/mL  were used.  The final concentration of ANS was 100 mmol/L.

2 Results

2.1  Both preheated  Hsp16.3  and a-crystallin exhibited greatly enhanced chaperone-like activities

a-crystallin did not aggregate even after being preheated at  100°C . Hsp16.3 remained clear even after being preincubated at  100  °C . Figure 1 clearly show that nearly 20% of the aggregation of DTT-treated insulin B chains was suppressed by untreated a-crystallin with 50% suppressed by preheated a-crystallin, while nearly 50% of the aggregation was inhibited by untreated  Hsp16.3  protein with 80% inhibited by preheated  Hsp16.3 .  These results strongly indicate that the chaperone-like activities of  Hsp16.3  and a-crystallin were strongly enhanced after serious preheat treatment.

2.2  Both preheated Hsp16.3 and a-crystallin regained their secondary structures rather than their tertiary structures

The influences of the structural changes in the preheated  Hsp16.3  and a-crystallin on the dramatically increased chaperone-like activities were then investigated. Circular dichroism (CD) spectroscopy was used to investigate the structural differences between the untreated and the preheated proteins. As shown in Fig. 2, for both Hsp16.3 and a-crystallin, the far-UV CD spectra of untreated and preheated proteins were almost identical, which suggests that the preheated proteins almost regained their secondary structure.  However, results shown in Fig. 3 show that there were significant differences in the near-UV CD spectra between the untreated and preheated proteins (curves 1 and 2 for Hsp16.3, curves 3 and 4 for a-crystallin). These results indicate that both preheated a-crystallin and  Hsp16.3  might have suffered irreversible changes in their tertiary structures.

2.3     Both preheated Hsp16.3 and a-crystallin regained their hydrophobic properties

Hydrophobic interactions play an important role for molecular chaperones to bind to their denaturing substrates.  The hydrophobic surface of Hsp16.3 and a-crystallin were measured using ANS as a hydrophobic probe.  The results showed that the ANS-binding fluorescence spectra of untreated and preheated proteins almost always overlapped (curves 1 and 2 for Hsp16.3, curves 3 and 4 for a-crystallin, Fig. 4).  These results suggest that the ANS-binding capacity of Hsp16.3 and a-crystallin were recovered after the preheating treatments, indicating almost unchanged hydrophobic surfaces.

3 Discussion

There is considerable interest in the effect of heat treatment on the chaperone-like activities of sHsps in vitro. Previous studies revealed that the capability of sHsps (and other molecular chaperones) to bind to partially denatured substrate proteins was greatly enhanced at elevated temperatures[8-10].  Here two members of the sHsp family, Hsp16.3 from M. tuberculosis and a-crystallin from vertebrate eye lens, exhibited dramatically increased chaperone-like activities after heat treatment at  100  °C . Further investigation showed that the change in the chaperone activity of preheated sHsps did not accompany with change in secondary structures (measured by far-UV CD spectra) and surface hydrophobicity (detected by using ANS as probe). Nevertheless, near-UV CD spectroscopy studies revealed significant differences in their tertiary structures between the untreated and the preheated small heat shock proteins.

Raman et al. reported that the 8 mol/L urea-denatured a-crystallin refolded rapidly upon dilution and showed native-like structure[11].  Similarily, in this study Hsp16.3 was denatured in 8 mol/L urea/GdnHCl and renatured by dialysis.  No significant difference was found in the chaperone-like activity between the denatured and the renatured proteins. Also, the secondary, tertiary, and quaternary structures were almost unchanged (data not shown). Furthermore, Hsp16.3 preheated at temperatures lower than  60  °C  also exhibited almost unchanged chaperone-like activity and secondary, tertiary as well as quaternary structures (unpublished data). In light of these results, it is plausible to conclude that the irreversible changed tertiary structures of Hsp16.3 and a-crystallin after heat treatment may have caused an energetic barrier at high temperatures and were directly involved in the enhanced chaperone-like activity.

Tertiary structures and chaperone-like activity of Hsp16.3 changed only after being heated to over  62  °C .  This phenomenon has great significance in physiological terms.  Generally, temperatures on earth are not this extreme, so irreversible structural and functional changes of sHsps would not occur when the heat shock condition was removed.  Thus, life could avoid the formation of too tight a complex between sHsps and non-native substrate proteins (such as nascent peptides), which could lead to difficult renaturation of the substrates.

Acknowledgements

We thank Ms. Xiaonan Ding in the Department of Biological Sciences and Biotechnology at Tsinghua University for her technical help. 

References

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