Heat‐induced changes in the conformation of α‐ and β‐crystalline: Unique thermal stability of α‐crystallin

Maiti, Motilal; Kono, Masahiro; Chakrabarti, Buddhapriya

doi:10.1016/0014-5793(88)80295-3

Cited by 96 publications

(51 citation statements)

References 25 publications

(13 reference statements)

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“…The observed change in ellipticity at 217 nm is larger at higher protein and salt concentrations. A similar increase in ellipticity has been observed for the native heteroaggregate of ␣-crystallin (39,40). However the reports differ in the extent of this increase.…”

Section: Resultssupporting

confidence: 70%

“…These differences can be explained on the basis of our results on individual subunits. In the earlier reports, ␣-crystallin used was isolated from the lens cortex (39) or the whole lens (40). Since the composition of ␣-crystallin varies from the outer cortex to the nucleus, with ␣B-crystallin being higher in the cortex, differential composition of ␣-crystallin could lead to the observed differences.…”

Section: Resultsmentioning

confidence: 99%

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Differential Temperature-dependent Chaperone-like Activity of αA- and αB-crystallin Homoaggregates

Datta

Rao

1999

Journal of Biological Chemistry

103

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␣-Crystallin, a heteromultimeric protein made up of ␣A-and ␣B-crystallins, functions as a molecular chaperone in preventing the aggregation of proteins. We have shown earlier that structural perturbation of ␣-crystallin can enhance its chaperone-like activity severalfold. The two subunits of ␣-crystallin have extensive sequence homology and individually display chaperonelike activity. We have investigated the chaperone-like activity of ␣A-and ␣B-crystallin homoaggregates against thermal and nonthermal modes of aggregation. We find that, against a nonthermal mode of aggregation, ␣B-crystallin shows significant protective ability even at subphysiological temperatures, at which ␣A-crystallin or heteromultimeric ␣-crystallin exhibit very little chaperone-like activity. Interestingly, differences in the protective ability of these homoaggregates against the thermal aggregation of ␤ L -crystallin is negligible. To investigate this differential behavior, we have monitored the temperature-dependent structural changes in both the proteins using fluorescence and circular dichroism spectroscopy. Intrinsic tryptophan fluorescence quenching by acrylamide shows that the tryptophans in ␣B-crystallin are more accessible than the lone tryptophan in ␣A-crystallin even at 25°C. Protein-bound 8-anilinonaphthalene-1-sulfonate fluorescence demonstrates the higher solvent accessibility of hydrophobic surfaces on ␣B-crystallin. Circular dichroism studies show some tertiary structural changes in ␣A-crystallin above 50°C. ␣B-crystallin, on the other hand, shows significant alteration of tertiary structure by 45°C. Our study demonstrates that despite a high degree of sequence homology and their generally accepted structural similarity, ␣B-crystallin is much more sensitive to temperaturedependent structural perturbation than ␣A-or ␣-crystallin and shows differences in its chaperone-like properties. These differences appear to be relevant to temperature-dependent enhancement of chaperone-like activity of ␣-crystallin and indicate different roles for the two proteins both in ␣-crystallin heteroaggregate and as separate proteins under stress conditions. ␣-Crystallin is a major protein of the mammalian lens and constitutes as much as 50% of its dry weight. Studies over the past few years have shown that ␣-crystallin is expressed in several nonlenticular tissues such as heart, brain, and kidney, and its expression is enhanced severalfold during stress and disease conditions (1-6). ␣-Crystallin is shown to have homology with small heat shock proteins (7-10). Horwitz (11) shows that ␣-crystallin can prevent the thermal aggregation of ␤-and ␥-crystallins and a few other proteins like a molecular chaperone. Demonstration of chaperone-like activity of ␣-crystallin has provided an excellent opportunity to investigate the mechanistic aspects of chaperone function in general and the role of ␣-crystallin under stress conditions in particular. It is possible that, in the lens, ␣-crystallin may chaperone the formation of the transparent and appropriately ...

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Section: Resultssupporting

confidence: 70%

Section: Resultsmentioning

confidence: 99%

Differential Temperature-dependent Chaperone-like Activity of αA- and αB-crystallin Homoaggregates

Datta

Rao

1999

Journal of Biological Chemistry

103

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“…5A) is a remarkable observation because the wild-type and the other mutants are very thermosoluble. In addition, native ␣-crystallin and mutants of ␣A-crystallin that were analyzed previously, including the elongated ␣A ins -polypeptide, do not precipitate up to at least 75°C (30,44,52). The CD and NMR experiments do not provide any evidence that precipitation of ALWKG mutant at around 55°C is preceded by precocious unfolding of domain structures.…”

Section: Resultsmentioning

confidence: 69%

“…However, none of these quaternary structure models is based on substantial experimental evidence. The subunits mainly comprise ␤-sheets (29,30), and several biophysical studies endorse a tertiary structure composed of two compact domains and an unstructured C-terminal region (31)(32)(33)(34). Indeed, as shown by NMR spectroscopy, ␣-crystallin (35) and hsp25 (36) have unstructured, flexible, and solvent-exposed C-terminal extensions.…”

mentioning

confidence: 99%

Immobilization of the C-terminal Extension of Bovine αA-Crystallin Reduces Chaperone-like Activity

Smulders

Carver

Lindner

et al. 1996

Journal of Biological Chemistry

123

110

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␣-Crystallins occur as multimeric complexes, which are able to suppress precipitation of unfolding proteins. Although the mechanism of this chaperone-like activity is unknown, the affinity of ␣-crystallin for aggregationprone proteins is probably based on hydrophobic interactions. ␣-Crystallins expose a considerable hydrophobic surface to solution, but nevertheless they are very stable and highly soluble. An explanation for this paradox may be that ␣-crystallin subunits have a polar and unstructured C-terminal extension that functions as a sort of solubilizer. In this paper we have described five ␣A-crystallins in which charged and hydrophobic residues were inserted in the C-terminal extension. Introduction of lysine, arginine, and aspartate does not substantially influence chaperone-like activity. In contrast, introduction of a hydrophobic tryptophan greatly diminishes functional activity. CD experiments indicate that this mutant has a normal secondary structure and fluorescence measurements show that the inserted tryptophan is located in a polar environment. However, NMR spectroscopy clearly demonstrates that the presence of the tryptophan residue dramatically reduces the flexibility of the C-terminal extension. Furthermore, the introduction of this tryptophan results in a considerably decreased thermostability of the protein. We conclude that changing the polarity of the C-terminal extension of ␣A-crystallin by insertion of a highly hydrophobic residue can seriously disturb structural and functional integrity.The composition of the vertebrate eye lens is dominated by a group of structural proteins known as crystallins. The largest of these, ␣-crystallin, is a dynamic multimeric complex composed of two types of homologous subunits, ␣A-and ␣B-crystallin (1). A few years ago, it was discovered that these subunits are also constitutively expressed in various non-lenticular tissues, suggesting that their function is more than merely structural (2-5). In addition, increased expression of ␣B-crystallin has been observed in a variety of neurodegenerative disorders such as multiple sclerosis (6), Alexander's disease (7,8), and Alzheimer's disease (9, 10). Although the physiological significance of this extralenticular expression is unknown, it certainly relates to the fact that ␣-crystallins belong to the family of small heat shock proteins (hsp) 1 (11, 12). Among the shared features of ␣-crystallins and other small hsp are the conferring of thermotolerance (13,14), interaction with actin (15, 16), phosphorylation (17, 18), and intracellular relocalization upon stress (19,20). Furthermore, in vitro experiments have shown that ␣A-and ␣B-crystallin as well as hsp25 can act as molecular chaperones by suppressing aggregation of denaturing proteins (21-23). Unfortunately, the mechanism of this functional activity is unclear because the three-dimensional structure of ␣-crystallin is unknown.Native ␣-crystallins occur as polydisperse particles with an average molecular mass of about 800 kDa. Understanding the multimeric arra...

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“…Even thermal denaturation led to divergent results. No thermal transition below 100°C could be found by means of circular dichroism [4]. In contrast, a thermal transition at about 60°C was observed by scanning microcalorimetry, however, the enthalpy change remained divergent [5 7].…”

Section: Introductionmentioning

confidence: 99%

The conformational stability of α‐crystallin is rather low: Calorimetric results

Gesierich

Pfeil

1996

FEBS Letters

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The eye lens protein and chaperonin, ct-crystallin, was studied by differential scanning microcalorimetry, spectroscopy and size exclusion chromatography. The thermal transition of c¢-crystallin proceeds at Ttrs --59.8 _+ 0.6°C with an enthalpy change of AH=336+9 kJ per mol subunit. Disagreement between previous AH values could be attributed to a side reaction that leads, depending on the scan rate, to the formation of a nonproductive folding form. The conformational stability of acrystallin is rather low (AG= 24+5 kJ/mol of subunit). The minimal cooperative unit of ot-crystallin is the monomeric subunit.

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Heat‐induced changes in the conformation of α‐ and β‐crystalline: Unique thermal stability of α‐crystallin

Cited by 96 publications

References 25 publications

Differential Temperature-dependent Chaperone-like Activity of αA- and αB-crystallin Homoaggregates

Differential Temperature-dependent Chaperone-like Activity of αA- and αB-crystallin Homoaggregates

Immobilization of the C-terminal Extension of Bovine αA-Crystallin Reduces Chaperone-like Activity

The conformational stability of α‐crystallin is rather low: Calorimetric results

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