Interaction of ATP and lens alpha crystallin characterized by equilibrium binding studies and intrinsic tryptophan fluorescence spectroscopy

Palmisano, David V.; Groth-Vasselli, Barbara; Farnsworth, Patricia N.; Reddy, M. Chandra Sekhar

doi:10.1016/0167-4838(94)00176-h

Cited by 45 publications

(48 citation statements)

References 27 publications

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“…An ATP-binding model seems to explain the results. Equilibrium studies of the ATP binding with ␣-crystallin reported a binding constant of 8 ϫ 10 3 M Ϫ1 at 37°C (24 (22). Binding of ATP would neutralize the charge cluster pulling them inside the globular structure, which may reorient some of these hydrophobic residues to the surface of ␣-crystallin to trigger the association with the substrate.…”

Section: Discussionmentioning

confidence: 99%

“…Thus, ␣-crystallin has been reported to have autophosphorylation activity by some workers (29,30), whereas others failed detect significant ATPase activity (23). Some spectroscopic (16,24) and proteolytic (31) studies suggested interaction between ATP and ␣-crystallin with concomitant conformational changes, but the nature of the changes and its functional implications are far from clear. According to a totally different viewpoint existing in the literature, most sHSPs having the conserved ␣-crystallin domain have no dependence on ATP for their functions (22).…”

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confidence: 99%

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Role of ATP on the Interaction of α-Crystallin with Its Substrates and Its Implications for the Molecular Chaperone Function

Biswas

Das

2004

Journal of Biological Chemistry

110

160

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ATP plays a significant role in the function of molecular chaperones of the large heat shock protein families. However, its role in the functions of chaperones of the small heat shock protein families is not understood very well. We report here a study on the role of ATP on the structure and function of the major eye lens chaperone ␣-crystallin. Our in vitro study shows that at physiological temperature, ATP induces the association of ␣-crystallin with substrate proteins. The association process is reversible and low affinity in nature with unit binding stoichiometry. 4,4 -Dianilino-1,1 -binaphthyl-5,5-disulfonic acid, dipotassium salt, binding studies show that ATP induces the exposure of additional hydrophobic sites on ␣-crystallin, but no appreciable enhancement of the same was observed for the substrate protein ␥-crystallin or carbonic anhydrase. An equilibrium unfolding study reveals that ATP at 3 mM concentration stabilizes the ␣-crystallin structure by 4.5 kJ/mol. The compactness induced by ATP makes it more resistant to tryptic cleavage. ATP-induced association of chaperone ␣-crystallin with substrate enhanced its aggregation prevention ability and also enhanced the refolding yield of lactate dehydrogenase from the unfolded state. Our results suggest that the binding of ATP to ␣-crystallin and not its hydrolysis is required for all these effects, as replacement of ATP by its nonhydrolyzable analogue adenosine-5 -O-(3-thiotriphosphate), tetralithium salt, reproduced all the results faithfully. The implication of the ATP-induced reversible protein-protein association at physiological temperatures on the functional role of ␣-crystallin in vivo is discussed.␣-Crystallin is the major protein component of the vertebrate eye lens and is composed of two subunits, ␣A and ␣B, 20 kDa each having 57% sequence homology between them. Both subunits associate to form a large oligomer with an average molecular mass of 800 kDa. It is a key member of the small heat shock protein (sHSP) 1 superfamily having a conserved core

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Role of ATP on the Interaction of α-Crystallin with Its Substrates and Its Implications for the Molecular Chaperone Function

Biswas

Das

2004

Journal of Biological Chemistry

110

160

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“…However, -crystallin does bind ATP [21]. The functional consequences of this property are not understood although there have been claims that ATP binding and hydrolysis enhance the chaperone ability of -crystallin [22].…”

Section: Parallels Between the Structure And Mechanism Of Chaperone Amentioning

confidence: 99%

Small Heat‐shock Proteins and Clusterin: Intra‐ and Extracellular Molecular Chaperones with a Common Mechanism of Action and Function?

et al. 2003

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Small heat-shock proteins (sHsps) and clusterin are molecular chaperones that share many functional similarities despite their lack of significant sequence similarity. These functional similarities, and some differences, are discussed. sHsps are ubiquitous intracellular proteins whereas clusterin is generally found extracellularly. Both chaperones potently prevent the amorphous aggregation and precipitation of target proteins under stress conditions such as elevated temperature, reduction and oxidation. In doing so, they act on the slow off-folding protein pathway. The conformational dynamism and aggregated state of both proteins may be crucial for their chaperone function. Subunit exchange is likely to be important in regulating chaperone action; the dissociated form of the protein is probably the chaperone-active species rather than the aggregated state. They both exert their chaperone action without the need for hydrolysis of ATP and have little ability to refold target proteins. Increased expression of sHsp and clusterin accompanies a range of diseases, e.g. Alzheimer's, Creutzfeldt-Jakob and Parkinson's diseases, that arise from protein misfolding and deposition of highly structured protein aggregates known as amyloid fibrils. The interaction of sHsps and clusterin with fibril-forming species is discussed along with their ability to prevent fibril formation, probably via utilization of their chaperone ability. Disciplines Life Sciences | Physical Sciences and Mathematics | Social and Behavioral Sciences Publication DetailsThis article was originally published as Carver, JA, Rekas, A, Thorn, DC and Wilson, MR, Small heat-shock proteins and clusterin: intra-and extracellular molecular chaperones with a common mechanism of action and function, IUBMB Life, 55, 2003, 661-668 AbstractSmall heat-shock proteins (sHsps) and clusterin are molecular chaperones that share many functional similarities despite their lack of significant sequence similarity. These functional similarities, and some differences, are discussed. sHsps are ubiquitous intracellular proteins whereas clusterin is generally found extracellularly. Both chaperones potently prevent the amorphous aggregation and precipitation of target proteins under stress conditions such as elevated temperature, reduction and oxidation. In doing so, they act on the slow off-folding protein pathway. The conformational dynamism and aggregated state of both proteins may be crucial for their chaperone function. Subunit exchange is likely to be important in regulating chaperone action; the dissociated form of the protein is probably the chaperone-active species rather than the aggregated state. They both exert their chaperone action without the need for hydrolysis of ATP and have little ability to refold target proteins. Increased expression of sHsp and clusterin accompanies a range of diseases, e.g. Alzheimer's, Creutzfeldt-Jakob and Parkinson's diseases, that arise from protein misfolding and deposition of highly structured protein aggregates known as amyloid f...

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“…Several publications have supported a structural and functional relationship between ATP and the sHSPs. Equilibrium binding studies, intrinsic tryptophan fluorescence, and 31 P nuclear magnetic resonance spectroscopy demonstrated an interaction between ATP and total bovine ␣-crystallin (36,37). Quenching of intrinsic fluorescence in the presence of ATP has been reported for both total ␣-crystallin and for ␣B-crystallin, suggesting ATP-induced conformational changes (31,37).…”

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confidence: 99%

ATP and the Core “α-Crystallin” Domain of the Small Heat-shock Protein αB-crystallin

Muchowski¹,

Hays²,

Yates³

et al. 1999

Journal of Biological Chemistry

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Electrospray ionization mass spectrometry (ESI-LC/ MS) of tryptic digests of human ␣B-crystallin in the presence and absence of ATP identified four residues located within the core "␣-crystallin" domain, Lys 82 , Lys 103 , Arg 116 , and Arg 123 , that were shielded from the action of trypsin in the presence of ATP. In control experiments, chymotrypsin was used in place of trypsin. The chymotryptic fragments of human ␣B-crystallin produced in the presence and absence of ATP were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS , located near or within the core ␣-crystallin domain, were shielded from the action of chymotrypsin in the presence of ATP. Chemically similar analogs of ATP were less protective than ATP against proteolysis by trypsin or chymotrypsin. ATP had no effect on the enzymatic activity of trypsin and the K m for trypsin was 0.031 mM in the presence of ATP and 0.029 mM in the absence of ATP. The results demonstrated an ATP-dependent structural modification in the core ␣-crystallin domain conserved in nearly all identified small heatshock proteins that act as molecular chaperones.␣-Crystallin is a protein that is expressed at high levels in all vertebrate eye lenses and is composed of two M r 20,000 subunits, ␣A and ␣B, which associate to form high molecular mass complexes with an average molecular mass of 800 kDa (1-3). In 1992 it was demonstrated that ␣A-and ␣B-crystallin function as molecular chaperones by suppressing the aggregation of other polypeptides in vitro (4). A large body of experimental evidence confirmed that ␣B-crystallin is a prototype for the small heat-shock protein (sHSP) 1 superfamily, which is characterized by the presence of a conserved core "␣-crystallin" domain that spans 80 -100 amino acids in the C-terminal domain (2, 5, 6). sHSPs are expressed ubiquitously in nature (5, 6), are up-regulated in several neurodegenerative diseases that include multiple sclerosis (7), Alzheimer's (8, 9), and Creutzfeldt-Jacob disease (10), and are mutated in inherited diseases including a desmin-related myopathy and cataract (11,12). Initially thought to be lens-specific structural proteins, both ␣A-and ␣B-crystallin have been identified in a variety of non-lens tissues (13,14). While ␣-crystallins and sHSPs function as molecular chaperones both in vitro and in vivo (15-35) a function for the conserved core ␣-crystallin domain has yet to be demonstrated. In this report trypsin proteolysis identified regions in the core domain that may be involved with the effect of ATP on chaperone activity of sHSPs.In contrast to the large heat-shock protein families HSP60, HSP70, HSP90, and HSP100, the effect of ATP on the molecular chaperone functions of sHSPs has only been reported recently (31). Several publications have supported a structural and functional relationship between ATP and the sHSPs. Equilibrium binding studies, intrinsic tryptophan fluorescence, and 31 P nuclear magnetic resonance spectroscopy demonstrated an interaction between ATP and total bovine ␣-crystallin (36...

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Interaction of ATP and lens alpha crystallin characterized by equilibrium binding studies and intrinsic tryptophan fluorescence spectroscopy

Cited by 45 publications

References 27 publications

Role of ATP on the Interaction of α-Crystallin with Its Substrates and Its Implications for the Molecular Chaperone Function

Role of ATP on the Interaction of α-Crystallin with Its Substrates and Its Implications for the Molecular Chaperone Function

Small Heat‐shock Proteins and Clusterin: Intra‐ and Extracellular Molecular Chaperones with a Common Mechanism of Action and Function?

ATP and the Core “α-Crystallin” Domain of the Small Heat-shock Protein αB-crystallin

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