Characteristics of super αA‐crystallin, a product of in vitro exon shuffling

Rijk, Anke F. van; Hurk, M.J.J. van den; Renkema, Wouter; Boelens, Wilbert C.; Jong, Wilfried W. de; Bloemendal, H.

doi:10.1016/s0014-5793(00)01908-6

Cited by 6 publications

(5 citation statements)

References 32 publications

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“…However, this is not free of controversy (29,47). In the present study we see a complete loss in the chaperone-like function of mutant ␣A-crystallin at and slightly above physiological temperatures despite an increase in hydrophobicity.…”

Section: F71g Mutant Of ␣A-crystallinmentioning

confidence: 40%

“…They concluded that there is no correlation between surface hydrophobicity and chaperone-like activity. Experiments with super-␣A-crystallin have indicated that the disappearance of chaperone-like activity may be independent of hydrophobicity (47). Further, Reddy et al (18) have shown recently that hydrophobicity is not the sole determinant of chaperone-like activity in ␣-crystallin.…”

Section: F71g Mutant Of ␣A-crystallinmentioning

confidence: 99%

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Phe71 Is Essential for Chaperone-like Function in αA-crystallin

Santhoshkumar¹,

Sharma²

2001

Journal of Biological Chemistry

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Experiments with mini-␣A-crystallin (KFVIFLD-VKHFSPEDLTVK) showed that Phe 71 in ␣A-crystallin could be essential for the chaperone-like action of the protein (Sharma, K. K., Kumar, R. S., Kumar, G. S., and Quinn, P. T. (2000) J. Biol. Chem. 275, 3767-3771). In the present study we replaced Phe 71 in rat ␣A-crystallin with Gly by site-directed mutagenesis and then compared the structural and functional properties of the mutant protein with the wild-type protein. There were no differences in molecular size or intrinsic tryptophan fluorescence between the proteins. However, 1,1-bi(4-anilino)naphthalene-5,5-disulfonic acid interaction indicated a higher hydrophobicity for the mutant protein.Both wild-type and mutant proteins displayed similar secondary structure during far UV CD experiments. Near UV CD signal showed a slight difference in the tertiary structure around the 285-295 region for the two proteins. The mutant protein was totally inactive in suppressing the aggregation of reduced insulin, heat-denatured citrate synthase, and alcohol dehydrogenase. However, a marginal suppression of ␤ L -crystallin aggregation was observed when mutant ␣A-crystallin was included. These results suggest that Phe 71 contributes to the chaperone-like action of ␣A-crystallin. Therefore we conclude that the 70 -88-region in ␣A-crystallin, identified by us earlier, is the functional chaperone site in ␣A-crystallin.␣-Crystallins are major refractive proteins in the vertebrate eye lens. When isolated from the lens they exist as polydisperse aggregates having an average molecular mass of Ϸ800 kDa (1, 2). ␣-Crystallin is composed of two subunits, ␣A and ␣B, which have considerable sequence homology between them and with other heat shock proteins (3, 4). Recently ␣-crystallin subunits were also reported to be present in nonlenticular tissues like heart, brain, and kidney (5-8). The significance of their presence in nonlenticular tissues is not clear. However, the increased expression of ␣B-crystallin observed in a variety of neurological disorders has drawn significant medical attention (9 -11). Like other small heat shock proteins, ␣-crystallin can sequester certain unfolding proteins in vitro, by preventing their aggregation and insolubilization (12-15). Both subunits of ␣-crystallin show chaperone-like activity to different extents (16 -18). Complex formation with ␤ L -and ␥-crystallin and decreased chaperone-like function during aging has indicated the importance of ␣-crystallin in maintaining the transparency of the lens (2, 19 -21). During chaperone-like action, hydrophobic surfaces in ␣-crystallin interact with specific sites in non-native target proteins (22-24). Earlier we were able to map the site in ␣A-and ␣B-crystallin responsible for chaperone-like action using photoactive cross-linkers and hydrophobic probes (25-27). Our studies with bis-ANS 1 and the hydrophobic protein mellitin have shown that there is an overlapping of chaperone site and hydrophobic site in ␣A-crystallin. Further, using a synthetic peptide (mini-␣A-crys...

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Section: F71g Mutant Of ␣A-crystallinmentioning

confidence: 40%

Section: F71g Mutant Of ␣A-crystallinmentioning

confidence: 99%

Phe71 Is Essential for Chaperone-like Function in αA-crystallin

Santhoshkumar¹,

Sharma²

2001

Journal of Biological Chemistry

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“…In other words, ANS binding studies did not show the correlation between the probe binding properties and chaperone activity as shown by bis-ANS and pyrene. Our own studies 2 and those from another laboratory (61) show that ANS binding and chaperone activity of some mutants of ␣-crystallin do not correlate. Our earlier study (18,28) on temperature-dependent chaperone activity and ANS binding or pyrene solubilization of the wild type ␣-crystallin showed good correlation.…”

Section: Figmentioning

confidence: 74%

Role of the C-terminal Extensions of α-Crystallins

Pasta¹,

Raman

Ramakrishna

et al. 2002

Journal of Biological Chemistry

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Several small heat shock proteins contain a well conserved ␣-crystallin domain, flanked by an N-terminal domain and a C-terminal extension, both of which vary in length and sequence. The structural and functional role of the C-terminal extension of small heat shock proteins, particularly of ␣A-and ␣B-crystallins, is not well understood. We have swapped the C-terminal extensions between ␣A-and ␣B-crystallins and generated two novel chimeric proteins, ␣ABc and ␣BAc. We have investigated the domain-swapped chimeras for structural and functional alterations. We have used thermal and non-thermal models of protein aggregation and found that the chimeric ␣B with the C-terminal extension of ␣A-crystallin, ␣BAc, exhibits dramatically enhanced chaperone-like activity. Interestingly, however, the chimeric ␣A with the C-terminal extension of ␣B-crystallin, ␣ABc, has almost lost its activity. Pyrene solubilization and bis-1-anilino-8-naphthalenesulfonate binding studies show that ␣BAc exhibits more solventexposed hydrophobic pockets than ␣A, ␣B, or ␣ABc. Significant tertiary structural changes are revealed by tryptophan fluorescence and near-UV CD studies upon swapping the C-terminal extensions. The far-UV CD spectrum of ␣BAc differs from that of ␣B-crystallin whereas that of ␣ABc overlaps with that of ␣A-crystallin. Gel filtration chromatography shows alteration in the size of the proteins upon swapping the C-terminal extensions. Our study demonstrates that the unstructured C-terminal extensions play a crucial role in the structure and chaperone activity, in addition to generally believed electrostatic "solubilizer" function.

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“…As a case in point, oligomers lacking chaperone activity arise from chimeric α‐crystallins [63]. Also, insertion of a peptide [41,64] and change of a single residue [10,11,13,65] lead to enlarged oligomers with curtailed chaperone action in vitro. In other work, dissociation of oligomers was a prerequisite for chaperoning in vitro [66], and disassembly of active units from an oligomeric (storage) state of α‐crystallin was proposed, upon structural analysis of αA‐crystallin by site‐directed spin labelling, as a model for chaperone function [67].…”

Section: Discussionmentioning

confidence: 99%

“…Chaperoning is thought to depend upon formation of oligomers that reach 800 kDa in mass and possess quaternary structure modifiable by environmental parameters [8,18,20,22,39,40]. Oligomers exhibit dynamic equilibrium with constituent subunits, which can affect chaperoning but is not in itself sufficient to ensure chaperone activity [25,41,42]. A small heat shock/a-crystallin protein from Methanococcus jannaschii, termed Mj hspl6.5, has been crystallized, revealing highly ordered oligomers of 24 subunits with a hollow center [9].…”

mentioning

confidence: 99%

Functional analysis of a small heat shock/α‐crystallin protein from Artemia franciscana

Crack

Mansour

Sun

et al. 2002

European Journal of Biochemistry

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Oviparously developing embryos of the brine shrimp, Artemia franciscana, synthesize abundant quantities of a small heat shock/a-crystallin protein, termed p26. Wild-type p26 functions as a molecular chaperone in vitro and is thought to help encysted Artemia embryos survive severe physiological stress encountered during diapause and anoxia. Full-length and truncated p26 cDNA derivatives were generated by PCR amplification of p26-3-6-3, then cloned in either pET21(+) or pRSETC and expressed in Escherichia coli BL21(DE3). All constructs gave a polypeptide detectable on Western blots with either p26 specific antibody, or with antibody to the His 6 epitope tag encoded by pRSETC. Full-length p26 in cell-free extracts of E. coli was about equal in mass to that found in Artemia embryos, but p26 lacking N-and C-terminal residues remained either as monomers or small multimers. All p26 constructs conferred thermotolerance on transformed E. coli, although not all formed oligomers, and cells expressing N-terminal truncated derivatives of p26 were more heat resistant than bacteria expressing p26 with C-terminal deletions. The C-terminal extension of p26 is seemingly more important for thermotolerance than is the N-terminus, and p26 protects E. coli against heat shock when oligomer size and protein concentration are low. The findings have important implications for understanding the functional mechanisms of small heat shock/a-crystallin proteins.

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Characteristics of super αA‐crystallin, a product of in vitro exon shuffling

Cited by 6 publications

References 32 publications

Phe71 Is Essential for Chaperone-like Function in αA-crystallin

Phe71 Is Essential for Chaperone-like Function in αA-crystallin

Role of the C-terminal Extensions of α-Crystallins

Functional analysis of a small heat shock/α‐crystallin protein from Artemia franciscana

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