Phe71 is essential for chaperone-like function in alpha A-crystallin.
Experiments with mini-alphaA-crystallin (KFVIFLDVKHFSPEDLTVK) showed that Phe(71) in alphaA-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 alphaA-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 beta(L)-crystallin aggregation was observed when mutant alphaA-crystallin was included. These results suggest that Phe(71) contributes to the chaperone-like action of alphaA-crystallin. Therefore we conclude that the 70-88-region in alphaA-crystallin, identified by us earlier, is the functional chaperone site in alphaA-crystallin.
Santhoshkumar
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Department of Ophthalmology, 1 Hospital Dr., University of Missouri, Columbia, MO 65212, USA.
Sharma
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KK
D000595:Amino Acid Sequence; D000817:Anilino Naphthalenesulfonates / Q000494:pharmacology; D000818:Animals; D002942:Circular Dichroism; D002950:Citrate (si)-Synthase / Q000737:chemistry; D003001:Cloning, Molecular; D003459:Crystallins / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005456:Fluorescent Dyes / Q000494:pharmacology; D007328:Insulin / Q000378:metabolism; D018832:Molecular Chaperones / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000737:chemistry* / Q000502:physiology*; D011485:Protein Binding; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry; D014466:Ultraviolet Rays
D000817:Anilino Naphthalenesulfonates; D003459:Crystallins; D005456:Fluorescent Dyes; D007328:Insulin; D018832:Molecular Chaperones; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D010649:Phenylalanine; C035763:5,5'-bis(8-(phenylamino)-1-naphthalenesulfonate); D014364:Tryptophan; D002950:Citrate (si)-Synthase