The interaction between αA- and αB-crystallin issequence-specific

[摘要] Purpose: We have previously shown that residue 42-57(TSLSPFYLRPPSFLRA; recognition sequence 1 or RS-1) and residue 60-71(WFDTGLSEMRLE; recognition sequence 2 or RS-2) in αB-crystallinplay a role in oligomerization and subunit interaction with αA-crystallin. When we created multiple mutations in αB-crystallinin RS-1 and RS-2 at S53(T), F54(G), L55(G), W60(R), and F61(N), we foundthat these mutations destabilized the protein, and the proteinprecipitated. When the individual mutations were created at F54, W60,and F61 in αB-crystallin, protein stability was not affected, butthe mutations had an effect on oligomerization and subunit interactionwith αA-crystallin. To find out whether the sequence specificityof these residues is important for the overall function of αB-crystallin, we inverted the 54-60 sequence such that 54FLRAPSW60became 54WSPARLF60 using site-directed mutagenesis. We studied theeffect of inversion on oligomerization and subunit interaction withαA-crystallin.Methods: Mutations were introduced using site-directed mutagenesisand the mutant protein, expressed in Escherichia coli BL21(DE3)pLysScells, was purified by ion-exchange and gel filtration chromatography.The mutation was confirmed by mass spectrometry. The structure andhydrophobicity were analyzed by spectroscopic methods. Thechaperone-like activities of wild-type and mutant proteins were comparedusing alcohol dehydrogenase and citrate synthase. Subunit exchangebetween αA- and αB-crystallin was monitored by fluorescenceresonance energy transfer (FRET). For this purpose, purified αB-and αBinvert-crystallin were labeled with Alexa fluor 350 whereasAlexa fluor 488 was used to label αA-crystallin.Results: The inversion of residues 54-60 led to homooligomers thatwere 38% smaller in size than their wild-type counterparts. Theinversion also reduced the tryptophan fluorescence intensity by 50%, ascompared to that of wild-type αB-crystallin. This suggests thatTrp54 is less exposed than Trp60. Inversion of residues did not affectthe total hydrophobicity in αB-crystallin. Secondary structuralanalysis revealed a slight increase in the α-helical content ofαBinvert-crystallin protein as compared to wild-type αB-crystallin. Except for an increase in the ellipticity of the αBinvert-crystallin mutant, no change was observed in the tertiarystructure, as compared with that of wild-type αB-crystallin.Chaperone-like function was similar in the αBinvert-crystallinmutant and wild-type αB-crystallin. The inversion of residuesdecreased the subunit exchange rate with αA-crystallin by twofold.Conclusions: This study establishes for the first time that properorientation of residues contributing to RS-1 and RS-2 sites in αB-crystallin is important for homooligomerization and optimal subunitinteraction with αA-crystallin.

[学科分类] 生物化学/生物物理