The interaction between alphaA- and alphaB-crystallin is sequence-specific.
OBJECTIVE
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 alphaB-crystallin play a role in oligomerization and subunit interaction with alphaA-crystallin. When we created multiple mutations in alphaB-crystallin in RS-1 and RS-2 at S53(T), F54(G), L55(G), W60(R), and F61(N), we found that these mutations destabilized the protein, and the protein precipitated. When the individual mutations were created at F54, W60, and F61 in alphaB-crystallin, protein stability was not affected, but the mutations had an effect on oligomerization and subunit interaction with alphaA-crystallin. To find out whether the sequence specificity of these residues is important for the overall function of alphaB-crystallin, we inverted the 54-60 sequence such that 54FLRAPSW60 became 54WSPARLF60 using site-directed mutagenesis. We studied the effect of inversion on oligomerization and subunit interaction with alphaA-crystallin.


METHODS
Mutations were introduced using site-directed mutagenesis and the mutant protein, expressed in Escherichia coli BL21(DE3)pLysS cells, was purified by ion-exchange and gel filtration chromatography. The mutation was confirmed by mass spectrometry. The structure and hydrophobicity were analyzed by spectroscopic methods. The chaperone-like activities of wild-type and mutant proteins were compared using alcohol dehydrogenase and citrate synthase. Subunit exchange between alphaA- and alphaB-crystallin was monitored by fluorescence resonance energy transfer (FRET). For this purpose, purified alphaB- and alphaBinvert-crystallin were labeled with Alexa fluor 350 whereas Alexa fluor 488 was used to label alphaA-crystallin.


RESULTS
The inversion of residues 54-60 led to homooligomers that were 38% smaller in size than their wild-type counterparts. The inversion also reduced the tryptophan fluorescence intensity by 50%, as compared to that of wild-type alphaB-crystallin. This suggests that Trp54 is less exposed than Trp60. Inversion of residues did not affect the total hydrophobicity in alphaB-crystallin. Secondary structural analysis revealed a slight increase in the alpha-helical content of alphaBinvert-crystallin protein as compared to wild-type alphaB-crystallin. Except for an increase in the ellipticity of the alphaBinvert-crystallin mutant, no change was observed in the tertiary structure, as compared with that of wild-type alphaB-crystallin. Chaperone-like function was similar in the alphaBinvert-crystallin mutant and wild-type alphaB-crystallin. The inversion of residues decreased the subunit exchange rate with alphaA-crystallin by two fold.


CONCLUSIONS
This study establishes for the first time that proper orientation of residues contributing to RS-1 and RS-2 sites in alphaB-crystallin is important for homooligomerization and optimal subunit interaction with alphaA-crystallin.
Sreelakshmi
Yellamaraju
Y
Department of Ophthalmology, University of Missouri, Columbia, MO 65212, USA. yellamarajusr@health.missouri.edu
Sharma
K Krishna
KK
D000595:Amino Acid Sequence; D004347:Drug Interactions; D004926:Escherichia coli / Q000378:metabolism; D005453:Fluorescence; D018014:Gene Transfer Techniques; D006801:Humans; D018832:Molecular Chaperones / Q000502:physiology; D008969:Molecular Sequence Data; D015394:Molecular Structure; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D014364:Tryptophan; D038202:alpha-Crystallin A Chain / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D038203:alpha-Crystallin B Chain / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
D018832:Molecular Chaperones; D038202:alpha-Crystallin A Chain; D038203:alpha-Crystallin B Chain; D014364:Tryptophan