Association properties of betaB1- and betaA3-crystallins: ability to form heterotetramers.
As major constituents of the mammalian lens, beta-crystallins associate into dimers, tetramers, and higher-order complexes to maintain lens transparency and refractivity. A previous study has shown that dimerization of betaB2- and betaA3-crystallins is energetically highly favored and entropically driven. While heterodimers further associate into higher-order complexes in vivo, a significant level of reversibly associated tetrameric crystallin has not been previously observed in vitro. To enhance our understanding of the interactions between beta-crystallins, we characterized the association of betaB1-crystallin, a major component of large beta-crystallin complexes (beta-high), with itself and with betaA3-crystallin. Mouse betaB1-crystallin and human betaA3-crystallin were expressed in Escherichia coli and purified chromatographically. Their association was then characterized using size-exclusion chromatography, native gel electrophoresis, isoelectric focusing, and analytical sedimentation equilibrium centrifugation. When present alone, each beta-crystallin associates into homodimers; however, no tetramer formation is seen. Once mixing has taken place, formation of a heterocomplex between betaB1- and betaA3-crystallins is observed using size-exclusion chromatography, native gel electrophoresis, isoelectric focusing, and sedimentation equilibrium. In contrast to results previously obtained after betaB2- and betaA3-crystallins had been mixed, mixed betaB1- and betaA3-crystallins show a dimer-tetramer equilibrium with a K d of 1.1 muM, indicating that these two beta-crystallins associate predominantly into heterotetramers in vitro. Thus, while each purified beta-crystallin associates only into homodimers and under the conditions studied mixed betaB2- and betaA3-crystallins form a mixture of homo- and heterodimers, mixed betaB1- and betaA3-crystallins associate predominantly into heterotetramers in equilibrium with heterodimers. These findings suggest a unique role for betaB1-crystallin in promoting higher-order crystallin association in the lens.
Chan
May P
MP
National Eye Institute and National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
Dolinska
Monika
M
Sergeev
Yuri V
YV
Wingfield
Paul T
PT
Hejtmancik
J Fielding
JF
D002850:Chromatography, Gel; D003459:Crystallins / Q000737:chemistry* / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D007525:Isoelectric Focusing; D008970:Molecular Weight; D046912:Multiprotein Complexes; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D014461:Ultracentrifugation; D038205:beta-Crystallin A Chain / Q000737:chemistry* / Q000235:genetics; D038206:beta-Crystallin B Chain
C489108:CRYBA1 protein, human; C491363:Crybb1 protein, mouse; D003459:Crystallins; D046912:Multiprotein Complexes; D011994:Recombinant Proteins; D038205:beta-Crystallin A Chain; D038206:beta-Crystallin B Chain