Monomer−Dimer Equilibrium of Normal and Modified βA3-Crystallins:  Experimental Determination and Molecular Modeling

• Published in: Biochemistry (Easton) 2000-12, Vol.39 (51), p.15799-15806
• Main Authors: Sergeev, Y. V, Wingfield, P. T, Hejtmancik, J. F
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Cattle; Chromatography, Gel; Computer Simulation; Crystallins - chemistry; Crystallins - genetics; Crystallography, X-Ray; Dimerization; Mice; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Protein Binding - genetics; Protein Conformation; Recombinant Proteins - chemistry; Sequence Alignment; Sequence Homology, Amino Acid; Solvents; Ultracentrifugation
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi001882h

β- and γ-Crystallins are major protein constituents of the mammalian lens, where their stability and association into higher order complexes are critical for clarity and refraction. Two regions of the βγ-crystallins have been suggested to modulate protein association, namely, the flexible N-terminal extensions and the intramolecular domain interfaces. The oligomeric state of wild-type recombinant murine βA3-crystallin (rβA3) was compared to that of modified βA3-crystallins with either an N-terminal deletion of residues 1 to 29 (rβA3tr) or with residues 114 to 123 of the interdomain linker replaced with the analogous linker from murine γB-crystallin (rβA3cp). All three proteins exhibited reversible monomer−dimer formation. The modifications to the N-terminus and domain linker resulted in tighter dimer formation as compared to wild-type protein as indicated by disassociation constants determined by sedimentation equilibrium:  6.62 × 10-6 M (rβA3), 0.86 × 10-6 M (rβA3cp), and 1.83 × 10-7 M (rβA3tr). Homology modeling of βA3-crystallins and solvation energy calculations also predicted tighter binding of the modified crystallins consistent with the centrifugation results. The findings suggest that under physiological conditions βA3 crystallin exists in a dynamic equilibrium between monomeric and dimeric protein and that modification, especially to the N-terminal extension, can promote self-association.