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Kirsten J Lampi, Matthew R Murray, Matthew Petterson, Bryce Eng, Eileen Yue, Stephanie Finet, Elisar Barbar, Larry L David; Dynamic conformational changes of β-crystallins upon hetero-oligomer formation contribute to stability. Invest. Ophthalmol. Vis. Sci. 2014;55(13):4052.
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© ARVO (1962-2015); The Authors (2016-present)
The structure of βB2 has been extensively studied, but little is known about its structure in the native complex interactions with other β-crystallins found in vivo. The purpose of this research is to determine the solution dynamics of βB2 and βA3 and their conformational changes in the hetero-dimer associated with stability changes.
Recombinant β-crystallins were expressed and cross-linked with isotope coded CyanurBiotinDimercaptoPropionylSuccinimide (CBDPS), digested with trypsin, biotinylated peptides purified, and samples analyzed by LC/MS using an Orbitrap Fusion mass spectrometer. β-crystallins were also analyzed by nuclear magnetic resonance (NMR) spectroscopy using a 700 MHz Bruker spectrometer to acquire 1H-15N HSQC spectra. The global shape of βB2 and βA3 was further determined by small angle X-ray scattering (SAXS) at the National Synchrotron facility in Gif-sur-Yvette, France.
The global shapes of βB2 and βA3 determined by SAXS suggest small different overall conformations in solution, close to the compact βB1 conformation and suggesting that βB2 does not adopt in solution the extended structure of the known crystal structure. Little change in NMR signal from the extensions was detected upon hetero-oligomer formation, in contrast to a decrease in signal that was attributed to the compact domains. The crosslinking data show two predominant intramolecular βB2 linking sites between residues 120 and 139, as well as 95 and 106. When plotted onto the known crystal structures, the data more closely fit the compact βB1 with a bent linker, as compared to the βB2 with an extended linker. The hetero-oligomer also is more resistant to heat-induced precipitation.
Formation of the more compact hetero-oligomers may be an important mechanism for increasing their stability needed for their long lifespan in vivo. The different conformations of the β-crystallins and difficulty in crystallizing hetero-oligomers suggest that this polydispersity is necessary to maintain lens transparency.
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