Abstract
Purpose :
While much is known about the structures of the homodimer β-crystallin subunits, little is known about the higherorder structure of the hetero-oligomers that exist in the lens. In this study we have determined the oligomer sizes and compared their stabilities.
Methods :
Hetero-oligomers were formed by incubating equalmolar amounts of homodimers. The native oligomer states, hetero-oligomerization kinetics, and oligomer structures were determined using native Ion Mobility-Mass Spectrometry (IMMS). Differences in solvent accessible regions were measured by hydrogen/deuterium exchange mass spectrometry (HDMS). Thermal stability was determined by measuring turbidity at 405 nm during heat-denaturation at 55°C, followed by determining molar sizes by static light scattering.
Results :
The major forms of both βB2 and βA3 homodimers, as well as the heterologous protein, were dimers. After 120 min of mixing the respective homodimers, about 90% of the heterodimer had formed. A higher protein concentrations, small amounts of higher-order oligomers, hexamers and octamers, were also detected by IMMS. Similar results were obtained for the βB1 and βA3, only formation of the heterodimer was 3x slower. Next, uptake of deuterium was compared between the homo- and hetero-dimer whole proteins. After 60 min, there were 20 less deuteriums incorporated into βA3 in the βB1:βA3 heterodimer than in the homodimer, suggesting less solvent accessibility and a more compact structure of the heterodimer. The heterodimers were also more resistant to heat-denaturation. During heating, dimer formation persisted until a threshold was reached where large light scattering aggregates were detected, with little to no intermediate molecular weight forms.
Conclusions :
Spontaneous formation of both βB1:βA3 and βB2:βA3 heterodimers, decreased deuterium exchange in βA3 within the βB1:βA3 heterodimer, and increased heat stability of βB1:βA3 heterodimer compared to each protein’s homodimeric form, support that heterologous interactions in the β-crystallins are important mediators of their long term stability. This study reports the first use of IMMS to analyze β-crystallin oligomers and measure the kinetics of subunit exchange.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.