The Quaternary Structure of Bovine alpha-Crystallin. Size and Charge Microheterogeneity: More than 1000 Different Hybrids?

Abstract

Cortical a-crystallin was size-fractionated by gel filtration on Ultrogel AcA22 and chargefractionated by anion-exchange chromatography on DE-52 DEAE-cellulose using gradient elution.Electron microscopy demonstrates that both native and reassociated a-crystallin are heterogeneous populations of spherical or slightly ellipsoidal molecules with diameters of 13.5 -16.0 nm (maximum at 14.0-15.0 nm) for native a-crystallin and 8.5-12.5 nm (maximum at 10.0-10.5 nm) for reassociated a-crystallin.An enormous charge heterogeneity of native a-crystallin was detected, which is shown to arise from variations in the stoichiometry of the 5 main types of subunits. The molar ratio of acidic chains (Az, A1 and to basic chains (B2 and BI) varies from 70/30-80/20 (averaging about 3/1) and the amount of deamidated chains (Al and B1) varies from 7-37%. Recombination of the subunits, after dissociation in 6 M urea, leads to a charge heterogeneity of reassociated a-crystallin very similar to that of native a-crystallin. Therefore, specific formation of pure A or B chain aggregates is not preferred. Instead, random combination of subunits is theoretically shown to be sufficient to describe the observed charge microheterogeneity of both reassociated and native a-crystallin.No obvious relationship exists between size and charge heterogeneity. Within these ranges of molecular weight and subunit composition there are more than 1000 different combinations of A2. A1, A:-151, B2 and B1 conceivable.Towards a better understanding of the eye lens architecture and its function on a molecular level, a thorough structural characterization of the main protein constituents, the crystallins, is required, in particular their quaternary structure, packing arrangement and interactions in vivo. Additional interest in one of the major multisubunit lens proteins, acrystallin, arises from its usefulness as a marker for studies of such fundamental processes as growth, differentiation and aging [l -31. We investigate various aspects of the quaternary structure of bovine low-M, a-crystallin in this and following papers in this series, with the purpose of providing a sound basis for further studies of aging and higher-order structures.The subunit composition and physical characteristics of bovine a-crystallin change with development and aging of the lens, which results, even at a very early stage, in a distinct size and charge heterogeneity.

Differential Temperature-dependent Chaperone-like Activity of αA- and αB-crystallin Homoaggregates

“…The molecular mass of the refolded ␣-crystallin, however, may vary depending on conditions like ionic strength, pH, and temperature (30). The size of the homoaggregates thus obtained are comparable to refolded heteromultimeric ␣-crystallin (31,32). ␣B-crystallin homoaggregates obtained in this way have a similar molecular mass as ␣B-crystallin homoaggregates isolated from nonlenticular tissue such as the heart (10, 33, 34).…”

Unique Structural Features of a Novel Class of Small Heat Shock Proteins

“…Two different plant smHSPs (HSP18.1 and HSP17.7) have been shown to form globular and mixed round and triangular 10-nm structures containing 12 subunits, respectively (3). In contrast, ␣-crystallins and mammalian smHSPs usually form larger complexes of 400 -800 kDa (up to ϳ40 subunits), and appear as 10 -18-nm globular or torus-like heterogeneous structures as observed by electron microscopy (23)(24)(25)(26). Furthermore, the oligomeric state of the mammalian smHSPs depends on various factors, such as the state of phosphorylation and temperature (11,27,28).…”

The Quaternary Structure of Bovine alpha-Crystallin. Size and Shape Studies by Sedimentation, Small-angle X-Ray Scattering and Quasi-elastic Light Scattering

“…Yet its exact role in lens structure is not clear, mainly because very little is known about its three-dimensional arrangement in vivo, its interactions with other lens proteins and lens membranes, or even its size, shape, quaternary structure and subunit symmetry. In this paper of the series various physicochemical parameters pertaining to size and shape of bovine a-crystallin are obtained by several independent techniques.Bovine a-crystallin cannot be isolated as a homogeneous protein, since postsynthetic alterations cause considerable size and charge heterogeneity [4]. It is The first paper in this series is [4], the preceding paper.…”

“…Bovine a-crystallin cannot be isolated as a homogeneous protein, since postsynthetic alterations cause considerable size and charge heterogeneity [4]. It is The first paper in this series is [4], the preceding paper.…”