C. Slingsby

Protein interactions in the calf eye lens: interactions between β-crystallins are repulsive whereas in γ-crystallins they are attractive

Non-specific interactions in β- and γ-crystallins have been studied by solution X-ray scattering and osmotic pressure experiments. Measurements were carried out as a function of protein concentration at two ionic strengths. The effect of temperature was tested between 7°C and 31°C. Two types of interactions were observed. With β-crystallin solutions, a repulsive coulombic interaction could be inferred from the decrease of the normalized X-ray scattering intensity near the origin with increasing protein concentration and from the fact that the osmotic pressure increases much more rapidly than in the ideal case. As was previously observed with α-crystallins, such behaviour is dependent upon ionic strength but is hardly affected by temperature. In contrast, with γ-crystallin solutions, the normalized X-ray scattering intensity near the origin increases with increasing protein concentration and the osmotic pressure increases less rapidly than in the ideal case. Such behaviour indicates that attractive forces are predominant, although we do not yet know their molecular origin. Under our experimental conditions, the effect of temperature was striking whereas no obvious contribution of the ionic strength could be seen, perhaps owing to masking by the large temperature effect. The relevance of the different types of non-specific interactions for lens function is discussed.

Rapid separation of bovine β-crystallin subunits βB1, βB2, βB3, βA3 and βA4

Abstract Bovine β-crystallin aggregates, βH-, βL1- and βL2-crystallins, prepared by rapid gel filtration, are each subjected to anion-exchange chromatography in deaggregating media using a Pharmacia Fast Protein Liquid Chromatography System. BetaB1, βB2 and βA4 subunits are rapidly isolated using a one step Mono Q column from βH-, βL2- and βL1-crystallin, respectively. BetaB3 and βA3 are separated from each other using a second Mono Q column starting from βL2- and βL1-crystallin respectively. Whereas βB2, βB3 and βA4 are common to all sizes of aggregate, βB1 is restricted to βH-crystallin and βA3 is absent from βL2-crystallin.

Close packing of an oligomeric eye lens β-crystallin induces loss of symmetry and ordering of sequence extensions

beta-Crystallins are oligomeric eye lens proteins that are related to monomeric gamma-crystallins. The main sequence difference between the two families is the presence of sequence extensions in the beta-crystallins. A major question concerns the role that these extensions play in mediating interactions at the high protein concentrations found in the lens. The predominant beta-crystallin polypeptide, beta B2, can be crystallized in two different space groups, I222 and C222. The I222 crystal structure revealed that the protein packed as a tetramer with perfect 222 symmetry but that the extensions were disordered. The X-ray structure of the C222 lattice of beta B2 has now been refined at 3.3 A, the structure analysed and compared with the I222 lattice. The protein is also a tetramer with 222 symmetry in the C222 lattice but differs in that parts of the N-terminal extensions have been visualized. In the asymmetric unit of the C222 lattice there are four subunits, each comprising a single polypeptide chain, in which certain flexible loops in the N-terminal domains and the N-terminal extensions have various conformations. The tetramers in the C222 lattice are more tightly packed than in the I222 form. Analysis of the tetramer contacts shows that the sites of interaction break the 222 symmetry of the tetramers. The N-terminal extensions play a major role in directing interactions between tetramers. One of the N-terminal extensions interacts with a hydrophobic patch on the N-terminal domain of another tetramer. These crystallographic observations obtained over a physiological concentration range indicate how, in beta-crystallin oligomers, the N-terminal extensions of beta B2 can switch from interacting with water to interacting with protein depending on their relative concentrations. This could be useful in maintaining a gradient of refractive index.

The avian eye lens protein δ-crystallin shows a novel packing arrangement of tetramers in a supramolecular helix

BACKGROUND Little is known of the intermolecular organization of crystallins in the protein-packed eye lens. The tetrameric structure of the 200,000 Da avian delta-crystallin, which is closely related to the enzyme argininosuccinate lyase and is characteristic of the accommodating, soft lens of birds, has recently been solved at atomic resolution at acidic pH. To help understand how delta-crystallin remains soluble at the very high concentrations found in the avian lens we have now crystallized turkey delta-crystallin at around neutral pH and examined its intermolecular interactions. RESULTS Turkey delta-crystallin has been crystallized around neutral pH. The X-ray structure has been solved at 4.5 A resolution in space group C2 with three and a half tetramers in the asymmetric unit. The symmetrical 222 tetramers have a novel packing arrangement consisting of continuous helices, with 7(3)2 non-crystallographic symmetry, in an approximately hexagonal close-packed array. The internal 222 symmetry of the tetramers allows different polymeric chains to be constructed, based on the tetramer-tetramer association observed in the crystalline helix. It is possible to build a model of a tubule of diameter 212 A that is very similar to observed tubules of bovine argininosuccinate lyase. CONCLUSIONS Elements of helical organization may occur in the concentrated solution of the avian eye lens where delta-crystallin is the prominent protein. The symmetry of the tetramer provides a choice in the direction of growth of a helix at each link so that highly hydrated irregular polymers may be formed rather than large compact regular structures that would not be compatible with a transparent lens.

Structural studies on βH-crystallin from bovine eye lens

Abstract Bovine lens βH-crystallin, isolated at pH 6·7, undergoes reversible dissociation into dimers and an intermediate size of oligomer (peak A) at pH 5·4. Peak A is enriched in the βB1 subunit but lacks βB2, whereas βB2 is a major component of the dimers. A method for isolation of βB1 from peak A is described. The pH dependence of the dissociation-reassociation suggests that histidines on the surface of the dimers become buried in the assembly of βH-crystallin. The positions of the four histidines on the surface of the compact domains of each subunit of the βB2 homodimer are shown. The βB1-enriched oligomer has a much lower solubility compared with the βB2 containing βH-crystallin. It is possible that βB2 plays a role in solubilizing β-crystallin aggregates.

Motifs involved in protein-protein interactions.

SummaryInteractions between proteins are extremely variable. However, in the dimeric proteins comprised of regular motifs, interface interactions are similar to those that stabilize monomers. Additional stability is gained by converting loops within motifs or domains to linkers across interfaces. In multi-domain proteins, interactions can be greatly effected by the conformation of linkers between domains. Complex association of subunits, involving higher rotational symmetry or cubic symmetry, frequently involves motif sharing across interfaces.

Molecular Interactions of Crystallins in Relation to Optical Properties

The transparency of the lens depends on an even distribution of protein and water over distances comparable to the wavelength of light, while the degree of refraction is controlled partly by the ability of the lens to change shape. The core regions of certain lenses such as carp and rat have an extremely high refractive index as a result of high protein concentration, which confers rigidity on that region of the lens. By contrast the outer regions of these lenses, like the complete human lens, have a lower proportion of protein to water and are malleable (van Heyningen, 1976; Philipson, 1969; Fagerholm et al., 1981). Furthermore, there is an increasing protein concentration gradient from the periphery to the core of the lens, leading to a gradient of refractive index that almost abolishes spherical aberration (Fernald and Wright, 1983; Sivak, 1985).