Calf lens alpha-crystallin quaternary structure. A three-layer tetrahedral model.

Calf lens alpha-crystallins are polydisperse globular particles made of a large number of two types of subunits, A and B, both of molecular weight congruent to 20,000. alpha-Crystallin populations consisting on average of 40 subunits or more were subjected to various changes in pH, ionic strength, temperature and urea concentration. Modifications in quaternary structure induced by variation of these physicochemical parameters were followed by means of X-ray and quasi-elastic light-scattering and quantified in terms of weight average molecular weight (M), radius of gyration (Rg) and hydrodynamic radius (Rh). High-pressure liquid chromatography was used as a control of polydispersity. Increasing the pH, decreasing the ionic strength and incubating at temperatures from 20 degrees C to 45 degrees C all resulted in the formation of particles of decreasing M, Rg and Rh values. These effects are cumulative. All monomodal alpha-crystallin populations encountered in this study, which covers a wide range of sizes and molecular weights, may be accounted for by a three-layer model with partial filling up of the layers. Applying basic principles of symmetry and postulating specific contacts between protein subunits to construct this three-layer model leads to tetrahedral symmetry, with 12, 24 and 24 sites in the first, second and third layers, respectively. Variations in probabilities of site occupancy account for both the observed quaternary structure modifications and the intrinsic polydispersity of alpha-crystallins

Self-assembly of brome mosaic virus capsids. Kinetic study using neutron and X-ray solution scattering.

The self-assembly of brome mosaic virus capsid has been studied kinetically by means of X-ray and neutron scattering. It appears to be a very fast process: for the concentrations used (5 to 8 mg/ml) the forward scattering reaches 50% of its maximal value in less than one second. Further, the assembly seems to proceed through intermediate states whose nature is still speculative.

Protein interactions in the calf eye lens: interactions between beta-crystallins are repulsive whereas in gamma-crystallins they are attractive.

Non-specific interactions in beta- and gamma-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 degrees C and 31 degrees C. Two types of interactions were observed. With beta-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 alpha-crystallins, such behaviour is dependent upon ionic strength but is hardly affected by temperature. In contrast, with gamma-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.

A laboratory evaluation of standard leakage tests for surgical & examination gloves.

Analysis of leakage tests for surgical and examination gloves is reported. Six widely applicable tests were evaluated, using samples from 16 glove manufacturers. Various techniques for inducing sub-millimeter holes were developed and employed for creating test holes in a wide variety of gloves. The prescribed tests were performed and the results compared to calculations of expected sensitivities. In general, there was reasonable agreement between calculation and measurement (within about a factor of 2), although there were some exceptions. Minimum detectable hole sizes, before the holes were stretched by the tests, were generally in the range of 25-100 microns in diameter. Theoretical predictions of minimum detectable hole sizes for the various tests ranged from about 100-200 microns (in the stretched condition). Because of inherent limitations of the test protocols, it is clear that much larger holes can go undetected under certain circumstances, especially in examination gloves. Some test procedures had important procedural ambiguities; some tests were patently impossible to perform. All tests had inherent limitations resulting in sensitivities that varied from glove to glove or that were quite nonuniform over the surface of the glove being tested or both.

X-ray analysis of the kinetics of Escherichia coli lipid and membrane structural transitions.

Synchrotron radiation was used to follow the time course of the transitions, induced by temperature jump, in Escherichia coli membranes and their lipid extracts isolated from a fatty acid auxotroph grown with different fatty acids. We measured the relaxation times associated with the phase transitions as well as with the conformational transition of the hydrocarbon chains and observed different behavior as a function of chemical composition. Relaxation times of about 1-2 s were found at a hexagonal to lamellar phase transition and within a lamellar phase whose parameters display important variations with temperature when the conformational transition takes place. On the other hand, no delay was observed for a phase transition where large lipid or water diffusion was not needed. We have shown that phase transitions and conformational transitions are, to a large extent, uncoupled and that the relaxation times corresponding to the latter transition could be related to the size of the ordered domains. In all cases, the order to disorder conformational transition is more rapid than the disorder to order transition. Finally, the relaxation times of the disorder to order transition observed with the membranes and with their lipid extracts were found to be strongly correlated, indicating that the proteins do not play a role in this transition.

Effect of calcium on the calf lens cytoplasm.

Opacification was induced in calf lens cytoplasmic extracts by addition of calcium. The sample turbidity was shown to increase with calcium molarity, incubation time and temperature and to decrease with the protein cytoplasmic concentration. Although this turbidity was enhanced when membrane fragments were left over in the cytoplasmic extracts, it did show up in the absence of any detectable vesicular fragment. Scattering techniques (X-ray and light) showed that the calcium-induced opacification is linked to enhanced light scattering, which results from the formation of additional scatterers, a few tens of nm in diameter. Additional structures were indeed visualized by freeze-fracture electron microscopy (FFEM): they appear as molecular clusters with diameters ranging from 20 to 90 nm, made of densely packed particles, with heterogeneous sizes. The turbidity expected from these clusters, as well as the expected variation of turbidity with cytoplasmic concentration, was calculated to be in agreement with the measurements. When compared with cold cataract, these results illustrate that similar opacities may result from completely different biophysical mechanisms.