Influence of the ionic strength on the heat-induced aggregation of the globular protein beta-lactoglobulin at pH 7

The influence of the ionic strength on the structure of beta-lactoglobulin aggregates formed after heating at pH 7 has been studied using static and dynamic light scattering. The native protein depletion has been monitored using size exclusion chromatography. Above a critical association concentration (CAC) well-defined clusters are formed containing about 100 monomers. The CAC increases with decreasing ionic strength. The so-called primary aggregates associate to form self similar semi-flexible aggregates with a large scale structure that is only weakly dependent on the ionic strength. The local density of the aggregates increases with increasing ionic strength. At a critical gel concentration, Cg, the size of the aggregates diverges. Cg decreases from 100 g/l without added salt to 1 g/l at 0.4M NaCl. For C > Cg the system gels except at high ionic strength close to Cg where the gels collapse under gravity and a precipitate is formed.     

Cumulative deamidations of the major lens protein γS‐crystallin increase its aggregation during unfolding and oxidation

Age‐related lens cataract is the major cause of blindness worldwide. The mechanisms whereby crystallins, the predominant lens proteins, assemble into large aggregates that scatter light within the lens, and cause cataract, are poorly understood. Due to the lack of protein turnover in the lens, crystallins are long‐lived. A major crystallin, γS, is heavily modified by deamidation, in particular at surface‐exposed N14, N76, and N143 to introduce negative charges. In this present study, deamidated γS was mimicked by mutation with aspartate at these sites and the effect on biophysical properties of γS was assessed via dynamic light scattering, chemical and thermal denaturation, hydrogen‐deuterium exchange, and susceptibility to disulfide cross‐linking. Compared with wild type γS, a small population of each deamidated mutant aggregated rapidly into large, light‐scattering species that contributed significantly to the total scattering. Under partially denaturing conditions in guanidine hydrochloride or elevated temperature, deamidation led to more rapid unfolding and aggregation and increased susceptibility to oxidation. The triple mutant was further destabilized, suggesting that the effects of deamidation were cumulative. Molecular dynamics simulations predicted that deamidation augments the conformational dynamics of γS. We suggest that these perturbations disrupt the native disulfide arrangement of γS and promote the formation of disulfide‐linked aggregates. The lens‐specific chaperone αA‐crystallin was poor at preventing the aggregation of the triple mutant. It is concluded that surface deamidations cause minimal structural disruption individually, but cumulatively they progressively destabilize γS‐crystallin leading to unfolding and aggregation, as occurs in aged and cataractous lenses.     

Cardiac alpha-crystallin

Rat hearts were perfused in the working heart or Langendorff mode and then subjected to total normothermic ischemia. The content of alpha-crystallin in the water soluble protein fraction obtained from these hearts diminished in a time-dependent manner during ischemia. The protein was recovered in the low g pellet of the homogenate. The redistribution was dramatic, selective for alpha-crystallin and irreversible. Large crystallin clumps formed also when exposing the soluble protein fraction of control hearts to slightly acidic pH (6.5-7.0). Electron microscopic analysis showed that aggregation of the globular homo-oligomeric units of crystallin occurred. The aggregates probably represented denatured protein and were similar in appearance to lenticular alpha H-crystallin. In purified form, however, cardiac crystallin particles did not cluster at pH 6.5. Aggregation only occurred in the presence of other protein components (including, probably, cytosolic actin) of the soluble fraction. A direct and selective interaction between actin and cardiac crystallin could be demonstrated using actin-Sepharose affinity chromatography procedures. The results suggest that large aggregates of cardiac crystallin form very early during ischemia, due to acidification of the cytosol. Cardiac crystallin is highly homologous to stress proteins and is localized on the Z-disks, where it plays probably a structural or protective role. Its rapid and complete denaturation could be involved in the genesis of the irreversible structural damages occurring during ischemia.     

Crystallins and hereditary cataracts: molecular mechanisms and potential for therapy

Hereditary childhood cataracts can arise from single-point mutations in genes encoding crystallins, the major protein components of the lens. The cataracts are most commonly inherited by an autosomal dominant mechanism. The nature of the changes in the lens resulting from these point mutations in crystallin genes has not been fully characterised. While aggregation and light scattering associated with expression of the mutant crystallin protein may be an end point, it is also necessary to determine the progression of changes induced at the level of development and differentiation. A key finding in recent work is that cell death or cytotoxicity is associated with mutations in alpha A-crystallin. The variable morphology or localisation of the cataract in different pedigrees, even with the identical crystallin gene mutation, has led to the idea that other environmental or genetic factors interact to give the final lens phenotype. The study of mechanisms of formation of hereditary cataracts may lead to a greater understanding of the mechanisms that lead to age-related cataracts, a very common cause of blindness in the ageing population.     

Translocation and induction of alpha B crystallin by heat shock in rat glioma (GA-1) cells.

Response to heat shock of alpha B crystallin expressed in rat astrocytoma GA-1 cells was analysed quantitatively using an immunoassay method. GA-1 cells contained a considerable amount of alpha B crystallin at growing phase. When the growing cells were heated at 45 degrees C, concentrations of alpha B crystallin in cell extracts were decreased to less than one-fifth of the original level within 15 min, with an increase in the insoluble fraction which was detected by immunoblotting. The low level of alpha B crystallin in the cytoplasm, that was observed for a few hours after heat shock, gradually recovered to the control level within several hours. At 10 h following heat shock (45 degrees C for 15 min), the concentration of alpha B crystallin in the soluble extract was about twice that of the control level, with little detectable amounts in the insoluble fraction. These results are additional evidence that suggest that alpha B crystallin is a small heat shock protein.     

Covalent change in alpha crystallin during human senile cataractogenesis

The high molecular weight aggregates (HMWA) obtained from normal and cataractous human lens nuclei have been resolved by SDS-polyacrylamide gel electrophoresis, and the alpha crystallin band has been probed with antisera made against the whole alpha crystallin molecule and with antisera made against synthetic peptides of alpha crystallin (alpha A2 147-161 and alpha A2 163-173). Quantitation of these antisera binding demonstrated that the anti-alpha A2 163-173 serum and the anti-alpha whole sera bound equally well to the alpha crystallin band from the HMWA fraction from normal and cataractous lenses. In contrast, the anti-alpha A2 147-161 serum bound little, if at all, to alpha crystallin from normal lenses, while it bound well to alpha crystallin from cataractous lenses. These results demonstrate a covalent alteration in the alpha crystallin molecule, and suggest a possible location of a covalent change that may occur during the cataractogenic process in the aged human lens.     

Expression of the murine small heat shock proteins hsp 25 and alpha B crystallin in the absence of stress

Stress induces the synthesis of several large and small heat shock proteins (hsp's). Two related small hsp's, hsp25 and alpha B crystallin exist in mice. alpha B crystallin is an abundant protein in several tissues even in the absence of stress. Particularly high amounts accumulate in the eye lens. Here we show that hsp25 is likewise constitutively expressed in many normal adult tissues. In the absence of stress the protein is most abundant in the eye lens, heart, stomach, colon, lung, and bladder. The stress-independent expression pattern of the two small hsp's is distinct. In several tissues the amount of hsp25 exceeds that accumulating in NIH 3T3 fibroblasts in response to heat stress. hsp25, like alpha B crystallin, exists in a highly aggregated form in the eye lens. The expression of hsp25 and alpha B crystallin in normal tissues suggests an essential, but distinct function of the two related proteins under standard physiological conditions.     

Dynamic light scattering by kappa- and lambda-carrageenan solutions

The intensity correlation functions of kappa- and lambda-carrageenan in various salt solutions and at different concentrations have been determined with the help of dynamic light scattering. From the first cumulant of these correlation functions the values of the translational diffusion coefficients D have been derived. They increase with macromolecular concentration. The extrapolated values to infinite dilution of the diffusion coefficients increase with increasing salt concentration as expected from the salt concentration dependence of the r.m.s. radii of gyration determined previously by static light scattering. The translational diffusion coefficient of lambda-carrageenan in 0.1 M NaCl is smaller than the corresponding value for the kappa species. This is consistent with the difference in contour length and linear charge density of the two samples used. No satisfactory interpretation for the concentration dependence of the diffusion coefficient seems to be possible at present. Although current theories for the macromolecular and salt concentration dependence of D, taking into account charge effects, seem to be applicable, they do not allow for a consistent interpretation of the data. No specific difference between the solution behaviour of kappa- and lambda-carrageenan has been detected.     

Structure-based analysis of the beta8 interactive sequence of human alphaB crystallin

The functional importance of the beta8 sequence ((131)LTITSSLS(138)), which is on the surface of the alpha crystallin core domain of human alphaB crystallin, was evaluated using site-directed mutagenesis. Ultraviolet circular dichroism determined that mutating the surface-exposed, nonconserved residues, Leu-131, Thr-132, Thr-134, Ser-135, Ser-136, and Ser-138 individually or in combination (alphaAbeta8 and CEbeta8), had no measurable effect on secondary and tertiary structure. Size exclusion chromatography determined the size of the complexes formed by the beta8 mutants to be 6-8 subunits larger than wt alphaB crystallin. In chaperone assays, the protective effect of the L131S, T132A, and S135C mutants of the beta8 sequence was similar to wt alphaB crystallin when beta(L) crystallin and alcohol dehydrogenase were the chaperone substrates and decreased to 66% when citrate synthase was the chaperone substrate. In contrast, the chaperone activity for all three substrates was dramatically reduced for the T134K, S138A, S136H, and CEbeta8 mutants. The prominent location of Thr-134, Ser-136, and Ser-138 on the exposed surface of the alpha crystallin core domain could account for the effect on complex assembly and chaperone activity. Modulation of chaperone activity by the exposed residues of the beta8 sequence in the alpha crystallin core domain was independent of complex size. The results established the beta3-beta8-beta9 surface of the alpha crystallin core domain as an interface for complex assembly and chaperone activity.     

Genetic factors influence cataract formation in alpha 3 connexin knockout mice

Connexin alpha 3 (Cx46 or Gja3) gene targeted null mice developed lens nuclear cataracts shortly after birth. A large variance in the cataracts was observed in alpha 3 null sibs on a mixed 129SvJae X C57BL/6J F3 background. This suggested that the genetic background might influence the cataract phenotype. Therefore, we placed the alpha 3 null mutation into a 129SvJae background, and also backcrossed the mutation for six generations into 129SvJ and C57BL/6J backgrounds. While alpha 3 nulls on the two 129 backgrounds contained severe cataracts associated with gamma crystallin cleavage, alpha 3 nulls on the C57B16 background had far milder cataracts with no detectable gamma crystallin cleavage. These findings suggest that a genetic modifier exists that influences gamma crystallin stability, and that gamma crystallin breakdown is associated with severe nuclear cataracts.     

Focussed beam reflectance measurement (FBRM) monitoring of particle size and morphology in suspension cultures of Morinda citrifolia and Centaurea calcitrapa

Laser light scattering technology, as applied in the Lasentec focussed beam reflectance measurement (FBRM) system, was used to characterise two morphologically dissimilar plant cell suspension cultures, Morinda citrifolia and Centaurea calcitrapa. Shake-flask suspensions were analysed in terms of biomass concentration and aggregate size/shape over the course of typical batch growth cycles. For the heavily aggregated C. calcitrapa, biomass levels [from 10-160 g fresh weight (fw) l(-1))] were linearly correlated with FBRM counts. For M. citrifolia, which grows in unbranched chains of 2-10 elongated cells, linear correlation of biomass concentration with FBRM counts was applicable in the range 0-100 g fw l(-1); at higher levels (100-300 g fw l(-1)), biomass was non-linearly correlated with FBRM counts and length-weighted average FBRM chord length. For both cell systems, particle morphology (size/shape) was quantified using semi-automated digital image analysis. The average aggregate equivalent diameter (C. calcitrapa) and average chain length (M. citrifolia), determined using image analysis, closely tracked the FBRM average chord length. The data clearly demonstrate the potential for applying the FBRM technique for rapid characterisation of plant cell suspension cultures.     

Quantitative characterization of the concentration-dependent interaction between molecules of Dextran 70 in aqueous solution: Measurement and analysis in the context of thermodynamic and compressible sphere models

The static light scattering and sedimentation equilibrium of solutions of Dextran 70 were measured as functions of concentration up to 100 g/L in pH 7.4 phosphate-buffered saline at temperatures between 5 and 37 °C. The concentration dependence of scattering intensity and the apparent molar mass obtained from sedimentation equilibrium were found to be nearly independent of temperature over this range to within the uncertainty of measurement. Global analysis of the concentration dependence of both properties yielded a reliable estimate of the concentration-dependent thermodynamic activity coefficient, a quantitative measure of the free energy of self-interaction. The self-interaction between Dextran molecules is compared with that of a globular protein (BSA) and a highly crosslinked polymer of similar molar mass (Ficoll 70). The observed concentration dependence of the free energy of Dextran self-interaction may be quantitatively accounted for by a semi-empirical model in which the polymer molecule is represented by a compressible sphere.     

Association of actin with alpha crystallins

The alpha crystallins are cytosolic proteins that co-localize and co-purify with actin-containing microfilaments. Affinity column chromatography employing both covalently-coupled actin or alpha crystallin was used to demonstrate specific and saturable binding of actin with alpha crystallin. This conclusion was confirmed by direct visualization of alpha aggregates bound to actin polymerized in vitro. The significance of this interaction in relation to the functional properties of these two polypeptides will be discussed.     

Study of a lipopolysaccharide-protein complex from Yersinia pseudotuberculosis in aqueous solutions by light scattering

Lipopolysaccharide-protein complex isolated from Yersinia pseudotuberculosis forms aggregates in aqueous solutions. Dissociation of aggregates was found by light scattering method for 1 M sodium chloride, 1 M guanidine chloride, and urea solutions. Very large particles were detected also in these solutions and acid media. Little light scattering alterations have been observed for 0.27 g/l solution, and pronounced effect of temperature has been detected for more diluted solutions.     

The Effect of Attractive Interactions and Macromolecular Crowding on Crystallins Association

In living systems proteins are typically found in crowded environments where their effective interactions strongly depend on the surrounding medium. Yet, their association and dissociation needs to be robustly controlled in order to enable biological function. Uncontrolled protein aggregation often causes disease. For instance, cataract is caused by the clustering of lens proteins, i.e., crystallins, resulting in enhanced light scattering and impaired vision or blindness. To investigate the molecular origins of cataract formation and to design efficient treatments, a better understanding of crystallin association in macromolecular crowded environment is needed. Here we present a theoretical study of simple coarse grained colloidal models to characterize the general features of how the association equilibrium of proteins depends on the magnitude of intermolecular attraction. By comparing the analytic results to the available experimental data on the osmotic pressure in crystallin solutions, we identify the effective parameters regimes applicable to crystallins. Moreover, the combination of two models allows us to predict that the number of binding sites on crystallin is small, i.e. one to three per protein, which is different from previous estimates. We further observe that the crowding factor is sensitive to the size asymmetry between the reactants and crowding agents, the shape of the protein clusters, and to small variations of intermolecular attraction. Our work may provide general guidelines on how to steer the protein interactions in order to control their association.     

Static light scattering from concentrated protein solutions, I: General theory for protein mixtures and application to self-associating proteins

Exact expressions for the static light scattering of a solution containing up to three species of point-scattering solutes in highly nonideal solutions at arbitrary concentration are obtained from multicomponent scattering theory. Explicit expressions for thermodynamic interaction between solute molecules, required to evaluate the scattering relations, are obtained using an equivalent hard particle approximation similar to that employed earlier to interpret scattering of a single protein species at high concentration. The dependence of scattering intensity upon total protein concentration is calculated for mixtures of nonassociating proteins and for a single self-associating protein over a range of concentrations up to 200 g/l. An approximate semiempirical analysis of the concentration dependence of scattering intensity is proposed, according to which the contribution of thermodynamic interaction to scattering intensity is modeled as that of a single average hard spherical species. Simulated data containing pseudo-noise comparable in magnitude to actual experimental uncertainty are modeled using relations obtained from the proposed semiempirical analysis. It is shown that by using these relations one can extract from the data reasonably reliable information about underlying weak associations that are manifested only at very high total protein concentration.     

Ubiquitin-protein conjugates and alpha B crystallin are selectively present in cells undergoing major cytomorphological reorganisation in early chicken embryos.

Ubiquitin-protein conjugates and alpha B crystallin are detected immunohistochemically in cells undergoing extensive morphological reorganisation in early chicken embryos. Cytoplasmic ubiquitinated proteins and alpha B crystallin are coordinately found in cells of the lens, notochord and myotome. The antigens appear in the myotome cells precisely at the point at which the cells begin to migrate from the dorsomedial lip of the dermamyotome. The findings indicate that ubiquitin and alpha B crystallin may have a coordinate role in the extensive architectural remodeling which occurs in these developing tissues in the early chick embryo. Some form of functional association between protein ubiquitination and alpha B crystallin in cells may explain why alpha B crystallin is found with ubiquitin-protein deposits in some neurodegenerative diseases.     

Increased light scattering resolution facilitates multidimensional flow cytometric analysis

Multidimensional flow cytometry identifies cell populations as clusters in a space created by the analysis of multiple parameters simultaneously. Optimal use of this multidimensional space requires each of the individual parameters to provide additional information for cell population discrimination as well as maximum utilization of the dynamic range available for each parameter. In this study we improve the visualization of the information present in light scattering signals from leukocytes to facilitate multidimensional flow cytometric analysis. Optimization of cell preparation techniques are essential to obtain high resolution light scattering signals that give complete separation of the granulocytes, monocytes, and granular and nongranular lymphocytes. The angle at which the forward scattered light was collected was modified to enhance the separation between leukocyte populations. Although orthogonal light scattering signals separate granular and nongranular lymphocytes, the resolution and dynamic range could not be displayed using linear or logarithmic functions. By applying a polynomial function to the orthogonal light scattering signals, all leukocyte populations could be displayed while maintaining high resolution. The combination of high resolution light scattering with a nonlinear display resulted in an equally spaced distribution of the cell populations distinguished by correlating forward and orthogonal light scattering signals. Using this approach, peripheral blood neutrophils, eosinophils, basophils, monocytes, and granular and nongranular lymphocytes were shown to occupy distinct locations in the correlation of orthogonal and forward light scattering. Surprisingly, the basophilic granulocytes were located close to granular lymphocytes and monocytes rather than near neutrophils and eosinophils.     

The thermal structural transition of alpha-crystallin modulates subunit interactions and increases protein solubility

BACKGROUND: Alpha crystallin is an oligomer composed of two types of subunits, alpha-A and alpha-B crystallin, and is the major constituent of human lens. The temperature induced condensation of alpha-crystallin, the main cause for eye lens opacification (cataract), is a two step-process, a nucleation followed by an aggregation phase, and a protective effect towards the aggregation is exhibited over the alpha crystallin phase transition temperature (Tc = 318.16 K). METHODS/RESULTS: To investigate if a modulation of the subunit interactions over Tc could trigger the protective mechanism towards the aggregation, we followed, by using simultaneously static and dynamic light scattering, the temperature induced condensation of alpha-crystallin. By developing a mathematical model able to uncouple the nucleation and aggregation processes, we find a previously unobserved transition in the nucleation rate constant. Its temperature dependence allows to determine fundamental structural parameters, the chemical potential (Δμ) and the interfacial tension (γ) of the aggregating phase, that characterize subunit interactions. CONCLUSIONS/GENERAL SIGNIFICANCE: The decrease of both Δμ and γ at Tc, and a relative increase in solubility, reveal a significative decrease in the strenght of alpha-crystallin subunits interactions, which protects from supramolecolar condensation in hypertermic conditions. On the whole, we suggest a general approach able to understand the structural and kinetic mechanisms involved in aggregation-related diseases and in drugs development and testing.