Calcium-induced decrease of the thermal stability and chaperone activity of alpha-crystallin

Alpha-crystallin, one of the major proteins in the vertebrate eye lens, acts as a molecular chaperone, like the small heat-shock proteins, by protecting other proteins from denaturing under stress or high temperature conditions. alpha-Crystallin aggregation is involved in lens opacification, and high [Ca(2+)] has been associated with cataract formation, suggesting a role for this cation in the pathological process. We have investigated the effect of Ca(2+) on the thermal stability of alpha-crystallin by UV and Fourier-transform infrared (FTIR) spectroscopies. In both cases, a Ca(2+)-induced decrease in the midpoint of the thermal transition is detected. The presence of high [Ca(2+)] results also in a marked decrease of its chaperone activity in an insulin-aggregation assay. Furthermore, high Ca(2+) concentration decreases Cys reactivity towards a sulfhydryl reagent. The results obtained from the spectroscopic analysis, and confirmed by circular dichroism (CD) measurements, indicate that Ca(2+) decreases both secondary and tertiary-quaternary structure stability of alpha-crystallin. This process is accompanied by partial unfolding of the protein and a clear decrease in its chaperone activity. It is concluded that Ca(2+) alters the structural stability of alpha-crystallin, resulting in impaired chaperone function and a lower protective ability towards other lens proteins. Thus, alpha-crystallin aggregation facilitated by Ca(2+) would play a role in the progressive loss of transparency of the eye lens in the cataractogenic process.     

Quaternary interactions in eye lens beta-crystallins: basic and acidic subunits of beta-crystallins favor heterologous association

beta-Crystallins are complex eye lens proteins made up of several related basic and acidic subunits that combine to form differently sized oligomers each displaying extensive polydispersity. As the sequences are homologous to the X-ray-determined bilobal structure of gamma-crystallin, beta-subunits are visualized as having a similar structure with additional N- and C-terminal extensions. Two basic (beta B2 and beta B3) and two acidic (beta A3 and beta A4) subunits have been isolated in deaggregating media, refolded, and reassociated in various combinations to determine which components favor dimers or higher oligomers. Homopolymers were compared with beta B2 homodimer in terms of charge, using Mono Q fast protein liquid chromatography, and size, using Superose 12 chromatography. Heterooligomeric formations were monitored by their intermediate charge properties compared with homooligomers. beta B2 associates with either beta B3- or beta A4-forming heterodimers whereas a larger oligomer is formed with beta A3. Naturally occurring beta-crystallin oligomers were analyzed by Mono Q chromatography and PhastGel electrophoresis. Whereas beta B2, beta B3, and beta A4 can each be reassociated to homodimers, beta A4 dimers are not found in native beta-crystallins. beta B2-beta A3 is a major component of intermediate-sized beta L1-crystallin and is absent from dimeric beta L2-crystallin. It is suggested that the pH dependence of the size of beta L1-crystallin is due to a dimer to tetramer equilibrium. By following dimer interactions using Superose 12 chromatography, beta B2-beta A4 was shown to interact with beta B2-beta A3. A model of beta-crystallin structure is proposed based on beta-subunits forming dimers with the next level of organization requiring an acidic subunit, beta A3, with a long N-terminal extension.     

Substrate binding changes conformation of the alpha-, but not the beta-subunit of mitochondrial processing peptidase

Lifetime analysis of tryptophan fluorescence of the mitochondrial processing peptidase (MPP) from Saccharomyces cerevisiae clearly proved that substrate binding evoked a conformational change of the alpha-subunit while presence of substrate influenced neither the lifetime components nor the average lifetime of the tryptophan excited state of the beta-MPP subunit. Interestingly, lifetime analysis of tryptophan fluorescence decay of the alpha-MPP subunit revealed about 11% of steady-state fractional intensity due to the long-lived lifetime component, indicating that at least one tryptophan residue is partly buried at the hydrophobic microenvironment. Computer modeling, however, predicted none of three tryptophans, which the alpha-subunit contains, as deeply buried in the protein matrix. We conclude this as a consequence of a possible dimeric (oligomeric) structure.     

Age-related changes of alpha-crystallin aggregate in human lens

Summary. Lens alpha-crystallin, composed of two subunits alpha A- and alpha B-crystallin, forms large aggregates in the lens of the eye. The present study investigated the aggregate of human lens alpha-crystallin from elderly and young donors. Recombinant alpha A- and alpha B-crystallins in molar ratios of alpha A to alpha B at 1:1, corresponding to the aged sample, were also studied in detail. We found by ultra-centrifugation analysis that the alpha-crystallin aggregate from elderly donors was large and heterogeneous with an average sedimentation coefficient of 30 S and a range of 20–60 S at 37 °C. This was higher compared to the young samples that had an average sedimentation coefficient of 17 S. The sedimentation coefficients of recombinant alpha A- and alpha B-crystallins were approximately 12 S and 15 S, respectively. Even when recombinant alpha-crystallins were mixed in molar ratios equivalent to those found in vivo, similar S values as the native aged alpha-crystallin aggregates were not obtained. Changes in the self-association of alpha-crystallin aggregate were correlated to changes in chaperone activity. Alpha-crystallin from young donors, and recombinant alpha A- and alpha B-crystallin and their mixtures showed chaperone activity, which was markedly lost in samples from the aged alpha-crystallin aggregates.     

Metal-based affinity separation of alpha- and gamma-chymotrypsin and thermal stability analysis of isolates.

Chymotrypsin preparations are contaminated by autolysis products and other post-translational products derived from the zymogen. Some prevalent contaminants are difficult to detect with activity assays and molecular weight separation. However, our differential scanning calorimetry (DSC) studies of alpha-chymotrypsin have revealed that gamma-chymotrypsin is a common contaminant in commercial preparations, and the alpha- and gamma-species differ significantly in thermal stability. Thus, DSC analysis provides a sensitive and rapid means to assess the homogeneity of preparations. Moreover, free metal ion binding studies conducted indicate that the alpha- and gamma-species differ substantially in the number of metal binding sites and metal affinity. Therefore, to attempt to repurify a commercial preparation of alpha-chymotrypsin, a resolubilized sample of alpha-chymotrypsin was subjected to immobilized metal (Cu+2) affinity chromatography with pH elution and the fractions were subjected to DSC analysis. The process successfully removed the majority of the contaminating gamma-chymotrypsin.     

Redox- and pH-dependent association of plastocyanin with lipid bilayers: effect on protein conformation and thermal stability

The effect of electrostatic interactions on the conformation and thermal stability of plastocyanin (Pc) was studied by infrared spectroscopy. Association of any of the two redox states of the protein with positively charged membranes at neutral pH does not significantly change the secondary structure of Pc. However, upon membrane binding, the denaturation temperature decreases, regardless of the protein redox state. The extent of destabilization depends on the proportion of positively charged lipid headgroups in the membrane, becoming greater as the surface density of basic phospholipids increases. In contrast, at pH 4.8 the membrane binding-dependent conformational change becomes redox-sensitive. While the secondary structures and thermal stabilities of free and membrane-bound oxidized Pc are similar under acidic conditions, the conformation of the reduced form of the protein drastically rearranges upon membrane association. This rearrangement does not depend on electrostatic interactions to occur, since it is also observed in the presence of uncharged lipid bilayers. The conformational transition, only observed for reduced Pc, involves the exposure of hydrophobic regions that leads to intermolecular interactions at the membrane surface. Membrane-mediated partial unfolding of reduced Pc can be reversed by readjusting the pH to neutrality, in the absence of electrostatic interactions. This redox-dependent behavior might reflect specific structural requirements for the interaction of Pc with its redox partners.     

Effects of monofunctional platinum binding on the thermal stability and conformation of a self-complementary 22-mer

We investigated the effect of various monofunctional platinum complexes on the thermal stability and conformation of a self-complementary 22-mer duplex oligonucleotide by means of CD and UV melting profiles. We studied several families of triamine complexes of the general formula PtA2AmCl where A2=(NH3)2 and ethylenediamine and where Am=N1-4-methyl-pyridine, N7-guanosine, and 9-ethyl-guanine. Platination by the N1-4-methyl-pyridine and 9-ethyl-guanine complexes led to a decrease in the Tm of the oligonucleotide by 2-11.5 degrees C while platination with the N7-guanosine complexes led to a rise in the melting temperature of the oligonucleotides by 4.5 degrees C. A similar inverse correlation between the two groups of platinum compounds was found in the CD spectra. In all cases, the cis isomer had a more pronounced effect on both the melting curve and the CD spectrum. The cis isomer was found to have a more destabilizing effect than its trans counterpart. This indicates that the cis geometry in fact forces a greater structural constraint on the backbone of the double helix. We have also found that the sugar of the guanosine has a significant influence on both the Tm and CD spectra; the sugar moiety contributes to the stability of the double helix, probably through the formation of hydrogen bonds.     

Acid-induced changes in thermal stability and fusion activity of influenza hemagglutinin

The conformational and thermal stability of full-length hemagglutinin (HA) of influenza virus (strain X31) has been investigated using a combination of differential scanning calorimetry (DSC), analytical ultracentrifugation, fluorescence, and circular dichroism (CD) spectroscopy as a function of pH. HA sediments as a rosette comprised of 5-6 trimers (31-35 S) over the pH range of 7.4-5.4. The DSC profile of HA in the native state at pH 7.4 is characterized by a single cooperative endotherm with a transition temperature (Tm) of 66 degrees C and unfolding enthalpy (DeltaH(cal)) of 800 kcal x (mol of trimer)(-1). Upon acidification to pH 5.4, there is a significant decrease in the transition temperature (from 66 to 45 degrees C), unfolding enthalpy [from 800 to 260 kcal x (mol of trimer)(-1)], and DeltaH(cal)/DeltaH(vH) ratio (from 3.0 to approximately 1.3). Whereas the far- and near-UV ellipticities are maintained over this pH range, there is an acid-induced increase in surface hydrophobicity and decrease in intrinsic tryptophanyl fluorescence. The major contribution to the DSC endotherm arises from unfolding HA1 domains. The relationship between acid-induced changes in thermal stability and the fusion activity of HA has been examined by evaluating the kinetics and extent of fusion of influenza virus with erythrocytes over the temperature and pH range of the DSC measurements. Surprisingly, X31 influenza virus retains its fusion activity at acidic pH and temperatures significantly below the unfolding transition of HA. This finding is consistent with the notion that the fusion activity of influenza virus may involve structural changes of only a small fraction of HA molecules.     

Zn2+ enhances the molecular chaperone function and stability of alpha-crystallin

Alpha-crystallin, the major eye lens protein, is a molecular chaperone that plays a crucial role in the suppression of protein aggregation and thus in the long-term maintenance of lens transparency. Zinc is a micronutrient of the eye, but its molecular interaction with alpha-crystallin has not been studied in detail. In this paper, we present results of in vitro experiments that show bivalent zinc specifically interacts with alpha-crystallin with a dissociation constant in the submillimolar range (Kd approximately 0.2-0.4 mM). We compared the effect of Zn2+ with those of Ca2+, Cu2+, Mg2+, Cd2+, Pb2+, Ni2+, Fe2+, and Co2+ at 1 mM on the structure and chaperoning ability of alpha-crystallin. An insulin aggregation assay showed that among the bivalent metal ions, only 1 mM Zn2+ improved the chaperone function of alpha-crystallin by 30% compared to that in the absence of bivalent metal ions. Addition of 1 mM Zn2+ increased the yield of alpha-crystallin-assisted refolding of urea-treated LDH to its native state from 33 to 38%, but other bivalent ions had little effect. The surface hydrophobicity of alpha-crystallin was increased by 50% due to the binding of Zn2+. In the presence of 1 mM Zn2+, the stability of alpha-crystallin was enhanced by 36 kJ/mol, and it became more resistant to tryptic cleavage. The implications of enhanced stability and molecular chaperone activity of alpha-crystallin in the presence of Zn2+ are discussed in terms of its role in the long-term maintenance of lens transparency and cataract formation.     

alpha- and beta-secretase: profound changes in Alzheimer's disease

The amyloid plaque, a neuropathological hallmark of Alzheimer's disease, is produced by the deposition of beta-amyloid (Abeta) peptide, which is cleaved from Amyloid Precursor Protein (APP) by the enzyme beta-secretase. Only small amounts of Abeta form in normal brain; more typically this is precluded by the processing of APP by alpha-secretase. Here, we describe a decrease in alpha-secretase (81% of normal) and a large increase in beta-secretase activity (185%) in sporadic Alzheimer's disease temporal cortex. Since alpha-secretase is present principally in neurons known to be vulnerable in Alzheimer's disease, and there is known competition between alpha- and beta-secretase for the substrate APP, it is significant that the majority of Alzheimer samples tested here were low in alpha-secretase. Eighty percent of Alzheimer brains examined had an increase in beta-secretase, a decrease in alpha-secretase, or both; which may account for the means by which the majority of people develop Alzheimer's disease.     

Differences in thermal stability of frog and rabbit alpha alpha- and alpha beta-tropomyosins determined by optical rotatory dispersion

Frog and rabbit alpha alpha- and alpha beta-tropomyosins were purified, and their thermal stabilities determined by use of optical rotatory dispersion. The tropomyosins were found to be virtually completely helical at 5 degrees C. Regions of different thermal stabilities were seen for all tropomyosins. Rabbit and frog alpha alpha-tropomyosin show very similar thermal properties, with main transitions near 47-49 degrees C. The main transition for frog alpha beta-tropomyosin is at 32 degrees C. The results show that the alpha beta-tropomyosins are less stable than the alpha alpha-forms. Only thermal transitions of the alpha beta-forms appear to be correlated with the body temperatures of the animals.     

Heat-induced changes in the mechanics of a collagenous tissue: isothermal, isotonic shrinkage.

We present data from isothermal, isotonic-shrinkage tests wherein bovine chordae tendineae were subjected to well-defined constant temperatures (from 65 to 90 degrees C), durations of heating (from 180 to 3600 s), and isotonic uniaxial stresses during heating (from 100 to 650 kPa). Tissue response during heating and "recovery" at 37 degrees C following heating was evaluated in terms of the axial shrinkage, a gross indicator of underlying heat-induced denaturation. There were three key findings. First, scaling the heating time via temperature and load-dependent characteristic times for the denaturation process collapsed all shrinkage data to a single curve, and thereby revealed a time-temperature-load equivalency. Second, the characteristic times exhibited an Arrhenius-type behavior with temperature wherein the slopes were nearly independent of applied load--this suggested that applied loads during heating affect the activation entropy, not energy. Third, all specimens exhibited a time-dependent, partial recovery when returned to 37 degrees C following heating, but the degree of recovery decreased with increases in the load imposed during heating. These new findings on heat-induced changes in tissue behavior will aid in the design of improved clinical heating protocols and provide guidance for the requisite constitutive formulations.     

Heat-induced changes in the finite strain viscoelastic behavioir of a collaagenous tissue

Supra-physiological temperatures are increasingly being used to treat many different soft need for injuries. To identify improved clinical treatments, however, there is a need for better information on the effect of the mechanics on the thermal damage process as well as the effect of the incurred damage on the subsequent mechanical properties. In this paper we report the first biaxial data on the stress relaxation behavior of a collagenous tissue before and after thermal damage. Based on a two-dimensional finite strain viscoelastic model, which incorporates an exponential elastic response, it is shown that the thermal damage can significantly decrease the characteristic time for stress relaxation and the stress residual.     

Heat-induced thermal resistance and its relationship to lysosomal response.

Two separate effects of hyperthermia on mouse splenic lysosomes have been reported, dependent on the severity of the treatment. Heating to temperatures below 42.5 degrees C causes a transient increase in lysosomal acid phosphatase activity which can be correlated with the ability of moderate hyperthermia to potentiate X-ray damage. Heating to temperatures above 42.5 degrees C results in an immediate increase in lysosomal membrane permeability which may be involved in tissue necrosis. By giving a priming heat treatment at 41.8 degrees C, induced thermal resistance was demonstrated for the lysosomal membrane effect, but not for the enzyme activation. The degree of induced thermal resistance observed is similar to that reported for the cell-killing effect of heat on tissues in vivo and cells in vitro and occurs over a similar time course. The relevance of these results to the understanding of fractionated hyperthermia in cancer therapy is discussed.     

Thermodynamics of thermal and athermal denaturation of gamma-crystallins: changes in conformational stability upon glutathione reaction

The conformational stabilities of bovine lens gamma-crystallin fractions II, IIIA, IIIB, and IVA and those modified with glutathione were compared by studying the thermal and guanidine hydrochloride (Gdn-HCl) denaturation behavior. The conformational state was monitored by both far-UV CD and fluorescence measurements. All the gamma-crystallins studied showed a sigmoidal order-disorder transition with varied melting temperatures. The thermal denaturation of these proteins is reversible up to a temperature 3 or 4 degrees C above T 1/2; above this temperature, irreversible aggregation occurs. The validity of a two-state approximation of both thermal and Gdn-HCl denaturation was tested for all four crystallins, and the presence of one or more intermediates was evident in the unfolding of IVA. delta GDH2O values of these crystallins range from 4 to 9 kcal/mol. Upon glutathione treatment IVA showed the maximum decrease in T 1/2 by approximately 9 degrees C and in delta GDH2O value by 29%; the smallest decrease in T 1/2 was for IIIA by 2 degrees C and in delta GDH2O by 15%. We have demonstrated that the glutathione reaction can dramatically reduce the conformational stability of gamma-crystallins and, thus, that the thermodynamic quantities of the unreacted crystallins can be used to evaluate the stability of these proteins when modified during cataract formation.