The small heat-shock chaperone protein, alpha-crystallin, does not recognize stable molten globule states of cytosolic proteins.

The small heat-shock protein (sHsp), alpha-crystallin, acts as a molecular chaperone by interacting with destabilized 'substrate' proteins to prevent their precipitation from solution under conditions of stress. alpha-Crystallin and all sHsps are intracellular proteins. Similarly to other chaperones, the 'substrate' protein is in an intermediately folded, partly structured molten globule state when it interacts and complexes with alpha-crystallin. In this study, stable molten globule states of the cytosolic proteins, gamma-crystallin and myoglobin, have been prepared. Within the lens, gamma-crystallin naturally interacts with alpha-crystallin and myoglobin and alpha-crystallin are present together in muscle tissue. The molten globule states of gamma-crystallin and myoglobin were prepared by reacting gamma-crystallin with glucose 6-phosphate and by removing the haem group of myoglobin. Following spectroscopic characterisation of these modified proteins, their interaction with alpha-crystallin was examined by a variety of spectroscopic and protein chemical techniques. In both cases, there was no interaction with alpha-crystallin that led to complexation. It is concluded that alpha-crystallin does not recognise stable molten globule states of cytosolic 'substrate' proteins and only interacts with molten globule states of proteins that are on the irreversible pathway towards an aggregated and precipitated form.

Non-oxidative modification of lens crystallins by kynurenine: a novel post-translational protein modification with possible relevance to ageing and cataract.

In humans, the crystallin proteins of the ocular lens become yellow-coloured and fluorescent with ageing. With the development of senile nuclear cataract, the crystallins become brown and additional fluorophores are formed. The mechanism underlying crystallin colouration is not known but may involve interaction with kynurenine-derived UV filter compounds. We have recently identified a sulphur-linked glutathionyl-3-hydroxykynurenine glucoside adduct in the lens and speculated that kynurenine may also form adducts with GSH and possibly with nucleophilic amino acids of the crystallins (e.g. Cys). Here we show that kynurenine modifies calf lens crystallins non-oxidatively to yield coloured (365 nm absorbing), fluorescent (Ex 380 nm/Em 450-490 nm) protein adducts. Carboxymethylation and succinylation of crystallins inhibited kynurenine-mediated modification by approx. 90%, suggesting that Cys, Lys and possibly His residues may be involved. This was confirmed by showing that kynurenine formed adducts with GSH as well as with poly-His and poly-Lys. NMR studies revealed that the novel poly-Lys-kynurenine covalent linkage was via the epsilon-amino group of the Lys side chain and the betaC of the kynurenine side chain. Analysis of tryptic peptides of kynurenine-modified crystallins revealed that all of the coloured peptides contained either His, Cys or an internal Lys residue. We propose a novel mechanism of kynurenine-mediated crystallin modification which does not require UV light or oxidative conditions as catalysts. Rather, we suggest that the side chain of kynurenine-derived lens UV filters becomes deaminated to yield an alpha,beta-unsaturated carbonyl which is highly susceptible to attack by nucleophilic amino acid residues of the crystallins. The inability of the lens fibre cells to metabolise their constituent proteins results in the accumulation of coloured/fluorescent crystallins with age.

Formation of betaA3/betaB2-crystallin mixed complexes: involvement of N- and C-terminal extensions.

The sequence extensions of the beta-crystallin subunits have been suggested to play an important role in the oligomerization of these eye lens proteins. This, in turn, may contribute to maintaining lens transparency and proper light refraction. In homo-dimers of the betaA3- and betaB2-crystallin subunits, these extensions have been shown by (1)H-NMR spectroscopy to be solvent-exposed and highly flexible. In this study, we show that betaA3- and betaB2-crystallins spontaneously form mixed betaA3/betaB2-crystallin complexes, which, from analytical ultracentrifugation experiments, are dimeric at low concentrations (<1 mg ml(-1)) and tetrameric at higher protein concentrations. (1)H-NMR spectroscopy reveals that in the betaA3/betaB2-crystallin tetramer, the N-terminal extensions of betaA3-crystallin remain water-exposed and flexible, whereas both N- and C-terminal extensions of betaB2-crystallin lose their flexibility. We conclude that both extensions of betaB2-crystallin are involved in protein-protein interactions in the betaA3/betaB2-crystallin hetero-tetramer. The extensions may stabilize and perhaps promote the formation of this mixed complex.

Macromolecular crowding: effects on actin polymerisation.

Dextran has been found to enhance the polymerisation of actin. This enhancement increases exponentially with increasing mass concentrations of dextran, in a manner that is consistent with excluded volume theory. Mathematical prediction of experimental results is difficult due to the fact that all participating species, namely F-actin, G-actin and dextran are best represented by differently shaped hard particles. Modelling dextran as a sphere of radius defined by an effective thermodynamic radius (Reff), we have predicted our experimental results to an acceptable degree, given the relative crudity of the model. The results imply that the highly crowded cellular environment may help to stabilise the filamentous actin network in vivo.

NMR spectroscopy of alpha-crystallin. Insights into the structure, interactions and chaperone action of small heat-shock proteins.

The subunit molecular mass of alpha-crystallin, like many small heat-shock proteins (sHsps), is around 20 kDa although the protein exists as a large aggregate of average mass around 800 kDa. Despite this large size, a well-resolved 1H NMR spectrum is observed for alpha-crystallin which arises from short, polar, highly-flexible and solvent-exposed C-terminal extensions in each of the subunits, alpha A- and alpha B-crystallin. These extensions are not involved in interactions with other proteins (e.g. beta- and gamma-crystallins) under non-chaperone conditions. As determined by NMR studies on mutants of alpha A-crystallin with alterations in its C-terminal extension, the extensions have an important role in acting as solubilising agents for the relatively-hydrophobic alpha-crystallin molecule and the high-molecular-weight (HMW) complex that forms during the chaperone action. The related sHsp, Hsp25, also exhibits a flexible C-terminal extension. Under chaperone conditions, and in the HMW complex isolated from old lenses, the C-terminal extension of the alpha A-crystallin subunit maintains its flexibility whereas the alpha B-crystallin subunit loses, at least partially, its flexibility, implying that it is involved in interaction with the 'substrate' protein. The conformation of 'substrate' proteins when they interact with alpha-crystallin has been probed by 1H NMR spectroscopy and it is concluded that alpha-crystallin interacts with 'substrate' proteins that are in a disordered molten globule state, but only when this state is on its way to large-scale aggregation and precipitation. By monitoring the 1H and 31P NMR spectra of alpha-crystallin in the presence of increasing concentrations of urea, it is proposed that alpha-crystallin adopts a two-domain structure with the larger C-terminal domain unfolding first in the presence of denaturant. All these data have been combined into a model for the quaternary structure of alpha-crystallin. The model has two layers each of approximately 40 subunits arranged in an annulus or toroid. A large central cavity is present whose entrance is ringed by the flexible C-terminal extensions. A large hydrophobic region in the aggregate is exposed to solution and is available for interaction with 'substrate' proteins during the chaperone action.

The molecular chaperone alpha-crystallin is in kinetic competition with aggregation to stabilize a monomeric molten-globule form of alpha-lactalbumin.

In vivo, alpha-crystallin and other small heat-shock proteins (sHsps) act as molecular chaperones to prevent the precipitation of 'substrate' proteins under stress conditions through the formation of a soluble sHsp-substrate complex. Using a range of different salt conditions, the rate and extent of precipitation of reduced alpha-lactalbumin have been altered. The interaction of alpha-crystallin with reduced alpha-lactalbumin under these various salt conditions was then studied using a range of spectroscopic techniques. Under conditions of low salt, alpha-lactalbumin aggregates but does not precipitate. alpha-Crystallin is able to prevent this aggregation, initially by stabilization of a monomeric molten-globule species of alpha-lactalbumin. It is proposed that this stabilization occurs through weak transient interactions between alpha-crystallin and alpha-lactalbumin. Eventually a stable, soluble high-molecular-mass complex is formed between the two proteins. Thus it appears that a tendency for alpha-lactalbumin to aggregate (but not necessarily precipitate) is the essential requirement for alpha-crystallin-alpha-lactalbumin interaction. In other words, alpha-crystallin interacts with a non-aggregated form of the substrate to prevent aggregation. The rate of precipitation of alpha-lactalbumin is increased significantly in the presence of Na2SO4 compared with NaCl. However, in the former case, alpha-crystallin is unable to prevent this aggregation and precipitation except in the presence of a large excess of alpha-crystallin, i.e. at mass ratios more than 10 times greater than in the presence of NaCl. It is concluded that a kinetic competition exists between aggregation and interaction of unfolding proteins with alpha-crystallin.

The interaction of the molecular chaperone, alpha-crystallin, with molten globule states of bovine alpha-lactalbumin.

Small heat shock proteins function in a chaperone-like manner to prevent the precipitation of proteins under conditions of stress (e. g. heat). alpha-Crystallin, the major mammalian lens protein, is a small heat shock protein. The mechanism of chaperone action of these proteins is poorly understood. In this paper, the conformational state of a protein when it forms a high molecular weight complex with alpha-crystallin is investigated by examining, using NMR spectroscopy and size exclusion high performance liquid chromatography, the interaction of alpha-crystallin with alpha-lactalbumin and its various intermediately folded (molten globule) states. The complex is formed following reduction of alpha-lactalbumin by dithiothreitol in the presence of alpha-crystallin, and this interaction has been monitored in real time by 1H NMR spectroscopy. It is concluded that alpha-crystallin interacts with a disordered molten globule state of alpha-lactalbumin while it is on an irreversible pathway toward aggregation and precipitation. alpha-Crystallin does not interact, however, with molten globule states of alpha-lactalbumin that are stable in solution, e.g. the reduced and carboxyamidated species. It is proposed that alpha-crystallin distinguishes between the various molten globule states of alpha-lactalbumin on the basis of the lifetimes of these states, i.e. the protein must be in a disordered molten globule state for a significant length of time and on the pathway to aggregation and precipitation for interaction to occur.

The molecular chaperone, alpha-crystallin, inhibits amyloid formation by apolipoprotein C-II.

Under lipid-free conditions, human apolipoprotein C-II (apoC-II) exists in an unfolded conformation that over several days forms amyloid ribbons. We examined the influence of the molecular chaperone, alpha-crystallin, on amyloid formation by apoC-II. Time-dependent changes in apoC-II turbidity (at 0.3 mg/ml) were suppressed potently by substoichiometric subunit concentrations of alpha-crystallin (1-10 microg/ml). alpha-Crystallin also inhibits time-dependent changes in the CD spectra, thioflavin T binding, and sedimentation coefficient of apoC-II. This contrasts with stoichiometric concentrations of alpha-crystallin required to suppress the amorphous aggregation of stressed proteins such as reduced alpha-lactalbumin. Two pieces of evidence suggest that alpha-crystallin directly interacts with amyloidogenic intermediates. First, sedimentation equilibrium and velocity experiments exclude high affinity interactions between alpha-crystallin and unstructured monomeric apoC-II. Second, the addition of alpha-crystallin does not lead to the accumulation of intermediate sized apoC-II species between monomer and large aggregates as indicated by gel filtration and sedimentation velocity experiments, suggesting that alpha-crystallin does not inhibit the relatively rapid fibril elongation upon nucleation. We propose that alpha-crystallin interacts stoichiometrically with partly structured amyloidogenic precursors, inhibiting amyloid formation at nucleation rather than the elongation phase. In doing so, alpha-crystallin forms transient complexes with apoC-II, in contrast to its chaperone behavior with stressed proteins.

Biological effects of electromagnetic fields--mechanisms for the effects of pulsed microwave radiation on protein conformation.

Microwave exposure under "athermal" conditions occurs when no temperature rise can be measured by conventional thermometry. The existence of biological effects arising from the athermal exposure is still controversial, partly because of a lack of the linear dose response relation. We propose a model in which pulsed microwave radiation causes a triggering of the heat shock or stress response by altering the conformation of proteins through a transient heating of the protein and its close environment. We support this by modelling using the heat diffusion equation and show that pulsed exposure even when athermal can lead to transient temperature excursions outside the normal range. We propose that the power window phenomenon in which biological effects are observed at low power levels may be caused by an incomplete triggering of the heat shock response.

Structural alterations of alpha-crystallin during its chaperone action.

The small heat-shock protein, alpha-crystallin, has chaperone ability whereby it stabilises proteins under stress conditions. In this study, alterations in the structure of alpha-crystallin during its interaction with a variety of substrate proteins (insulin, alpha-lactalbumin, ovotransferrin and serum albumin) under stress conditions have been examined using visible absorption, 31P-NMR and 1H-NMR and fluorescence spectroscopy. The fluorescence and 31P-NMR data imply that during the chaperone action of alpha-crystallin under reducing conditions, there is a slight increase in hydrophilicity of its N-terminal region and an alteration in flexibility of its C-terminal region, but overall, alpha-crystallin does not undergo a gross structural change. The fluorescence data suggest that substrate proteins interact with alpha-crystallin in a molten globule or intermediately folded state. The same conclusion is made from 1H-NMR spectroscopic monitoring of the interaction of alpha-crystallin with substrate proteins, e.g. the insulin B chain. The stoichiometry of interaction between alpha-crystallin and the various substrate proteins reveals that steric factors are important in determining the efficiency of interaction between the two proteins, i.e. on a molar subunit basis, alpha-crystallin is a more efficient chaperone protein with smaller substrate proteins. Comparison is also made between the high-molecular-mass (HMM) complexes formed between alpha-crystallin and ovotransferrin when reduced and heat stressed. Under heating conditions, fluorescence spectroscopy indicates that the HMM complex has a greater exposure of hydrophobicity to solution than that formed by reduction. Furthermore, in interacting with heated ovotransferrin, the C-terminal extension of the alphaB-crystallin subunit preferentially loses its flexibility suggesting that it is involved in stabilising bound ovotransferrin. By contrast, this extension is only partially reduced in flexibility in the HMM complex formed after reduction of ovotransferrin. The functional role of the C-terminal extensions in the chaperone action and the overall quaternary structure of alpha-crystallin is discussed.

Immobilization of the C-terminal extension of bovine alphaA-crystallin reduces chaperone-like activity.

alpha-Crystallins occur as multimeric complexes, which are able to suppress precipitation of unfolding proteins. Although the mechanism of this chaperone-like activity is unknown, the affinity of alpha-crystallin for aggregation-prone proteins is probably based on hydrophobic interactions. alpha-Crystallins expose a considerable hydrophobic surface to solution, but nevertheless they are very stable and highly soluble. An explanation for this paradox may be that alpha-crystallin subunits have a polar and unstructured C-terminal extension that functions as a sort of solubilizer. In this paper we have described five alphaA-crystallins in which charged and hydrophobic residues were inserted in the C-terminal extension. Introduction of lysine, arginine, and aspartate does not substantially influence chaperone-like activity. In contrast, introduction of a hydrophobic tryptophan greatly diminishes functional activity. CD experiments indicate that this mutant has a normal secondary structure and fluorescence measurements show that the inserted tryptophan is located in a polar environment. However, NMR spectroscopy clearly demonstrates that the presence of the tryptophan residue dramatically reduces the flexibility of the C-terminal extension. Furthermore, the introduction of this tryptophan results in a considerably decreased thermostability of the protein. We conclude that changing the polarity of the C-terminal extension of alphaA-crystallin by insertion of a highly hydrophobic residue can seriously disturb structural and functional integrity.

Identification of glutathionyl-3-hydroxykynurenine glucoside as a novel fluorophore associated with aging of the human lens.

A novel fluorophore was isolated from human lenses using high performance liquid chromatography (HPLC). The new fluorophore was well separated from 3-hydroxykynurenine glucoside (3-OHKG) and its deaminated isoform, 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid O-glucoside, which are known UV filter compounds. The new compound exhibited UV absorbance maxima at 260 and 365 nm, was fluorescent (Ex(360 nm)/Em(500 nm)), and increased in concentration with age. Further analysis of the purified compound by microbore HPLC with in-line electrospray ionization mass spectrometry revealed a molecular mass of 676 Da. This mass corresponds to that of an adduct of GSH with a deaminated form of 3-OHKG. This adduct was synthesized using 3-OHKG and GSH as starting materials. The synthetic glutathionyl-3-hydroxykynurenine glucoside (GSH-3-OHKG) adduct had the same HPLC elution time, thin-layer chromatography R(F) value, UV absorbance maxima, fluorescence characteristics, and mass spectrum as the lens-derived fluorophore. Furthermore, the (1)H and (13)C NMR spectra of the synthetic adduct were entirely consistent with the proposed structure of GSH-3-OHKG. These data indicate that GSH-3-OHKG is present as a novel fluorophore in aged human lenses. The GSH-3-OHKG adduct was found to be less reactive with beta-glucosidase compared with 3-OHKG, and this could be due to a folded conformation of the adduct that was suggested by molecular modeling.

Mouse Hsp25, a small shock protein. The role of its C-terminal extension in oligomerization and chaperone action.

Under conditions of cellular stress, small heat shock proteins (sHsps), e.g. Hsp25, stabilize unfolding proteins and prevent their precipitation from solution. 1H NMR spectroscopy has shown that mammalian sHsps possess short, polar and highly flexible C-terminal extensions. A mutant of mouse Hsp25 without this extension has been constructed. CD spectroscopy reveals some differences in secondary and tertiary structure between this mutant and the wild-type protein but analytical ultracentrifugation and electron microscopy show that the proteins have very similar oligomeric masses and quaternary structures. The mutant shows chaperone ability comparable to that of wild-type Hsp25 in a thermal aggregation assay using citrate synthase, but does not stabilize alpha-lactalbumin against precipitation following reduction with dithiothreitol. The accessible hydrophobic surface of the mutant protein is less than that of the wild-type protein and the mutant is also less stable at elevated temperature. 1H NMR spectroscopy reveals that deletion of the C-terminal extension of Hsp25 leads to induction of extra C-terminal flexibility in the molecule. Monitoring complex formation between Hsp25 and dithiothreitol-reduced alpha-lactalbumin by 1H NMR spectroscopy indicates that the C-terminal extension of Hsp25 retains its flexibility during this interaction. Overall, these data suggest that a highly flexible C-terminal extension in mammalian sHsps is required for full chaperone activity.

The mammalian small heat-shock protein Hsp20 forms dimers and is a poor chaperone.

Hsp20 is one of the newly described members of the mammalian small heat-shock protein (sHsp) family. It occurs most abundantly in skeletal muscle and heart. We isolated clones for Hsp20 from a rat heart cDNA library, and expressed the protein in Escherichia coli to characterize this little known sHsp. Recombinant Hsp20 displayed similar far-ultraviolet circular dichroism spectra as the most closely related sHsp, alpha B-crystallin, but was less heat stable, denaturing upon heating to 50 degrees C. While other mammalian recombinant sHsps form large multimeric complexes, Hsp20 occurs in two complex sizes, 43-kDa dimers and 470-kDa multimers. The ratio between the two forms depends on protein concentration. Moreover, Hsp20 has a much lower chaperone-like activity than alpha B-crystallin, as indicated by its relatively poor capacity to diminish the reduction-induced aggregation of insulin B chains. Hsp20 is considerably shorter at the C-terminus and less polar than other sHsps, but 1H-NMR spectroscopy reveals that the last 10 residues are flexible, as in the other sHsps. Our findings suggest that Hsp20 is a special member of the sHsp family in being less heat stable and tending to form dimers. These properties, together with the shorter and less polar C-terminal extension, may contribute to the less effective chaperone-like activity.