Robyn A. Lindner

Immobilization of the C-terminal extension of bovine alpha A-crystallin reduces chaperone-like activity

α-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 α-crystallin for aggregation-prone proteins is probably based on hydrophobic interactions. α-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 α-crystallin subunits have a polar and unstructured C-terminal extension that functions as a sort of solubilizer. In this paper we have described five αA-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 αA-crystallin by insertion of a highly hydrophobic residue can seriously disturb structural and functional integrity.

The Interaction of the Molecular Chaperone α-Crystallin with Unfolding α-Lactalbumin: A Structural and Kinetic Spectroscopic Study

The unfolding of the apo and holo forms of bovine α-lactalbumin (α-LA) upon reduction by dithiothreitol (DTT) in the presence of the small heat-shock protein α-crystallin, a molecular chaperone, has been monitored by visible and UV absorption spectroscopy, mass spectrometry and 1H NMR spectroscopy. From these data, a description and a time-course of the events that result from the unfolding of both forms of the protein, and the state of the protein that interacts with α-crystallin, have been obtained. α-LA contains four disulphide bonds and binds a calcium ion. In apo α-LA, the disulphide bonds are reduced completely over a period of ∼1500 seconds. Fully reduced α-LA adopts a partly folded, molten globule conformation that aggregates and, ultimately, precipitates. In the presence of an equivalent mass of α-crystallin, this precipitation can be prevented via complexation with the chaperone. α-Crystallin does not interfere with the kinetics of the reduction of disulphide bonds in apo α-LA but does stabilise the molten globule state. In holo α-LA, the disulphide bonds are less accessible to DTT, because of the stabilisation of the protein by the bound calcium ion, and reduction occurs much more slowly. A two-disulphide intermediate aggregates and precipitates rapidly. Its precipitation can be prevented only in the presence of a 12-fold mass excess of α-crystallin. It is concluded that kinetic factors are important in determining the efficiency of the chaperone action of α-crystallin. It interacts efficiently with slowly aggregating, highly disordered intermediate (molten globule) states of α-LA. Real-time NMR spectroscopy shows that the kinetics of the refolding of apo α-LA following dilution from denaturant are not affected by the presence of α-crystallin. Thus, α-crystallin is not a chaperone that is involved in protein folding per se. Rather, its role is to stabilise compromised, partly folded, molten globule states of proteins that are destined for precipitation.

The Interaction of the Molecular Chaperone, α-Crystallin, with Molten Globule States of Bovine α-Lactalbumin

Small heat shock proteins function in a chaperone-like manner to prevent the precipitation of proteins under conditions of stress (e.g. heat). α-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 α-crystallin is investigated by examining, using NMR spectroscopy and size exclusion high performance liquid chromatography, the interaction of α-crystallin with α-lactalbumin and its various intermediately folded (molten globule) states. The complex is formed following reduction of α-lactalbumin by dithiothreitol in the presence of α-crystallin, and this interaction has been monitored in real time by 1H NMR spectroscopy. It is concluded that α-crystallin interacts with a disordered molten globule state of α-lactalbumin while it is on an irreversible pathway toward aggregation and precipitation. α-Crystallin does not interact, however, with molten globule states of α-lactalbumin that are stable in solution, e.g. the reduced and carboxyamidated species. It is proposed that α-crystallin distinguishes between the various molten globule states of α-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.

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.

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 (Ex360 nm/Em500 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 chromatographyR F value, UV absorbance maxima, fluorescence characteristics, and mass spectrum as the lens-derived fluorophore. Furthermore, the 1H and 13C 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 β-glucosidase compared with 3-OHKG, and this could be due to a folded conformation of the adduct that was suggested by molecular modeling.

R120G αB-crystallin promotes the unfolding of reduced α-lactalbumin and is inherently unstable

α‐Crystallin is the principal lens protein which, in addition to its structural role, also acts as a molecular chaperone, to prevent aggregation and precipitation of other lens proteins. One of its two subunits, αB‐crystallin, is also expressed in many nonlenticular tissues, and a natural missense mutation, R120G, has been associated with cataract and desmin‐related myopathy, a disorder of skeletal muscles [Vicart P, Caron A, Guicheney P, Li Z, Prevost MC, Faure A, Chateau D, Chapon F, Tome F, Dupret JM, Paulin D & Fardeau M (1998) Nat Genet20, 92–95]. In the present study, real‐time 1H‐NMR spectroscopy showed that the ability of R120G αB‐crystallin to stabilize the partially folded, molten globule state of α‐lactalbumin was significantly reduced in comparison with wild‐type αB‐crystallin. The mutant showed enhanced interaction with, and promoted unfolding of, reduced α‐lactalbumin, but showed limited chaperone activity for other target proteins. Using NMR spectroscopy, gel electrophoresis, and MS, we observed that, unlike the wild‐type protein, R120G αB‐crystallin is intrinsically unstable in solution, with unfolding of the protein over time leading to aggregation and progressive truncation from the C‐terminus. Light scattering, MS, and size‐exclusion chromatography data indicated that R120G αB‐crystallin exists as a larger oligomer than wild‐type αB‐crystallin, and its size increases with time. It is likely that removal of the positive charge from R120 of αB‐crystallin causes partial unfolding, increased exposure of hydrophobic regions, and enhances its susceptibility to proteolysis, thus reducing its solubility and promoting its aggregation and complexation with other proteins. These characteristics may explain the involvement of R120G αB‐crystallin with human disease states.

The Quaternary Organization and Dynamics of the Molecular Chaperone HSP26 Are Thermally Regulated

The function of ScHSP26 is thermally controlled: the heat shock that causes the destabilization of target proteins leads to its activation as a molecular chaperone. We investigate the structural and dynamical properties of ScHSP26 oligomers through a combination of multiangle light scattering, fluorescence spectroscopy, NMR spectroscopy, and mass spectrometry. We show that ScHSP26 exists as a heterogeneous oligomeric ensemble at room temperature. At heat-shock temperatures, two shifts in equilibria are observed: toward dissociation and to larger oligomers. We examine the quaternary dynamics of these oligomers by investigating the rate of exchange of subunits between them and find that this not only increases with temperature but proceeds via two separate processes. This is consistent with a conformational change of the oligomers at elevated temperatures which regulates the disassembly rates of this thermally activated protein.

Formation of βA3/βB2-crystallin mixed complexes: involvement of N- and C-terminal extensions

The sequence extensions of the β-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 βA3- and βB2-crystallin subunits, these extensions have been shown by 1H-NMR spectroscopy to be solvent-exposed and highly flexible. In this study, we show that βA3- and βB2-crystallins spontaneously form mixed βA3/βB2-crystallin complexes, which, from analytical ultracentrifugation experiments, are dimeric at low concentrations (<1 mg ml−1) and tetrameric at higher protein concentrations. 1H-NMR spectroscopy reveals that in the βA3/βB2-crystallin tetramer, the N-terminal extensions of βA3-crystallin remain water-exposed and flexible, whereas both N- and C-terminal extensions of βB2-crystallin lose their flexibility. We conclude that both extensions of βB2-crystallin are involved in protein–protein interactions in the βA3/βB2-crystallin hetero-tetramer. The extensions may stabilize and perhaps promote the formation of this mixed complex.

Caerin 4.1, an Antibiotic Peptide from the Australian Tree Frog, Litoria caerulea. The N.M.R.-Derived Solution Structure.

Caerin 4.1 (GLWQK5IKSAA10GDLAS15GIEVG20IKS-NH2) is an antibiotic peptide isolated from the Australian tree frog Litoria caerulea. Unlike caerin 1.1, the major peptide isolated from this species, caerin 4.1 has a narrow spectrum of antibiotic activity, e.g. it shows selective activity against Pasteurella haemolytica and Escherichia coli. Caerin 4.1 consists of 23 amino acid residues and is comparable in size with other wide-spectrum antibiotic peptides isolated from Australian amphibians, e.g. caerin 1.1 and maculatin 1.1. An n.m.r. study in trifluoroethanol/water indicates that caerin 4.1 forms an amphipathic α-helix with distinct hydrophilic and hydrophobic zones. Two regions of well defined helicity (from Gln4 to Ala10 and from Ile17 to Ile21) are separated by a central helical region of greater conformational variability. The enhanced disorder in this region arises from the presence of two central glycine residues at positions 11 and 16. However, the degree of disorder and hence flexibility is much less than in caerin 1.1 where central proline residues are present instead. This reduced central flexibility may account for the narrow spectrum of biological activity of caerin 4.1, i.e. because biological membranes of the various bacteria have different composition and topology, their optimal interaction with the relatively rigid caerin 4.1 peptide is not possible.