Invited review: Modeling milk stability.

Novel insights into the stability of milk and milk products during storage and processing result from describing caseins near neutral pH as hydrophilic, intrinsically disordered, proteins. Casein solubility is strongly influenced by pH and multivalent ion binding. Solubility is high at neutral pH or above but decreases as casein net charge approaches zero, allowing a condensed casein phase or gel to form then increases at lower pH. Of particular importance for casein micelle stability near neutral pH is the proportion of free caseins in the micelle (i.e., caseins not bound directly to nanoclusters of calcium phosphate). Free caseins are more soluble and better able to act as molecular chaperones (to prevent casein and whey protein aggregation) than bound caseins. Some free caseins are highly phosphorylated and can also act as mineral chaperones to inhibit the growth of calcium phosphate phases and prevent mineralized deposits from forming on membranes or heat exchangers. Thus, casein micelle stability is reduced when free caseins bind to amyloid fibrils, destabilized whey proteins or calcium phosphate. The multivalent-binding model of the casein micelle quantitatively describes these and other factors affecting the stability of milk and milk protein products during manufacture and storage.

Structural differences between bovine A(1) and A(2) ß-casein alter micelle self-assembly and influence molecular chaperone activity.

Within each milk protein there are many individual protein variants and marked alterations to milk functionality can occur depending on the genetic variants of each protein present. Bovine A(1) and A(2) ß-casein (ß-CN) are 2 variants that contribute to differences in the gelation performance of milk. The A(1) and A(2) ß-CN variants differ by a single AA, the substitution of histidine for proline at position 67. ß-Casein not only participates in formation of the casein micelle but also forms an oligomeric micelle itself and functions as a molecular chaperone to prevent the aggregation of a wide range of proteins, including the other caseins. Micelle assembly of A(1) and A(2) ß-CN was investigated using dynamic light scattering and small-angle X-ray scattering, whereas protein functionality was assessed using fluorescence techniques and molecular chaperone assays. The A(2) ß-CN variant formed smaller micelles than A(1) ß-CN, with the monomer-micelle equilibrium of A(2) ß-CN being shifted toward the monomer. This shift most likely arose from structural differences between the 2 ß-CN variants associated with the adoption of greater polyproline-II helix in A(2) ß-CN and most likely led to enhanced chaperone activity of A(2) ß-CN compared with A(1) ß-CN. The difference in micelle assembly, and hence chaperone activity, may provide explain differences in the functionality of homozygous A(1) and A(2) milk. The results of this study highlight that substitution of even a single AA can significantly alter the properties of an intrinsically unstructured protein such as ß-CN and, in this case, may have an effect on the functionality of milk.

Invited review: Caseins and the casein micelle: their biological functions, structures, and behavior in foods.

A typical casein micelle contains thousands of casein molecules, most of which form thermodynamically stable complexes with nanoclusters of amorphous calcium phosphate. Like many other unfolded proteins, caseins have an actual or potential tendency to assemble into toxic amyloid fibrils, particularly at the high concentrations found in milk. Fibrils do not form in milk because an alternative aggregation pathway is followed that results in formation of the casein micelle. As a result of forming micelles, nutritious milk can be secreted and stored without causing either pathological calcification or amyloidosis of the mother's mammary tissue. The ability to sequester nanoclusters of amorphous calcium phosphate in a stable complex is not unique to caseins. It has been demonstrated using a number of noncasein secreted phosphoproteins and may be of general physiological importance in preventing calcification of other biofluids and soft tissues. Thus, competent noncasein phosphoproteins have similar patterns of phosphorylation and the same type of flexible, unfolded conformation as caseins. The ability to suppress amyloid fibril formation by forming an alternative amorphous aggregate is also not unique to caseins and underlies the action of molecular chaperones such as the small heat-shock proteins. The open structure of the protein matrix of casein micelles is fragile and easily perturbed by changes in its environment. Perturbations can cause the polypeptide chains to segregate into regions of greater and lesser density. As a result, the reliable determination of the native structure of casein micelles continues to be extremely challenging. The biological functions of caseins, such as their chaperone activity, are determined by their composition and flexible conformation and by how the casein polypeptide chains interact with each other. These same properties determine how caseins behave in the manufacture of many dairy products and how they can be used as functional ingredients in other foods.

Darwinian transformation of a 'scarcely nutritious fluid' into milk.

In an early challenge to an aspect of Darwin's theory of natural selection, Jackson Mivart contended that milk could not have evolved 'from a scarcely nutritious fluid from an accidentally hypertrophied cutaneous gland'. The evolutionary change from a gland secretion to milk involves an increase in calcium and protein concentrations by up to 100- and 1000-fold, respectively. Even so, the challenge, we suggest, is not just a problem of scale. An increase in the concentrations of calcium and phosphate brings an increased risk of calcification of the secretory gland because calcium phosphate is highly insoluble. In addition, two of the four constituent milk casein proteins (κ and α(S2)) aggregate to produce toxic amyloid fibrils. It is proposed that both problems were solved through the cosecretion of ancestral ß- and κ-caseins to form a stable amorphous aggregate of both proteins with sequestered amorphous calcium phosphate, that is, a primordial casein micelle. Evolutionarily, a gradual increase in the concentration of casein micelles could therefore produce progressively more nutritious fluids for the neonate without endangering the reproductive potential of the mother.

Investigation of gammaE-crystallin target protein binding to bovine lens alpha-crystallin by small-angle neutron scattering.

alpha-Crystallin, one of the main constituent proteins in the crystalline lens, is an important molecular chaperone both within and outside the lens. Presently, the structural relationship between alpha-crystallin and its target proteins during chaperone action is poorly understood. It has been hypothesised that target proteins bind within a central cavity. Small-angle neutron-scattering (SANS) experiments in conjunction with isotopic substitution were undertaken to investigate the interaction of a target lens protein (gammaE-crystallin) with alpha-crystallin (alpha(H)) and to measure the radius of gyration (Rg) of the proteins and their binary complexes in solution under thermal stress. The size of the alpha(H) in D(2)O incubated at 65 degrees C increased from 69+/-3 to 81+/-5 A over 40 min, in good agreement with previously published small-angle X-ray scattering (SAXS) and SANS measurements. Deuterated gammaE-crystallin in H(2)O buffer (gammaE(D)/H(2)O) and hydrogenous gammaE-crystallin in D(2)O buffer (gammaE(H)/D(2)O) free in solution were of insufficient size and/or too dilute to provide any measurable scattering over the angular range used, which was selected primarily to investigate gammaE:alpha(H) complexes. The evolution of the aggregation size/shape as an indicator of alpha(H) chaperone action was monitored by recording the neutron scattering in different H:D solvent contrasts under thermally stressed conditions (65 degrees C) for binary mixtures of alpha(H), gammaE(H), and gammaE(D). It was found that Rg(alpha(H):gammaE(D)/D(2)O)>Rg(alpha(H):gammaE(H)/D(2)O)>Rg(alpha(H)/D(2)O) and that Rg(alpha(H):gammaE(H)/D(2)O) approximately Rg(alpha(H)/D(2)O). The relative sizes observed for the complexes weighted by the respective scattering powers of the various components imply that gammaE-crystallin binds in a central cavity of the alpha-crystallin oligomer, during chaperone action.

Structure/function studies of dogfish alpha-crystallin, comparison with bovine alpha-crystallin.

PURPOSE: alpha-Crystallin is the major protein of the mammalian lens where it contributes to the refractive properties needed for vision and possibly to the stability of the tissue. The aim of this study was to determine whether the properties of alpha-crystallin have changed during the course of evolution. METHODS: Dogfish alpha-crystallin, which appeared over 420 million years ago, has been contrasted with bovine alpha-crystallin, which emerged around 160 million years later, by comparing their sizes, the microenvironments of their cysteine and tryptophan residues, their chaperone-like activities and the flexibility of their COOH-terminal extensions. RESULTS: Dogfish alpha-crystallin consists of alphaA- and alphaB-polypeptides, in a 1:5 ratio, and has a molecular mass of around 400 kDa. By contrast, the bovine protein is around 600-800 kDa in mass and has a 3:1 subunit ratio. Cysteine residues in the proteins were equally accessible to reaction with 5,5'-dithiobis-(2-nitrobenzoic acid). Quenching of fluorescence with acrylamide indicated tryptophan residues in the two proteins were in similar environments. The chaperone activity of dogfish alpha-crystallin was comparable to that of bovine alpha-crystallin in preventing the heat-induced precipitation of beta(L)-crystallin but the dogfish protein was three times more effective at preventing insulin precipitation after reduction at 37 C. (1)H nuclear magnetic resonance spectroscopic studies showed that the last 17 amino acids of the dogfish alphaB polypeptide (V162-K178) have great conformational flexibility, are highly exposed to solvent and adopt little ordered conformation. This is comparable to, but slightly longer in length, than the COOH-terminal extension observed in mammalian alpha-crystallins. CONCLUSIONS: The structure and properties of alpha-crystallin have changed relatively little during the evolutionary period from the emergence of sharks and mammals.

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.

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.

Clusterin is an ATP-independent chaperone with very broad substrate specificity that stabilizes stressed proteins in a folding-competent state.

We recently reported that the ubiquitous, secreted protein clusterin has chaperone activity in vitro [Humphreys et al. (1999) J. Biol. Chem. 274, 6875-6881]. In this study, we demonstrate that clusterin (i) inhibits stress-induced precipitation of a very broad range of structurally divergent protein substrates, (ii) binds irreversibly via an ATP-independent mechanism to stressed proteins to form solubilized high molecular weight complexes, (iii) lacks detectable ATPase activity, (iv) when acting alone, does not effect refolding of stressed proteins in vitro, and (v) stabilizes stressed proteins in a state competent for refolding by heat shock protein 70 (HSP70). Furthermore, we show that, at physiological levels, clusterin inhibits stress-induced precipitation of proteins in undiluted human serum. Clusterin represents the first identified secreted mammalian chaperone. However, reports from others suggest that, at least under stress conditions, clusterin may be retained within cells to exert a protective effect. Regardless of the topological site(s) of action, the demonstration that clusterin can stabilize stressed proteins in a refolding-competent state suggests that, during stresses, the action of clusterin may inhibit rapid and irreversible protein precipitation and produce a reservoir of inactive but stabilized molecules from which other refolding chaperones can subsequently salvage functional proteins.

Polypeptide modification and cross-linking by oxidized 3-hydroxykynurenine.

3-Hydroxykynurenine (3OHKyn) is present in the mammalian lens as a UV filter and is formed from kynurenine in the tryptophan metabolic pathway. 3OHKyn is a readily autoxidized o-aminophenol which binds to proteins in vitro. The lens, particularly its central region, the nucleus, becomes increasingly oxidized with age. Under such conditions, the oxidation products of 3OHKyn may bind to lens proteins and contribute to nuclear cataract formation. The purpose of this study was to determine the structures of in vitro reaction products of 3OHKyn with model peptides as a general model for 3OHKyn modification of proteins. 3OHKyn was incubated with the dipeptide glycylglycine (GG) and the tetrapeptide tuftsin (sequence TKPR) under oxidizing conditions, and the reaction products were characterized by a variety of spectroscopic techniques. The major 3OHKyn-GG reaction product involves formation of a benzimidazole moiety between the GG N-terminus and the oxidized amino and/or phenol groups of 3OHKyn. In contrast, tuftsin, which has an N-terminal threonine, forms predominantly a cross-linked dimer with oxidized 3OHKyn. This product is analogous in structure to the dimeric reaction product, quinilinobenzoxamine, formed between oxidized 3OHKyn and glycyllysine [Aquilina, J. A., et al. (1999) Biochemistry 38, 11455-11464], which contains a benzoxazole moiety. The identification of a tuftsin dimer suggests that 3OHKyn can react with any peptide having a free alpha-amino group, via a general side chain elimination mechanism. The identification of both benzimidazole and benzoxazole adducts in peptides with a free N-terminus suggests that peptide amino groups can react initially at either the aromatic amino or hydroxyl group of oxidized 3OHKyn. The proportion of each adduct may change, however, depending on the amino acid sequence at the N-terminus.

The antibiotic and anticancer active aurein peptides from the Australian Bell Frogs Litoria aurea and Litoria raniformis the solution structure of aurein 1.2.

Seventeen aurein peptides are present in the secretion from the granular dorsal glands of the Green and Golden Bell Frog Litoria aurea, and 16 from the corresponding secretion of the related Southern Bell Frog L. raniformis. Ten of these peptides are common to both species. Thirteen of the aurein peptides show wide-spectrum antibiotic and anticancer activity. These peptides are named in three groups (aureins 1-3) according to their sequences. Amongst the more active peptides are aurein 1.2 (GLFDIIKKIAESF-NH2), aurein 2.2 (GLFDIVKKVVGALGSL-NH2) and aurein 3.1 (GLFDIVKKIAGHIAGSI-NH2). Both L. aurea and L. raniformis have endoproteases that deactivate the major membrane-active aurein peptides by removing residues from both the N- and C-termini of the peptides. The most abundant degradation products have two residues missing from the N-terminal end of the peptide. The solution structure of the basic peptide, aurein 1.2, has been determined by NMR spectroscopy to be an amphipathic alpha-helix with well-defined hydrophilic and hydrophobic regions. Certain of the aurein peptides (e.g. aureins 1.2 and 3.1) show anticancer activity in the NCI test regime, with LC50 values in the 10-5-10-4 M range. The aurein 1 peptides have only 13 amino-acid residues: these are the smallest antibiotic and anticancer active peptides yet reported from an anuran. The longer aurein 4 and 5 peptides, e.g. aurein 4.1 (GLIQTIKEKLKELAGGLVTGIQS-OH) and aurein 5. 1 (GLLDIVTGLLGNLIVDVLKPKTPAS-OH) show neither antibacterial nor anticancer activity.

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.

Solution structure and backbone dynamics of long-[Arg(3)]insulin-like growth factor-I.

Long-[Arg(3)]insulin-like growth factor-I (IGF-I) is a potent analog of insulin-like growth factor-I that has been modified by a Glu(3) --> Arg mutation and a 13-amino acid extension appended to the N terminus. We have determined the solution structure of (15)N-labeled Long-[Arg(3)]-IGF-I using high resolution NMR and restrained molecular dynamics techniques to a precision of 0.82 +/- 0.28 A root mean square deviation for the backbone heavy atoms in the three alpha-helices and 3.5 +/- 0.9 A root mean square deviation for all backbone heavy atoms excluding the 8 N-terminal residues and the 8 C-terminal eight residues. Overall, the structure of the IGF-I domain is consistent with earlier studies of IGF-I with some minor changes remote from the N terminus. The major variations in the structure, compared with IGF-I, occur at the N terminus with a substantial reorientation of the N-terminal three residues of the IGF-I domain. These results are interpreted in terms of the lower binding affinity for insulin-like growth factor-binding proteins. The backbone dynamics of Long-[Arg(3)]IGF-I were investigated using (15)N nuclear spin relaxation and the heteronuclear nuclear Overhauser enhancement (NOE). There is a considerable degree of flexibility in Long-[Arg(3)]IGF-I, even in the alpha-helices, as indicated by an average ((1)H)(15)N NOE of 0.55 for the regions. The largest heteronuclear NOEs are observed in the helical regions, lower heteronuclear NOEs are observed in the C-domain loop separating helix 1 from helix 2, and negative heteronuclear NOEs are observed in the N-terminal extension and at the C terminus. Despite these data indicating conformational flexibility for the N-terminal extension, slow amide proton exchange was observed for some residues in this region, suggesting some transitory structure does exist, possibly a molten helix. A certain degree of flexibility may be necessary in all insulin-like growth factors to enable association with various receptors and binding proteins.

Maculatin 1.1, an anti-microbial peptide from the Australian tree frog, Litoria genimaculata solution structure and biological activity.

The dorsal glands of Australian tree frogs from the Litoria species contain a diversity of antibiotic peptides that forms part of the defence system of the animal. Here, the antibiotic activity and structure of maculatin 1.1, a 21 amino acid peptide from Litoria genimaculata, are compared. The activity data on maculatin 1.1 and a series of its analogues imply that the mechanism of action of maculatin 1.1 involves binding to, and subsequent lysis of, the bacterial cell membrane. The structure of maculatin 1.1 was determined using NMR spectroscopy in a trifluoroethanol/water mixture and when incorporated into dodecylphosphocholine micelles. Under both conditions, the peptide adopts a very similar conformation, i.e. a helical structure with a central kink in the vicinity of Pro15. The kink allows the peptide to adopt a well-defined amphipathic conformation along its entire length. The similar structures determined under both solvent conditions imply that structures of membrane-interacting peptides in trifluoroethanol/water mixtures are representative of those adopted in a membrane environment, e.g. when incorporated into micelles. The synthetic Ala15 analogue of maculatin 1.1 has markedly reduced activity and its NMR-derived structure is a well-defined helix, which lacks the central kink and flexibility of the parent molecule. It is concluded that the kink is important for full biological activity of the peptide, probably because it allows maximum amphipathicity of the peptide to facilitate interaction with the membrane. The structure of maculatin 1.1 is compared with a related peptide, caerin 1.1 [Wong, H., Bowie, J.H. and Carver, J.A. (1997) Eur. J. Biochem. 247, 545-557], which has an additional central proline residue and enhanced central flexibility compared with maculatin 1.1. The role of central flexibility within antibiotic peptides in their interaction with bacterial membranes is discussed.

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.

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.

Host defence peptides from the skin glands of the Australian blue mountains tree-frog Litoria citropa. Solution structure of the antibacterial peptide citropin 1.1.

Nineteen citropin peptides are present in the secretion from the granular dorsal glands of the Blue Mountains tree-frog Litoria citropa; 15 of these peptides are also present in the secretion from the submental gland. Two major peptides, citropin 1.1 (GLFDVIKKVASVIGGL-NH2), citropin 1.2 (GLFDIIKKVASVVGGL-NH2) and a minor peptide, citropin 1.3 (GLFDIIKKVASVIGGL-NH2) are wide-spectrum antibacterial peptides. The amphibian has an endoprotease which deactivates these membrane-active peptides by removing residues from the N-terminal end: loss of three residues gives the most abundant degradation products. The solution structure of the basic peptide citropin 1.1 has been determined by NMR spectroscopy [in a solvent mixture of trifluoroethanol/water (1 : 1)] to be an amphipathic alpha-helix with well-defined hydrophobic and hydrophilic regions. The additional four peptides produced by the dorsal glands are structurally related to the antibacterial citropin 1 peptides but contain three more residues at their C-terminus [e.g. citropin 1.1.3 (GLFDVIKKVASVIGLASP-OH)]. These peptides show minimal antibacterial activity; their role in the amphibian skin is not known.

Elucidation of a novel polypeptide cross-link involving 3-hydroxykynurenine.

3-Hydroxykynurenine, a metabolite of tryptophan, is a powerful antioxidant and neurotoxin. The neurotoxicity results from the oxidation of 3-hydroxykynurenine, and hydroxyl radicals, formed via H(2)O(2), may also be implicated [Okuda, S., Nishiyama, N., Saito, H. , and Katsuki, H. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 12553-12558]. Oxidation of o-aminophenols, such as 3-hydroxykynurenine, also results in the formation of highly reactive quinonimines. Thus, one possible consequence of 3-hydroxykynurenine oxidation may be covalent modification of cellular macromolecules. Such a process could contribute to the neurotoxicity and may potentially be important in other tissues, such as the human lens, where 3-hydroxykynurenine functions as a UV filter. In this work, we demonstrate that 3-hydroxykynurenine can bind to protein amino groups and, further, that under oxidative conditions, 3-hydroxykynurenine can function to cross-link polypeptide chains. The structure of the cross-linked moiety, using the peptide glycyllysine, has been elucidated. The cross-link, which is both colored and fluorescent, involves the peptide alpha-amino groups. Proteins modified by 3-hydroxykynurenine become colored and fluorescent as well as cross-linked. LC-MS studies indicate that the cross-link is also present in gamma-crystallin, following incubation of this lens protein in the presence of 3-hydroxykynurenine. Similar posttranslational modifications of lens proteins accompany cataract formation, and knowledge of the precise mode of reaction of 3-hydroxykynurenine with proteins will assist in determining if 3-hydroxykynurenine is involved in degenerative conditions in which oxidation of such aminophenols is implicated.

The solution structure of uperin 3.6, an antibiotic peptide from the granular dorsal glands of the Australian toadlet, Uperoleia mjobergii.

Uperin 3.6 (GVIDA5AKKVV10NVLKN15LF-NH2) is a wide-spectrum antibiotic peptide isolated from the Australian toadlet, Uperoleia mjobergii. With only 17 amino acid residues, it is smaller than most other wide-spectrum antibiotic peptides isolated from amphibians. In 50% (by vol.) trifluoroethanol, an NMR study and structure calculations indicate that uperin 3.6 adopts a well-defined amphipathic alpha-helix with distinct hydrophilic and hydrophobic faces. Examination of the activities of synthetic modifications of uperin 3.6 reveal that the three lysine residues are essential for antibiotic activity.

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.