D. Balasubramanian

The bacterial pigment xanthomonadin offers protection against photodamage

Xanthomonas oryzae pv. oryzae is a bacterial pathogen that causes leaf blight, a serious disease of rice. Most members of the genus Xanthomonas produce yellow, membrane bound, brominated aryl polyene pigments called xanthomonadins whose functional role is unclear. We find that pigment‐deficient mutants of X. oryzae pv. oryzae exhibit hypersensitivity to photobiological damage. A clone containing the xanthomonadin biosynthetic gene cluster alleviates the hypersensitivity of the pigment‐deficient mutant. Extracts containing xanthomonadin provide protection against photodynamic lipid peroxidation in liposomes. These results lead us to suggest a role for the pigment, namely protection against photodamage.

β-Carbolines That Accumulate in Human Tissues May Serve a Protective Role against Oxidative Stress

β-Carbolines are tricyclic nitrogen heterocycles formed in plants and animals as Maillard reaction products between amino acids and reducing sugars or aldehydes. They are being detected increasingly in human tissues, and their physiological roles need to be understood. Two β-carboline carboxylates have been reported to accumulate in the human eye lens. We report here on the identification of another β-carboline, namely 1-methyl-1-vinyl -2,3,4-trihydro-β-carboline-3-carboxylic acid, in the lenses of some cataract patients from India. Analysis of these three lenticular β-carbolines using photodynamic and antioxidant assays shows all of them to be inert as sensitizers and effective as antioxidants; they quench singlet oxygen, superoxide and hydroxyl radicals and inhibit the oxidative formation of higher molecular weight aggregates of the test protein, eye lens γ-crystallin. Such antioxidative ability of β-carbolines is of particular relevance to the lens, which faces continual photic and oxidative stress. The β-carboline diacid IV is also seen to display an unexpected ability of inhibiting the thermal coagulation of γ-crystallin and the dithiothreitol-induced precipitation of insulin. These results offer experimental support to earlier suggestions that one of the roles that the β-carbolines have is to offer protection against oxidative stress to the human tissues where they accumulate.

The molten globular intermediate form in the folding pathway of human carbonic anhydrase B

The acid‐induced and guanidinium chloride‐induced conformational transitions in human carbonic anhydrase B have been. analyzed. A structural form was detected at pH 3, which has a higher secondary structural order than the native enzyme but little tertiary structure. The enzyme dissolved in an intermediate concentration of the denaturant guanidinium chloride (1 M at pH 7.5) also adopts a similar conformational state. This form, denoted as the intermediate form I, possesses most of the characteristics defined for the molten globular state of globular proteins and might serve as the embryonic structural intermediate during the self‐organization of the protein into its functional native form.

Protein-associated pigments that accumulate in the brunescent eye lens: Identification of a quinoline derivative

Brunescent (dark brown) cataract is particularly prevalent in the tropics. Enzymatic digestion of the insoluble protein fraction of brunescent cataractous eye lenses from India, followed by high performance liquid chromatographic separation of the pigments and spectroscopic investigations, have led to the identification of one of the pigments as 4‐hydroxyquinoline‐3‐[α‐aminoacetic acid] (compound A). The 4‐hydroxyquinoline moiety is shown to be a photodynamic agent that generates O•− 2 and leads to protein crosslinking. This suggests that the compound A may play a long‐term deleterious role in situ in the lens.

Hydrophobic surfaces in oligosaccharides: linear dextrims are amphiphilic chains

Polysaccharide chains are usually considered to be highly hydrophilic, since they have no obvious nonpolar moieties in them. Yet, it is possible to realise conformations in these chains wherein all the hydroxy groups are disposed in one side or face of the chain and the hydrogens disposed in the other. We experimentally demonstrate that such an amphiphilic surface is present in linear oligomeric dextrins, i.e., alpha-1,4-linked D-glucosides, but not in alpha-1,6-D-glucosides (dextrans) or in beta-1,4-D-glucosides (cellulose). This amphiphilicity is generated as a consequence of the stereochemical constraints, which vary with the structure of the sugar and with the type of linkage. Oligosaccharide chains that can adopt incipient helical structures might display amphiphilicity. This property might be relevant to intermolecular recognition on cell surfaces, lectin-sugar binding, antigen-antibody interactions and the like, and might be manifested more in heteromolecular recognition process than as homomolecular self-aggregation.

Studies on the interaction of the dye, Stains-all, with individual calcium-binding domains of calmodulin

We show that the calcium‐mimic dye, Stains‐all, is a convenient probe to study the structural features of the individual calcium‐binding sites of calmodulin (CaM) and related calcium‐binding proteins (CaBP). These peptides bind the dye in their calcium‐binding sites, and induce a circular dichroism (CD) band in the bound dye in the 620 nm (J band) region, which is abolished upon the addition of calcium. Replacement of Asp by Asn in the +x position of the weaker calcium‐binding site (site I of CaM) abolishes the dye binding, while the same change in the higher affinity site IV attenuates the binding of the dye and does not abolish it. Replacement of Tyr in site IV with Trp does not distort the geometry, although it increases the dye binding affinity.

Microenvironmental differences amongst micelles of various shapes and structures

Micelles of different amphiphiles adopt different shapes and internal packing arrangements in water, depending on their chemical structures and the conditions of the medium. Two microenvironmental features, namely the polarity and the microviscosity that the aggregate offers to a solubilized molecule, have been monitored using extrinsic fluorescence probes. While the differences between micelles of spherical and rod-like shapes are not always distinct, stacked micelles and peptide micelles offer distinctly lower polarity and higher microviscosity to solubilizates than the others.

Conformational studies on δ-crystallin, the core protein of the bird eye lens

Proteins that perform other functions elsewhere appear to be recruited for structural purposes in the eye lens. The lens being a tissue with very little metabolic activity and little or no turnover, the lens proteins, crystallins, are long lived. In an effort to understand whether their recruitment might be related to their conformation and structural stability, we have examined these features of the avian lens protein δ-crystallin. The native molecule is a tetramer (molecular mass 200 kDa) that is highly α-helical in conformation, and with an unusually blue tryptophan fluorescence (315,325 nm), which is only partially quenched by conventional quenchers. We show that the fluorescence doublet arises due to Trp residues that are effectively buried inside the rigid hydrophobic core of the tetrameric aggregate. The protein is heat stable up to 91°C. Guanidinium chloride (GuHCl) effects the complete denaturation of δ-crystallin, whereas heat or urea treatment results in only partial unfolding or dissociation. The initial transition is the disruption of the quaternary structure by perturbing the intersubunit interactions, leading to exposure of hydrophobic contact surfaces (as monitored by extrinsic probe fluorescence). This initial transition is seen upon heating to 60°C as well as in 1 M GuHCl and 4 M urea. We show that in 2.2 M GuHCl the molecule is swollen but is still largely helical with the Trp residues being present in a somewhat more polar environment than in the native molecule. Beyond 4 M GuHCl there is a gradual unfolding of the molecule, which is complete in 6 M GuHCl. This structural robustness of δ-crystallin might be important in its recruitment as the core protein of the avian lens.