Titanium dioxide photocatalyzed oxidation of proteins in biocontaminated waters.

The TiO2 photocatalyzed oxidation of the proteins serum albumin, ovalbumin and gamma globulin, is reported. All the amino acids were susceptible to photocatalytic oxidation. However, some were especially vulnerable. Tyrosine was particularly sensitive, as was the semiaromatic histidine, although to a lesser extent. The lack of an activating group on the aromatic ring in Phe, renders the system less amenable to degradation. The photocatalytic degradation of the aliphatic amino acids Gly and Asp, was particularly slow, like in the Fenton oxidation where production of glycine was observed during the cleavage of collagen induced by hydroxyl radicals. Intermediate degradation rate was noticed in Ser, Arg, Val, Cys and Phe.

A citrate-binding site in calmodulin.

Calmodulin (CaM) is a major Ca2+ messenger which, upon Ca2+ activation, binds and activates a number of target enzymes involved in crucial cellular processes. The dependence on Ca2+ ion concentration suggests that CaM activation may be modulated by low-affinity Ca2+ chelators. The effect on CaM structure and function of citrate ion, a Ca2+ chelator commonly found in the cytosol and the mitochondria, was therefore investigated. A series of structural and biochemical methods, including tryptic mapping, immunological recognition by specific monoclonal antibodies, CIDNP-NMR, binding to specific ligands and association with radiolabeled citrate, showed that citrate induces conformational modifications in CaM which affect the shape and activity of the protein. These changes were shown to be associated with the C-terminal lobe of the molecule and involve actual binding of citrate to CaM. Analyzing X-ray structures of several citrate-binding proteins by computerized molecular graphics enabled us to identify a putative citrate-binding site (CBS) on the CaM molecule around residues Arg106-His107. Owing to the tight proximity of this site to the third Ca(2+)-binding loop of CaM, binding of citrate is presumably translated into changes in Ca2+ binding to site III (and indirectly to site IV). These changes apparently affect the structural and biochemical properties of the conformation-sensitive protein.

CIDNP study of the aromatic side chain interactions in myotoxin alpha.

CIDNP and COSY measurements were applied to study aromatic side chain interactions and conformations in myotoxin alpha, a Crotalus venom toxin which acts as blocker of the Ca2+ influx in the sarcoplasmic reticulum calcium pump. New evidence for the existence of a hydrophobic aromatic cluster at the amino terminus was obtained. This cluster consists of Tyr1, His5, His10, and (possibly) F12. The CIDNP data clearly establish that the usual order of the tyrosine 2, 6 and 3, 5 proton signals of Tyr1 is inverted, because of the large diamagnetic shielding effects of one ring on the other. The lines of the 2, 6 ring protons of Tyr1 and proton 4 in each of His5 and His10 are significantly broadened, an outcome of the side-chain hydrophobic interaction. The aromatic cluster could possibly present a hydrophobic sticky patch for binding of toxin by Ca2+ ATPase.

Aromatic side-chain interactions as the origin of immunological diversity in the TyrTyrGluGlu and TyrGluTyrGlu epitopes: NMR and fluorescence evidence.

Spectroscopic methods have been applied to elucidate conformational differences responsible for the immunological diversity of two synthetic multichain copolymers, Tyr1Tyr2Glu3Glu4-poly-DL-Ala--poly-Lys and Tyr1Glu2Tyr3Glu4-poly-DL-Ala--poly-Lys. Despite their far-reaching structural similarity in the epitope peptide and complete identity in the poly-Ala--poly-Lys carrier, these two copolymers manifest a wide range of opposed immunological attributes. Different genetic control mechanisms govern their immunogenic properties, and their interactions with antigen presenting cells or T cells and B cells are mediated via different immunological routes. Following previous photoCIDNP (photoChemically Induced Dynamic Nuclear Polarization) investigations, we applied NMR and fluorescence measurements to these two copolymers in order to search for structural differences that could account for their opposed immunological behaviour. The differences between the two antigens are traced to the spatial orientation of the tyrosine residues. Hydrophobic Tyr1--Tyr3 intramolecular inter-side-chain interactions characterize the Tyr1Glu2Tyr3Glu4 polymer, whereas Tyr1 and Tyr2 in the Tyr1Tyr2Glu3Glu4 polymer are non-interacting and freely rotating. It is thus inferred that Tyr1 and Tyr2 are distant and point to different directions in space, whereas Tyr1 and Tyr3 are in close proximity, as was suggested by a previous CIDNP study and by molecular structure computations. We infer that these structural differences may relate to the different immunological behaviour of the TyrTyrGluGlu and TyrGluTyrGlu polymers.

Structural origin of the immunological diversity of two closely related tetrapeptides: CIDNP study of TyrTyrGluGlu and TyrGluTyrGlu epitopes.

Photochemically induced dynamic nuclear polarization (photoCIDNP) measurements, specific for exposed tyrosine residues, have been applied to elucidate conformational differences responsible for the immunological diversity of the synthetic multichain copolymers, Tyr1Tyr2Glu3Glu4-poly-DL-Ala-poly-Lys and Tyr1Glu2Tyr3Glu4-poly-DL-Ala-poly-LS. These two copolymers are essentially identical in their molecular weight, size, shape and composition, and differ only in the order of the two internal amino acid residues within the sequence of the tetrapeptide epitopes. Nonetheless, previous studies have shown that the two macromolecules behave differently, as evidenced by their immunological and immunogenic properties. As immunogens they act under different genetic control mechanisms, and differ in their interactions with antigen presenting cells, T cells and B cells. Antibodies elicited against these two antigens do not cross react. The photoCIDNP measurements of these two polymers, intended to elucidate discrete structural differences controlling immune recognition, showed that in the TyrTyrGluGlu polymer, Tyr1 and Tyr2 rings are free, non-interacting and undergo fast internal rotation. Computed minimum energy conformations confirm these conclusions and indicate that Tyr1 and Tyr2 point to different regions in space. In TyrGluTyrGlu, however, CIDNP measurements give rise to one broad tyrosine 3,5 proton signal, the result of a strong Tyr1-Tyr3 hydrophobic interaction. These two tyrosine residues are thus close in space, and undergo slow internal rotation. These results are in agreement with the computed minimum energy conformations.

A tale of two peptides, TyrTyrGluGlu and TyrGluTyrGlu, and their diverse immune behaviour.

Studies on a synthetic multichain polypeptide antigen, (T,G)-A--L, prepared by polymerization techniques, led to a better understanding of the molecular basis of antigenicity, and of many other immunological phenomena, as well as to the discovery of determinant-specific genetic control of immune response. In view of the intensity of studies with this polymer, we were interested in elucidating its major B and T cell epitopes. We investigated two tetrapeptides, TyrTyrGluGlu and TyrGluTyrGlu. Both were attached to multichain branched poly(DLalanine). Even though the two resulting synthetic immunogens are essentially identical in their molecular weight, size, shape and composition, and differ chemically only in the sequence of the tetrapeptide epitopes, the immunological differences observed were profound. Antibodies in the two systems do not cross-react. The major B cell epitope of (T,G)-A--L is TyrTyrGluGlu, whereas the major T cell epitope is TyrGluTyrGlu. The two antigens are under different genetic controls, and differ in their uptake by macrophages. The TyrTyrGluGlu polymer is thymus-dependent, whereas the TyrGluTyrGlu polymer is thymus-independent. Investigation of the two tetrapeptides in their polymeric form, by photochemically induced dynamic nuclear magnetic polarization techniques, shows that they differ strongly in their intra-epitope aromatic interactions. Phenolic groups in TyrGluTyrGlu interact with each other, whereas they are far apart in TyrTyrGluGlu, as seen also in computer-derived models.

Studies on the biotin-binding sites of avidin and streptavidin. A chemically induced dynamic nuclear polarization investigation of the status of tyrosine residues.

We applied the protein photochemically induced dynamic nuclear polarization (photo-c.i.d.n.p.) method to explore the conformation of the side chains of tyrosine, tryptophan and histidine residues in three biotin-binding proteins. The c.i.d.n.p. spectra of avidin, streptavidin and 'core' streptavidin were compared with those of their complexes with biotin and its derivatives. The data indicate that the single tyrosine residue (Tyr-33) of avidin is clearly inaccessible to the triplet flavin photo-c.i.d.n.p. probe. The same holds for all tryptophan and histidine side chains. Although the analogous Tyr-43 residue of streptavidin is also buried, at least three of the other tyrosine residues of this protein are exposed. The same conclusions apply to the truncated form of the protein, core streptavidin. As judged by the photo-c.i.d.n.p. results, complexing of avidin and streptavidin with biotin, N-epsilon-biotinyl-L-lysine (biocytin) or biotinyltyrosine has little or no effect on tyrosine accessibility in these proteins. Biotinyltyrosine can be used to probe the depth of the corresponding binding site. The accessibility of the tyrosine side chain of biotinyltyrosine in the complex demonstrates the exquisite fit of the biotin-binding cleft of avidin: only the biotin moiety appears to be accommodated, leaving the tyrosine side chain exposed.

Reactivities of tyrosine, histidine, tryptophan, and methionine in radical pair formation in flavin triplet induced protein nuclear magnetic polarization.

In order to check the validity of several basic assumptions of protein photochemically induced nuclear polarization (protein photo-CIDNP), we have investigated the quenching processes of the dye triplets by the side chains of tyrosine, histidine, and tryptophan in a variety of molecular systems and environments. The quenching (H atom or electron transfer) is the generating process of the triplet electron-spin-correlated radical pair, the evolution of which gives rise to nuclear polarization. At pH 7 the quenching of 10-(carboxyethyl)flavin triplets by tyrosine and tryptophan is almost diffusion controlled. Quenching by histidine is slower. We have also investigated the slow quenching (by electron transfer) by the side chains of methionine and could show that quenching by cysteine S derivatives is negligible. Quenching by tyrosine and histidine peptides and by the tyrosines of the pancreatic trypsin inhibitor protein is slightly slower than by free side chains. Quenching is strongly viscosity controlled, to be expected of a process requiring bimolecular contact. Reactivity trends at high viscosities resemble those observed in fluid aqueous solutions. Activation energies of quenching by tyrosine, tryptophan, and histidine are similar. No difference could be detected in the mechanism of quenching by these side chains. No fast static quenching was observed that could compete with the diffusional process.

Photochemically induced nuclear polarization study of exposed tyrosines, tryptophans, and histidines in postsynaptic neurotoxins and in membranotoxins of elapid and hydrophid snake venoms.

The accessibility of surface tyrosines, histidines, and tryptophans in snake venom neurotoxins (short and long) and in membranotoxins to excited triplet 10-(carboxyethyl)-flavin was studied by photochemically induced dynamic nuclear polarization at 270 MHz. Trp-29 is accessible in the short neurotoxins--erabutoxins a, b, and c and cobrotoxin--and also in the long neurotoxins--alpha-cobratoxin and alpha-bungarotoxin. Tyr-25 is practically inaccessible in all neurotoxins. Tyr-39 in cobrotoxin and Tyr-55 in alpha-bungarotoxin are accessible. His-6 (revised sequence) is inaccessible in the erabutoxins while His-26 is only very weakly accessible. His-22 of alpha-cobratoxin is inaccessible as are His-4 and -68 in alpha-bungarotoxin and His-4 of cobrotoxin. His-33 of cobrotoxin is accessible. The rigidity order alpha-bungarotoxin greater than or equal to alpha-cobratoxin greater than or equal to erabutoxins, with respect to the unfolding effect of 7 M urea, was deduced in this study. In the membranotoxins studied (cardiotoxin and its analogues I, II, and IV as well as cytotoxin I and II), the two tyrosines Tyr-25 and Tyr-58 are only weakly accessible. Tyr-14 is completely accessible and so is in all probability Tyr-29. These studies allow deductions to be made about the accessibilities in analogous systems. Thus, the accessibility of His-33 and the inaccessibility of His-4 in cobrotoxin can be used to deduce the conformations of these residues in a large group of neurotoxins including the alpha-toxin of Naja nigricollis, neurotoxin II of Naja naja oxiana, and neurotoxins I and III of Naja mossambica mossambica.(ABSTRACT TRUNCATED AT 250 WORDS)

Photochemically induced nuclear polarization study of the accessibility of tyrosines in insulin.

The accessible tyrosines of bovine insulin were studied by the photochemically induced dynamic nuclear polarization (photo-CIDNP) method. Tyrosine 1H nuclear polarization is observed in acidic, neutral, and basic solutions at all concentrations studied, in the absence of added salts as well as in the presence of 0.05-0.1 M chloride or phosphate. At pH 2.1 in the presence of chloride, at concentrations of 640 microM and above, most of the nuclear polarization at delta 6.82 originates from one group of tyrosines. On the basis of the crystallographic model, these are assumed to be the A14 tyrosines. We explored the possibility of a genuine concentration dependence of the photo-CIDNP intensity of insulin due to aggregation. In order to discern between such effects and trivial kinetic effects traceable to the optical irradiation method, the effects of concentration changes on polarization were examined in three apparently nonassociating trypsin inhibitor proteins. In insulin, the intensity of Tyr-A 14 polarization changes slowly at concentrations above 1 mM, suggesting that these residues are similarly accessible in all association states. At insulin concentrations below 320 microM, additional tyrosine emission signals were observed. These signals are probably due to B16 and B26 tyrosines of monomers. Polarization transfer effects from Tyr-A14 are evident in the tetramer and hexamer. Enhanced absorption effects in the two histidines (B5 and B10) of the insulin monomer were observed at pH 10 in the presence of 0.1 M phosphate.

Biochemical applications of chemically induced nuclear polarization in phenols, peptides, catecholamines and related molecules.

A photochemical method for producing proton nuclear polarization in phenolic biogenic molecules such as tyrosyl polypeptides and phenolic amines is described. The nuclear polarization is due to the reversible hydrogen-atom transfer from the phenolic hydroxy group to triplet photoexcited xanthene-dye molecules. Typical results obtained with insulin, catecholamines and other phenolic molecules are treated in detail.