STRUCTURE AND STABILITY OF γ-CRYSTALLINS-V. COVALENT AND NONCOVALENT PROTEIN-PROTEIN INTERACTIONS IN PHOTOSENSITIZED REACTIONS

Abstract— Irradiation of γ-crystallins with 300 nm light or with the photosensitizers riboflavin or methylene blue (MB) leads to intermolecular cross-linking and insolubilization. Sodium dodecyl sulfate polyacrylamide gel electrophoresis studies reveal that these cross-links are composed of nondisulfide covalent bonds. The water-insoluble phase is stabilized by noncovalent forces, as denaturants readily dissolve it. High-performance liquid chromatography and electrophoresis results further indicate that the higher multimers are part of this water-insoluble fraction only, with the exception of MB-sensitized reactions, which are also able to produce a water-soluble, high-molecular-weight protein of at least 1 million. Labeling the external sulfhydryl groups with iodoacetamide does not prevent the photoreac-tions; however, a reducing agent such as dithiothreitol does. A mechanism involving initial oxidation and interaction of sulfhydryl groups (forming an intramolecular disulfide) buried within the protein as a necessary precursor to polymerization and precipitation has been proposed in the preceding paper. The present study provides support for this mechanism.     

SENSITIZER-INDUCED CONFORMATIONAL CHANGES IN LENS CRYSTALLIN—II. PHOTODYNAMIC ACTION OF RIBOFLAVIN ON BOVINE α-CRYSTALLIN

Abstract— Photolysis of α-crystallin in the presence of riboflavin under both aerobic and anaerobic conditions causes a rapid decrease in Trp emission; photooxidation most likely occurs via non-covalent complex formation between the sensitizer molecule and the substrate. However, the change in the tertiary structure of the protein, as manifested in the near-UV CD, is very different between aerobic and anaerobic photolysis. Riboflavin-sensitized reaction under aerobic condition causes a major change in the microenvironments of thiol groups as well as in the near-UV CD, whereas under anaerobic condition the change in the near-UV CD is much less and SH-group environments remain unaltered. The sensitizer in this photoinduced change in conformation of the protein is very selective and specific.     

Design,construction and characterization of the compact ultrafast terahertz free-electron laser undulator

A compact ultrafast terahertz (CUTE) free-electron laser (FEL) is being developed at the Raja Ramanna Centre for Advanced Technology (RRCAT), Indore. The undulator required for the CUTE-FEL has recently been developed. We have designed, built and characterized a variable gap, 5 cm period, 2.5 m long pure permanent magnet undulator in two identical segments. The tolerable error in the magnetic field was 1% in rms, and we have measured it to be 0.7%. The obtained rms phase shake is around 2°. To ensure that the trajectories do not have an exit error in position or angle, corrector coils have been designed. Shimming coils have been applied for both the undulator segments to reduce the amplitude of the betatron oscillations in the vertical trajectory. Details of novel corrector coils and soft iron shims are given and their performance is discussed.      

Rapid solid-phase syntheses of a peptidic-aminoglycoside library

A library of mono- and di-amino acid peptidic-aminoglycosides (PAs), with kanamycin and neomycin as the model aminoglycosides, was systematically and rapidly synthesized via solid phase peptide synthesis. Aminoglycosides were first converted into N-Boc protected carboxylic acids and fifteen l-amino acids were then used in the diversification of the full library. The approach outlined describes a rapid synthetic procedure where >200?PA compounds can be synthesized in a few months with 85–95% purity. UV thermal denaturation assessed the binding stabilization by PAs to model human and bacterial A-site rRNA sequences. Significant differences were found in thermal melting profiles among PAs that were attributed to specific amino acid sequences. Neomycin PAs lead to a much larger variation in the stabilization of A-site rRNA sequences (ΔT_m?=?2.6–17.1?°C) as compared to kanamycin PAs (ΔTm?=?0.4–4.3?°C). Kanamycin PAs had little activity against Gram-negative and Gram-positive bacteria as compared with neomycin PAs that had significant antibacterial activity with MIC ranging from 2 to 16?μM.     

UV induced thermoluminescence of benzophenone

The UV induced thermoluminescence (TL) of benzophenone has been invetigated at 77 K. Intensity dependence studies of TL induced by ultraviolet light suggest that TL is due to a biphotonic ionization of the molecule by UV. The TL excitation and emission spectra are observed to coincide with the phosphorescene excitation and emission of the molecule. It is concluded that the TL of this molecule is a result of the ion-electron recombination leading to excitation of the triplet state.     

SENSITIZER-INDUCED CONFORMATIONAL CHANGES IN LENS CRYSTALLIN—I. PHOTODYNAMIC ACTION OF METHYLENE BLUE AND N-FORMYLKYNURENINE ON BOVINE α-CRYSTALLIN

Abstract— Fluorescence and circular dichroic properties of bovine a-crystallin have been monitored to detect changes in the structural integrity of the protein following photoreactions in the presence of sensitizer, either methylene blue or N-formylkynurenine. Methylene blue-sensitized photooxidation causes a change in the tertiary structure as manifested in the near-UV CD; this is observed within 0.5 h of irradiation during which time tryptophan emission decreases rapidly. Using inhibitors specific for active species of oxygen, it has been shown that singlet oxygen predominantly causes this change but the sensitizer molecules also have some role in this process. Upon 6 h of irradiation in the presence of methylene blue under both aerobic and anaerobic conditions, the thiol groups that were in a non-polar region of the protein are exposed to polar environments. In conformity with these fluorescence results. near-UV CD (tertiary structure) suffers a drastic alteration whereas the far-UV CD (secondary structure) remains virtually unchanged. The studies with inhibitors indicate that sensitizer molecule itself is primarily responsible for this process. This major change in the conformation has been explained by suggesting that a large portion of the protein unfolds in the photosensitized reaction, thereby altering microenviron-ments, orientation, and intermolecular interactions of different amino acids. N-formylkynurenine also shows some changes in the near-UV CD, presumably, caused by H₂O₂ generated in the photosensitized reaction. But the major alteration in the microenvironments of thiol groups and in the near-UV CD, as observed in the case of methylene blue, does not occur even when the protein is irradiated for 6 h in the presence of N-formylkynurenine and air.