Flavin-based Blue-Light photosensors: a photobiophysics update

This review deals with the biophysical aspects of flavin-based photosensors, comprising cryptochromes, LOV (Light, Oxygen and Voltage) and BLUF (Blue Light sensing Using FAD) proteins. Special emphasis is given to structural issues, photocycle quantum yields and energetics, mechanism of the light-triggered reactions, early stages in signal transduction and oligomeric states of the light sensing protein modules. For BLUF and LOV domains important parallels are emerging, despite their different alpha/beta fold arrangement, whereas there is increasing evidence for a mechanicistic and functional splitting of the cryptochrome family.     

Preparation of in-house calibration source for the use in radioactivity analysis of the environmental samples: consideration of homogeneity

An in-house reference material has been prepared in Kuwait Institute for Scientific Research radioecology laboratory, for quality control purposes of gamma spectrometer systems. The material contains a known amount of uranium ore reference material (prepared by the International Atomic Energy Agency and coded as IAEA-RGU-1) which is mixed with marine sediment collected from Kuwait bay. The IAEA-RGU-1 has been certified that it is in equilibrium state with the decay daughters, and stable to be used for quality control purposes. Nevertheless, the homogeneous distribution of the doped material with the prepared source should be verified. This has been examined using gamma spectrometry measurements in conjunction with analysis of variance statistical tools, Dixon, box plots and Grubbs tests. The calculated total uncertainty has been utilized to establish the recommended specific activity ranges of ²²⁶Ra, ²²⁴Th, ²¹⁴Pb, ²¹⁴Bi and ²¹⁰Pb radioisotopes in the prepared source. The obtained results showed that the estimated uncertainty arising from the sample inhomogeneity has a significant contribution in the total uncertainty. The stability control charts of the ultra-low background gamma spectrometry system demonstrated the suitability of the prepared material for the purpose of quality control. However, the emitted gamma-rays from the prepared source covers the required energy range for determination of natural and artificial radionuclides in different species of environmental samples such as marine sediment, soil samples, and samples contaminated by naturally occurring radioactive material produced by oil industry. In addition, the material might be used for system calibration in case its traceability is proven. The experimental data revealed the significance of the homogeneity in preparing environmental samples for radioactivity measurements; in particular when small sample quantities of environmental samples are required to be analyzed.     

Development of tyrosinase labile protecting groups for amines

[reaction: see text] The development of two novel protecting groups for amines is described. Thus, a range of amines have been converted to ureas, and the deprotection of these upon exposure to mushroom tyrosinase (E.C. 1.14.18.1) has been demonstrated.     

Inner-shell Eigenvalues from valence orbital only calculations

Methods are examined for calculating Inner-shell Eigenvalues from molecular orbital models which do not explicitly include core basis functions. If the “valence-orbital only” calculation is a good one, a rather straightforward method can be used to obtain core eigenvalues with a rms error of ±0.01 a.u. compared with ab-initio values. Even simpler methods can be used to reproduce trends among core eigenvalues. The AAMOM valence technique, and to a lesser extent, the INDO model, can be used to yield core eigenvalues for orbitals centered on carbon and nitrogen: for oxygen the agreement is poorer. Extended Hückel or Iterative Extended Hückel methods cannot be used for this purpose with any degree of confidence. Eigenvalues from ab-initio studies or from AAMOM and INDO can be used in assigning ionization processes (XPS) from orbitals localized on carbon and nitrogen: ionization processes from oxygen orbitals are not well treated. An attempt is made to explain this behaviour.     

First proficiency test for the determination of NORM in contaminated soil from the oil field

The Atomic Energy Commission of Syria (AECS), in cooperation with the Arabic Atomic Energy Agency (AAEA), has initiated the first proficiency test exercise for the determination of naturally occurring radioactive materials (NORM) in contaminated soil from the oil field. The soil sample was collected from one of the most highly radioactively contaminated lagoons with production water in the Syrian oil fields, which was then prepared, characterised and certified according to a standard procedure. Samples were dispatched to laboratories from eight Arab countries; Kuwait, Egypt, Yemen, Tunisia, Jordan, the Kingdom of Saudi Arabia, Libya and Syria. The results were evaluated using three statistical criteria; z-score, the U _test score and the relative bias. These statistical methods were used to evaluate the performance of each laboratory, in addition to the overall evaluation for each radionuclide. This evaluation has indicated that 57% and 86% of the results passed the criteria set for precision and accuracy applied for this test in relation to ²²⁶Ra and ²²⁸Ra, respectively. These two radionuclides are considered to be the most important radionuclides in the oil industry.     

Functional Characterization of a LOV‐Histidine Kinase Photoreceptor from Xanthomonas citri subsp. citri

The blue‐light (BL) absorbing protein Xcc‐LOV from Xanthomonas citri subsp. citri is composed of a LOV‐domain, a histidine kinase (HK) and a response regulator. Spectroscopic characterization of Xcc‐LOV identified intermediates and kinetics of the protein's photocycle. Measurements of steady state and time‐resolved fluorescence allowed determination of quantum yields for triplet (Φ_T = 0.68 ± 0.03) and photoproduct formation (Φ₃₉₀ = 0.46 ± 0.05). The lifetime for triplet decay was determined as τ_T = 2.4–2.8 μs. Fluorescence of tryptophan and tyrosine residues was unchanged upon light‐to‐dark conversion, emphasizing the absence of significant conformational changes. Photochemistry was blocked upon cysteine C76 (C76S) mutation, causing a seven‐fold longer lifetime of the triplet state (τ_T = 16–18.5 μs). Optoacoustic spectroscopy yielded the energy content of the triplet state. Interestingly, Xcc‐LOV did not undergo the volume contraction reported for other LOV domains within the observation time window, although the back‐conversion into the dark state was accompanied by a volume expansion. A radioactivity‐based enzyme function assay revealed a larger HK activity in the lit than in the dark state. The C76S mutant showed a still lower enzyme function, indicating the dark state activity being corrupted by a remaining portion of the long‐lived lit state.     

THE INTERACTION OF MELANIN WITH 8-METHOXYPSORALEN: EFFECT ON RADIATIVE AND NONRADIATTVE TRANSITIONS. A FLUORESCENCE AND PULSED PHOTOACOUSTIC STUDY

Abstract: The interaction of 8-methoxypsoralen (8-MOP) with synthetic eumelanin was investigated using static and time-resolved fluorescence and pulsed photoacoustic calorimetry. Due to the strong overlap of the absorption bands of melanin and 8-MOP, a method is presented to account for the systematic errors introduced by the optical filter effect exerted by each absorbing species in the fluorescence and the photoacoustic measurements. As a preliminary step to the understanding of the nonradiative behavior of the psoralen-melanin complexes, the photoacoustic parameters of 8-MOP in various solvents were determined. Spectroscopic data indicate the absence of interaction at the ground-state level, whereas the singlet excited state of 8-MOP is quenched by the pigment; the average fluorescence lifetimes are independent of the melanin concentration, thus indicating a static quenching mechanism. The photoacoustic data show that the quenching process involves an increased intersystem crossing probability, which is almost unaffected by the presence of oxygen, as expected for a molecule essentially acting as a type I photosensitizing agent.     

Isocyanide addition to pyridinium salts. Efficient entry into substituted nicotinonitrile derivatives

The addition of isocyanides to pyridinium salts is studied. The process takes place efficiently when a carboxamido group is present in the 3 position of the pyridine ring. The outcome of the reaction involves the stabilization of the nitrilium intermediate by the amide, which suffers a mild dehydration, leading regioselectively to beta-cyano-gamma-carbamoyl-1,4-dihydropyridines. In this way, a variety of nicotinamide derivatives were carbamoylated. Extension to quinolinium, isoquinolinium, and N-acylpyridinium salts is also reported. [reaction: see text]     

Tryptophan Fluorescence in the Bacillus subtilis Phototropin‐related Protein YtvA as a Marker of Interdomain Interaction¶

The Bacillus subtilis protein YtvA, related to plant phototropins (phot), binds flavin mononucleotide (FMN) within the N‐terminal light, oxygen and voltage (LOV) domain. The blue light‐triggered photocycle of YtvA and phot involves the reversible formation of a covalent photoadduct between FMN and a cysteine (cys) residue. YtvA contains a single tryptophan, W103, localized on the LOV domain and conserved in all phot‐LOV domains. In this study, we show that the fluorescence parameters of W103 in YtvA‐LOV are markedly different from those observed in the full‐length YtvA. The fluorescence quantum yields are ca 0.03 and 0.08, respectively. In YtvA‐LOV, the maximum is redshifted (ca 345 vs 335 nm) and the average fluorescence lifetime shorter (2.7 vs 4.7 ns). These data indicate that W103 is located in a site of tight contact between the two domains of YtvA. In the FMN‐cys adduct, selective excitation of W103 at 295 nm results in minimal changes of the fluorescence parameters with respect to the dark state. On 280 nm excitation, however, there is a detectable decrease in the fluorescence emitted from tyrosines, with concomitant increase in W103 fluorescence. This effect is reversible in the dark and might arise from a light‐regulated energy transfer process from a yet unidentified tyrosine to W103.