The LOV2 domain of phototropin: a reversible photochromic switch.

Light, oxygen, or voltage (LOV) domains constitute a new class of photoreceptor proteins that are sensitive to blue light through a noncovalently bound flavin chromophore. Blue-light absorption by the LOV2 domain initiates a photochemical reaction that results in formation of a long-lived covalent adduct between a cysteine and the flavin cofactor. We have applied ultrafast spectroscopy on the photoaccumulated covalent adduct state of LOV2 and find that, upon absorption of a near-UV photon by the adduct state, the covalent bond between the flavin and the cysteine is broken and the blue-light-sensitive ground state is regained on an ultrafast time scale of 100 ps.

Protein kinetics: structures of intermediates and reaction mechanism from time-resolved x-ray data.

We determine the number of authentic reaction intermediates in the later stages of the photocycle of photoactive yellow protein at room temperature, their atomic structures, and a consistent set of chemical kinetic mechanisms, by analysis of a set of time-dependent difference electron density maps spanning the time range from 5 micros to 100 ms. The successful fit of exponentials to right singular vectors derived from a singular value decomposition of the difference maps demonstrates that a chemical kinetic mechanism holds and that structurally distinct intermediates exist. We identify two time-independent difference maps, from which we refine the structures of the corresponding intermediates.

Differential localization of glutamate receptor subunits at the Drosophila neuromuscular junction.

The subunit composition of postsynaptic neurotransmitter receptors is a key determinant of synaptic physiology. Two glutamate receptor subunits, Drosophila glutamate receptor IIA (DGluRIIA) and DGluRIIB, are expressed at the Drosophila neuromuscular junction and are redundant for viability, yet differ in their physiological properties. We now identify a third glutamate receptor subunit at the Drosophila neuromuscular junction, DGluRIII, which is essential for viability.

Mutation in slowmo causes defects in Drosophila larval locomotor behaviour.

We have identified a mutant slowmotion phenotype in first instar larval peristaltic behaviour of Drosophila. By the end of embryogenesis and during early first instar phases, slowmo mutant animals show a marked decrease in locomotory behaviour, resulting from both a reduction in number and rate of peristaltic contractions. Inhibition of neurotransmitter release, using targeted expression of tetanus toxin light chain (TeTxLC), in the slowmo neurons marked by an enhancer-trap results in a similar phenotype of largely absent or uncoordinated contractions.

A radiologic study of the tympanic bone: anatomy and surgery.

Objective: The aim of this study was to obtain data on the anatomic structure of the tympanic bone using parasagittal reformatted images created from high-resolution axial computed tomographic scans. In particular, the thickness of the bone in the region of the temporomandibular joint and the floor of the external auditory canal was assessed. The findings are discussed with particular emphasis on the relevance to surgery in this area.

Effects of dietary improvement on bone metabolism in elderly underweight women with osteoporosis: a randomised controlled trial.

Malnutrition in elderly people contributes to osteoporosis and fracture. The aim of the study was to investigate the effects of nutritional improvement on bone metabolism in elderly community-dwelling women. A 12-month randomized controlled trial of 71 ambulant women aged > or =70 years with BMI < or =21 kg/m(2 )and osteoporosis at the hip was undertaken.

Effectiveness and comparison of citronella and scentless spray bark collars for the control of barking in a veterinary hospital setting.

This study was designed to evaluate the efficacy of a citronella bark collar and a new, scentless spray bark collar in a veterinary hospital or a similar kennel-type environment. Forty-one dogs were included in the study. The efficacy of each collar was evaluated independently in comparison to control as well as in a crossover design.

Assessing dissatisfaction with an out of hours service: reasons and remedies.

Objective: To identify characteristics associated with dissatisfaction following contact with an out of hours co-operative in Glasgow, and to identify reasons for this dissatisfaction. To make recommendations for good practice in the light of these findings.

Design: Survey of attenders during one week of operation of an out of hours co-operative.

General practitioners and the Internet--a questionnaire survey of Internet connectivity and use in Lothian.

Objective: To determine the extent of Internet connectivity and use by GPs in the Lothian Health Board area and to investigate which Internet medical information resources GPs find most useful for work related purposes.

Design: Postal questionnaire.

Setting: General Practice.

Primary reactions of the LOV2 domain of phototropin, a plant blue-light photoreceptor.

The phototropins constitute an important class of plant photoreceptor kinases that control a range of physiological responses, including phototropism, light-directed chloroplast movement, and light-induced stomatal opening. The LOV2 domain of phototropin binds a molecule of flavin mononucleotide (FMN) and undergoes a photocycle involving light-driven covalent adduct formation between a conserved cysteine residue and the C(4a) atom of FMN. This product state promotes C-terminal kinase activation and downstream signal transduction.

Application of singular value decomposition to the analysis of time-resolved macromolecular x-ray data.

Singular value decomposition (SVD) is a technique commonly used in the analysis of spectroscopic data that both acts as a noise filter and reduces the dimensionality of subsequent least-squares fits. To establish the applicability of SVD to crystallographic data, we applied SVD to calculated difference Fourier maps simulating those to be obtained in a time-resolved crystallographic study of photoactive yellow protein. The atomic structures of one dark state and three intermediates were used in qualitatively different kinetic mechanisms to generate time-dependent difference maps at specific time points.

Crystal structure of a photoactive yellow protein from a sensor histidine kinase: conformational variability and signal transduction.

Photoactive yellow protein (E-PYP) is a blue light photoreceptor, implicated in a negative phototactic response in Ectothiorhodospira halophila, that also serves as a model for the Per-Arnt-Sim superfamily of signaling molecules. Because no biological signaling partner for E-PYP has been identified, it has not been possible to correlate any of its photocycle intermediates with a relevant signaling state. However, the PYP domain (Ppr-PYP) from the sensor histidine kinase Ppr in Rhodospirillum centenum, which regulates the catalytic activity of Ppr by blue light absorption, may allow such issues to be addressed.

The frontiers of time-resolved macromolecular crystallography: movies and chirped X-ray pulses.

Three important frontiers of ultrafast time-resolved macromolecular crystallography are presented: extension of this technique to other biological systems; further developments in the elucidation of mechanism through the analysis of time-dependent movies to extract the underlying, time-independent, intermediate structures; and enhanced time resolution. The last is intimately linked with the nature of the pump-probe experiment itself, with the sources of random and, particularly, systematic experimental error, and with the factors that contribute to overall time resolution. All experiments to date have utilized the unchirped X-ray pulses that are emitted by synchrotron sources.

The LOV domain family: photoresponsive signaling modules coupled to diverse output domains.

For single-cell and multicellular systems to survive, they must accurately sense and respond to their cellular and extracellular environment. Light is a nearly ubiquitous environmental factor, and many species have evolved the capability to respond to this extracellular stimulus. Numerous photoreceptors underlie the activation of light-sensitive signal transduction cascades controlling these responses.

Genome sequencing of a 239-kb region of rice chromosome 10L reveals a high frequency of gene duplication and a large chloroplast DNA insertion.

In this study we describe a 239-kb region on the long arm of rice chromosome 10 that contains a high density (71%) of locally duplicated genes, including 24 copies of a glutathione S-transferase gene. Intriguingly, embedded within this cluster is a large insertion (approximately 33 kb) of rice (Oryza sativa) chloroplast DNA that is derived from two separate regions of the chloroplast genome. We used DNA fiber-based fluorescence in situ hybridization (fiber-FISH) analyses of O.

Radiation damage of protein crystals at cryogenic temperatures between 40 K and 150 K.

X-ray radiation damage of lysozyme single crystals by an intense monochromatic beam from the Advanced Photon Source is studied at cryogenic temperatures between 40 K and 150 K. The results confirm that primary radiation damage is both linearly dependent on the X-ray dose and independent of temperature. The upper limit for the primary radiation damage observed in our previous study [Teng & Moffat (2000), J.

Photoexcited structure of a plant photoreceptor domain reveals a light-driven molecular switch.

The phototropins are flavoprotein kinases that control phototropic bending, light-induced chloroplast movement, and stomatal opening in plants. Two flavin mononucleotide binding light, oxygen, or voltage (LOV) domains are the sites for initial photochemistry in these blue light photoreceptors. We have determined the steady state, photoexcited crystal structure of a flavin-bound LOV domain.

Protein conformational relaxation and ligand migration in myoglobin: a nanosecond to millisecond molecular movie from time-resolved Laue X-ray diffraction.

A time-resolved Laue X-ray diffraction technique has been used to explore protein relaxation and ligand migration at room temperature following photolysis of a single crystal of carbon monoxymyoglobin. The CO ligand is photodissociated by a 7.5 ns laser pulse, and the subsequent structural changes are probed by 150 ps or 1 micros X-ray pulses at 14 laser/X-ray delay times, ranging from 1 ns to 1.

A molecular movie at 1.8 A resolution displays the photocycle of photoactive yellow protein, a eubacterial blue-light receptor, from nanoseconds to seconds.

The photocycle of the bacterial blue-light photoreceptor, photoactive yellow protein, was stimulated by illumination of single crystals by a 7 ns laser pulse. The molecular events were recorded at high resolution by time-resolved X-ray Laue diffraction as they evolved in real time, from 1 ns to seconds after the laser pulse. The complex structural changes during the photocycle at ambient temperature are displayed in a movie of difference electron density maps relative to the dark state.

Structure of a flavin-binding plant photoreceptor domain: insights into light-mediated signal transduction.

Phototropin, a major blue-light receptor for phototropism in seed plants, exhibits blue-light-dependent autophosphorylation and contains two light, oxygen, or voltage (LOV) domains and a serine/threonine kinase domain. The LOV domains share homology with the PER-ARNT-SIM (PAS) superfamily, a diverse group of sensor proteins. Each LOV domain noncovalently binds a single FMN molecule and exhibits reversible photochemistry in vitro when expressed separately or in tandem.