RESUMO
X-ray free-electron laser (XFEL) light sources have enabled the rapid growth of time-resolved structural experiments, which provide crucial information on the function of macromolecules and their mechanisms. Here, the aim was to commission the SwissMX fixed-target sample-delivery system at the SwissFEL Cristallina experimental station using the PSI-developed micro-structured polymer (MISP) chip for pump-probe time-resolved experiments. To characterize the system, crystals of the light-sensitive protein light-oxygen-voltage domain 1 (LOV1) from Chlamydomonas reinhardtii were used. Using different experimental settings, the accidental illumination, referred to as light contamination, of crystals mounted in wells adjacent to those illuminated by the pump laser was examined. It was crucial to control the light scattering from and through the solid supports otherwise significant contamination occurred. However, the results here show that the opaque MISP chips are suitable for defined pump-probe studies of a light-sensitive protein. The experiment also probed the sub-millisecond structural dynamics of LOV1 and indicated that at Δt = 10â µs a covalent thioether bond is established between reactive Cys57 and its flavin mononucleotide cofactor. This experiment validates the crystals to be suitable for in-depth follow-up studies of this still poorly understood signal-transduction mechanism. Importantly, the fixed-target delivery system also permitted a tenfold reduction in protein sample consumption compared with the more common high-viscosity extrusion-based delivery system. This development creates the prospect of an increase in XFEL project throughput for the field.
RESUMO
Charge-transfer reactions in proteins are important for life, such as in photolyases which repair DNA, but the role of structural dynamics remains unclear. Here, using femtosecond X-ray crystallography, we report the structural changes that take place while electrons transfer along a chain of four conserved tryptophans in the Drosophila melanogaster (6-4) photolyase. At femto- and picosecond delays, photoreduction of the flavin by the first tryptophan causes directed structural responses at a key asparagine, at a conserved salt bridge, and by rearrangements of nearby water molecules. We detect charge-induced structural changes close to the second tryptophan from 1 ps to 20 ps, identifying a nearby methionine as an active participant in the redox chain, and from 20 ps around the fourth tryptophan. The photolyase undergoes highly directed and carefully timed adaptations of its structure. This questions the validity of the linear solvent response approximation in Marcus theory and indicates that evolution has optimized fast protein fluctuations for optimal charge transfer.
Assuntos
Desoxirribodipirimidina Fotoliase , Humanos , Animais , Desoxirribodipirimidina Fotoliase/química , Desoxirribodipirimidina Fotoliase/genética , Desoxirribodipirimidina Fotoliase/metabolismo , Triptofano/química , Elétrons , Drosophila melanogaster/metabolismo , Escherichia coli/genética , Transporte de Elétrons , Cristalografia por Raios XRESUMO
Resumen Objetivo: Comparar dos sistemas de puntuación para un test de fluidez verbal con el Modelo de Escalas de Calificación. Método: Se analizaron datos de 289 participantes, de los cuales 92 habían sido diagnosticados con Parkinson. Las puntuaciones se calcularon con dos sistemas de categorización: un procedimiento convencional y otro basado en percentiles. Resultados: Las puntuaciones Rasch procedentes de percentiles dan lugar a categorías adecuadas y medidas fiables; la correlación con las puntuaciones del test Minimental es evidencia de validez concurrente. Tras controlar estadísticamente el efecto de la edad, las medidas Rasch procedentes de percentiles discriminan entre ambos grupos, lo que evidencia validez predictiva. Conclusiones: El análisis de los dos procedimientos permite recomendar el uso de las categorías basadas en percentiles.
Abstract Objective: Two scoring systems for a verbal fluency test were compared using the Rasch Rating Scale Model. Method: The analysis was carried out on 289 participants, 92 of whom had had a Parkinson's disease diagnosis. Scores were calculated with two different category systems: a conventional procedure and a percentile-based one. Results: The percentile-based Rasch scores produce adequate categories and reliable measures, while the correlation with the Mini Mental State Examination evinces concurrent validity. After statistically controlling for age, percentile-based Rasch measures discriminated between both groups, demonstrating predictive validity. Conclusions: The analysis of the two procedures allows for the recommendation of the use of percentile-based categories.