RESUMEN
Attachment of human noroviruses to histo blood group antigens (HBGAs) is thought to be essential for infection, although how this binding event promotes infection is unknown. Recent studies have shown that 60% of all GII.4 epidemic strains may undergo a spontaneous post-translational modification (PTM) in an amino acid located adjacent to the binding pocket for HBGAs. This transformation proceeds with an estimated half-life of 1-2 days under physiological conditions, dramatically affecting HBGA recognition. The surface-exposed position of this PTM and its sequence conservation suggests a relevant role in immune escape and host-cell recognition. As a first step towards the understanding of the biological implications of this PTM at atomic resolution, we report the complete assignment of methyl resonances of a MILProSVProSA methyl-labeled sample of a 72 kDa protruding domain from a GII.4 Saga human norovirus strain. Assignments were obtained from methyl-methyl NOESY experiments combined with site-directed mutagenesis and automated assignment. This data provides the basis for a detailed characterization of the PTM-driven modulation of immune recognition in human norovirus on a molecular level.
Asunto(s)
Aminoácidos/química , Norovirus/metabolismo , Resonancia Magnética Nuclear Biomolecular , Proteínas Virales/química , Cristalografía por Rayos X , Humanos , Mutagénesis Sitio-Dirigida , Dominios Proteicos , Multimerización de ProteínaRESUMEN
Spontaneous activity shifts at constant experimental conditions represent a widespread regulatory mechanism in ion channels. The molecular origins of these modal gating shifts are poorly understood. In the K+ channel KcsA, a multitude of fast activity shifts that emulate the native modal gating behaviour can be triggered by point-mutations in the hydrogen bonding network that controls the selectivity filter. Using solid-state NMR and molecular dynamics simulations in a variety of KcsA mutants, here we show that modal gating shifts in K+ channels are associated with important changes in the channel dynamics that strongly perturb the selectivity filter equilibrium conformation. Furthermore, our study reveals a drastically different motional and conformational selectivity filter landscape in a mutant that mimics voltage-gated K+ channels, which provides a foundation for an improved understanding of eukaryotic K+ channels. Altogether, our results provide a high-resolution perspective on some of the complex functional behaviour of K+ channels.
Asunto(s)
Proteínas Bacterianas/metabolismo , Activación del Canal Iónico/fisiología , Canales de Potasio/metabolismo , Potasio/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Enlace de Hidrógeno , Activación del Canal Iónico/genética , Simulación de Dinámica Molecular , Mutación , Canales de Potasio/química , Canales de Potasio/genética , Conformación Proteica , Homología de Secuencia de AminoácidoRESUMEN
A small active in-vacuum beamstop has been developed to monitor the flux of intense third-generation synchrotron X-ray beams protecting the downstream detector from the direct beam. Standard active beamstops, where a built-in diode directly absorbs the beam, have limitations in size and lifetime. In the present design, a silicon PIN diode detects the photons back-scattered from a cavity in the beamstop. This approach drastically reduces the radiation dose on the diode and thus increases its lifetime. The beamstop with a diameter of 2â mm has been fabricated to meet the requirements for the P12 bioSAXS beamline of EMBL Hamburg at PETRAâ III (DESY). The beamstop is in regular user operation at the beamline and displays a good response over the range of energies tested (6-20â keV). Further miniaturization of the diode is easily possible as its size is not limited by the PIN diode used.
RESUMEN
The EMBL Hamburg Outstation currently operates five synchrotron beamlines for protein crystallography. The strongest of these beamlines is the fixed-energy beamline BW7B which receives about half of the radiation (1.5 mrad) from a 56 pole wiggler located at the DORIS III storage ring at the German synchrotron facility DESY. Over the last years this beamline has been upgraded and equipped with a fully automated crystallographic end-station and a robotic sample changer. The current set-up allows for remote operation, controlled from the user's area, of sample mounting, centering and data collection of pre-frozen crystals mounted in Hampton-type cryovials on magnetic caps. New software and intuitive graphical user interfaces have been developed that control the complete beamline set-up. Furthermore, algorithms for automatic sample centering based on UV fluorescence are being developed and combined with strategy programs in order to further automate the collection of entire diffraction data sets.