Action of Molecular Switches in GPCRs - Theoretical and Experimental Studies

G protein coupled receptors (GPCRs), also called 7TM receptors, form a huge superfamily of membrane proteins that, upon activation by extracellular agonists, pass the signal to the cell interior. Ligands can bind either to extracellular N-terminus and loops (e.g. glutamate receptors) or to the binding site within transmembrane helices (Rhodopsin-like family). They are all activated by agonists although a spontaneous auto-activation of an empty receptor can also be observed. Biochemical and crystallographic methods together with molecular dynamics simulations and other theoretical techniques provided models of the receptor activation based on the action of so-called “molecular switches” buried in the receptor structure. They are changed by agonists but also by inverse agonists evoking an ensemble of activation states leading toward different activation pathways. Switches discovered so far include the ionic lock switch, the 3-7 lock switch, the tyrosine toggle switch linked with the nPxxy motif in TM7, and the transmission switch. The latter one was proposed instead of the tryptophan rotamer toggle switch because no change of the rotamer was observed in structures of activated receptors. The global toggle switch suggested earlier consisting of a vertical rigid motion of TM6, seems also to be implausible based on the recent crystal structures of GPCRs with agonists. Theoretical and experimental methods (crystallography, NMR, specific spectroscopic methods like FRET/BRET but also single-molecule-force-spectroscopy) are currently used to study the effect of ligands on the receptor structure, location of stable structural segments/domains of GPCRs, and to answer the still open question on how ligands are binding: either via ensemble of conformational receptor states or rather via induced fit mechanisms. On the other hand the structural investigations of homo- and heterodimers and higher oligomers revealed the mechanism of allosteric signal transmission and receptor activation that could lead to design highly effective and selective allosteric or ago-allosteric drugs

Coherent diffractive imaging of microtubules using an X-ray laser

X-ray free electron lasers (XFELs) create new possibilities for structural studies of biological objects that extend beyond what is possible with synchrotron radiation. Serial femtosecond crystallography has allowed high-resolution structures to be determined from micro-meter sized crystals, whereas single particle coherent X-ray imaging requires development to extend the resolution beyond a few tens of nanometers. Here we describe an intermediate approach: the XFEL imaging of biological assemblies with helical symmetry. We collected X-ray scattering images from samples of microtubules injected across an XFEL beam using a liquid microjet, sorted these images into class averages, merged these data into a diffraction pattern extending to 2 nm resolution, and reconstructed these data into a projection image of the microtubule. Details such as the 4 nm tubulin monomer became visible in this reconstruction. These results illustrate the potential of single-molecule X-ray imaging of biological assembles with helical symmetry at room temperature

Flow-aligned, single-shot fiber diffraction using a femtosecond X-ray free-electron laser

A major goal for X-ray free-electron laser (XFEL) based science is to elucidate structures of biological molecules without the need for crystals. Filament systems may provide some of the first single macromolecular structures elucidated by XFEL radiation, since they contain one-dimensional translational symmetry and thereby occupy the diffraction intensity region between the extremes of crystals and single molecules. Here, we demonstrate flow alignment of as few as 100 filaments (Escherichia coli pili, F-actin, and amyloid fibrils), which when intersected by femtosecond X-ray pulses result in diffraction patterns similar to those obtained from classical fiber diffraction studies. We also determine that F-actin can be flow-aligned to a disorientation of approximately 5 degrees. Using this XFEL-based technique, we determine that gelsolin amyloids are comprised of stacked β-strands running perpendicular to the filament axis, and that a range of order from fibrillar to crystalline is discernable for individual α-synuclein amyloids

Potreba po mladinskem centru v občini Cerknica

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Wireless time synchronization of a collaborative brain-computer-inteface using Bluetooth low energy

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Experimental evaluation of a pairwise broadcast synchronization in a low-power cyber-physical system

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Ubiquitous amyloids

The common view of amyloids and prion proteins is that they are associated with many currently incurable diseases and present a great danger to an organism. This danger comes from the fact that not only prion proteins, but also the infectious form(s) of amyloids, as it has been shown recently, are able to transmit the disease. On the other hand, organisms take advantage of the strength and durability of specific forms of amyloids. Such forms do not spread any disease. Also, in nanotechnology there is a constantly growing need to employ amyloid fibrils in many industrial applications. With increasing knowledge about amyloids and prion proteins we are aware that the amyloidal state is inherent to any protein, making the problem of amyloid formation a central one in aging-related diseases. However, the "good" amyloids can be beneficial and even necessary for our health. Furthermore, because of their mechanical properties, the amyloids are of great interest to engineers

Yersinia effector protein (YopO)-mediated phosphorylation of host gelsolin causes calcium-independent activation leading to disruption of actin dynamics.

Pathogenic Yersinia bacteria cause a range of human diseases. To modulate and evade host immune systems, these yersiniae inject effector proteins into host macrophages. One such protein, the serine/threonine kinase YopO (YpkA in Yersinia pestis), uses monomeric actin as bait to recruit and phosphorylate host actin polymerization-regulating proteins, including the actin-severing protein gelsolin, to disrupt actin filaments and thus impair phagocytosis. However, the YopO phosphorylation sites on gelsolin and the consequences of YopO-mediated phosphorylation on actin remodeling have yet to be established. Here we determined the effects of YopO-mediated phosphorylation on gelsolin and identified its phosphorylation sites by mass spectrometry. YopO phosphorylated gelsolin in the linker region between gelsolin homology domains G3 and G4, which, in the absence of calcium, are compacted but adopt an open conformation in the presence of calcium, enabling actin binding and severing. Using phosphomimetic and phosphodeletion gelsolin mutants, we found that YopO-mediated phosphorylation partially mimics calcium-dependent activation of gelsolin, potentially contributing to a reduction in filamentous actin and altered actin dynamics in phagocytic cells. In summary, this work represents the first report of the functional outcome of serine/threonine phosphorylation in gelsolin regulation and provides critical insight into how YopO disrupts normal gelsolin function to alter host actin dynamics and thus cripple phagocytosis