Ultrafast structural changes direct the first molecular events of vision

視覚に関わるタンパク質の超高速分子動画 --薄暗いところで光を感じる仕組み--. 京都大学プレスリリース. 2023-03-23.Vision is initiated by the rhodopsin family of light-sensitive G protein-coupled receptors (GPCRs). A photon is absorbed by the 11-cis retinal chromophore of rhodopsin, which isomerizes within 200 femtoseconds to the all-trans conformation, thereby initiating the cellular signal transduction processes that ultimately lead to vision. However, the intramolecular mechanism by which the photoactivated retinal induces the activation events inside rhodopsin remains experimentally unclear. Here we use ultrafast time-resolved crystallography at room temperature to determine how an isomerized twisted all-trans retinal stores the photon energy that is required to initiate the protein conformational changes associated with the formation of the G protein-binding signalling state. The distorted retinal at a 1-ps time delay after photoactivation has pulled away from half of its numerous interactions with its binding pocket, and the excess of the photon energy is released through an anisotropic protein breathing motion in the direction of the extracellular space. Notably, the very early structural motions in the protein side chains of rhodopsin appear in regions that are involved in later stages of the conserved class A GPCR activation mechanism. Our study sheds light on the earliest stages of vision in vertebrates and points to fundamental aspects of the molecular mechanisms of agonist-mediated GPCR activation

Viscoelasticity and Interface Bending Properties of Lecithin Reverse Wormlike Micelles Studied by Diffusive Wave Spectroscopy in Hydrophobic Environment

Upon the addition of minute quantities of water into a phosphatidylcholine (PC) solution in certain organic solvents, PC micelles elongate into giant reverse wormlike micelles that entangle and form highly viscous microemulsions, called lecithin organogels. We investigated the microrheological properties of concentrated PC–cyclohexane reverse wormlike micellar systems by diffusive wave spectroscopy (DWS) in apolar medium, combined with bulk shear rheology. We applied DWS to our oil-continuous system by using hydrophobic poly­(hydroxystearic acid)-grafted PMMA particles as monodisperse tracer particles. Relevant parameters such as the micellar scission energy and persistence length were extracted from the microrheology data and interpreted according to the sphere-to-rod-to-sphere structural transition. On the basis of these quantities, we calculated the bending and saddle-splay moduli of the PC-covered water–cyclohexane interface. This approach represents a new method for the quantitative estimation of these fundamental parameters, which are thought to underpin the self-assembly of surfactants

A Reverse Micellar Mesophase of Face-Centered Cubic <i>Fm</i>3̅<i>m</i> Symmetry in Phosphatidylcholine/Water/Organic Solvent Ternary Systems

We report the formation of a <i>reverse</i> micellar cubic mesophase of symmetry <i>Fm</i>3̅<i>m</i> (Q²²⁵) in ternary mixtures of soy bean phosphatidylcholine (PC), water, and an organic solvent, including cyclohexane, (<i>R</i>)-(+)-limonene, and isooctane, studied by small-angle X-ray scattering (SAXS) and oscillatory shear rheology at room temperature. The mesophase structure consists of a compact packing of remarkably large reverse micelles in a face-centered cubic (fcc) lattice, a type of micellar packing not yet reported for reverse micellar mesophases. Form factor fitting in the pure L₂ phase and in the <i>Fm</i>3̅<i>m</i>–L₂ coexistence region yields quantitative estimations of the PC interface rigidity. The compact <i>Fm</i>3̅<i>m</i> structure results mainly from release of lipid tail frustration and hard-sphere interactions between monodisperse micelles, as suggested by a comparison with the <i>Fd</i>3̅<i>m</i> structure found in the PC/water/α-tocopherol system

Direct visualization of dispersed lipid bicontinuous cubic phases by cryo-electron tomography

Bulk and dispersed cubic liquid crystalline phases (cubosomes), present in the body and in living cell membranes, are believed to play an essential role in biological phenomena. Moreover, their biocompatibility is attractive for nutrient or drug delivery system applications. Here the three-dimensional organization of dispersed cubic lipid self-assembled phases is fully revealed by cryo-electron tomography and compared with simulated structures. It is demonstrated that the interior is constituted of a perfect bicontinuous cubic phase, while the outside shows interlamellar attachments, which represent a transition state between the liquid crystalline interior phase and the outside vesicular structure. Therefore, compositional gradients within cubosomes are inferred, with a lipid bilayer separating at least one water channel set from the external aqueous phase. This is crucial to understand and enhance controlled release of target molecules and calls for a revision of postulated transport mechanisms from cubosomes to the aqueous phase

Facile Dispersion and Control of Internal Structure in Lyotropic Liquid Crystalline Particles by Auxiliary Solvent Evaporation

Submicron sized, structured lyotropic liquid crystalline (LLC) particles, so-called hexosomes and cubosomes, are generally obtained by high energy input dispersion methods, notably ultrasonication and high-pressure emulsification. We present a method to obtain dispersions of such LLC particles with a significantly reduced energy input, by evaporation of an auxiliary volatile solvent immiscible with water, e.g. cyclohexane or limonene. The inner structure of the particles can be precisely controlled by the addition of a nonvolatile oil, such as α-tocopherol or tetradecane consistently with bulk phase diagrams,. Two different lyotropic surfactants were employed, industrial grade monolinoleine (MLO) and soy bean phosphatidylcholine (PC). The lyotropic surfactant and oil phase modifier were first dissolved in the volatile solvent to give a liquid reverse micellar (<i>L</i>₂) phase, which requires significantly less energy input to be dispersed in an aqueous solution of secondary emulsifier compared to the corresponding gel-like bulk mesophase. The auxiliary volatile solvent was then removed from the emulsion by evaporation at room temperature, yielding LLC particles of the desired inner structure, <i>Pn</i>3̅<i>m</i>, <i>H</i>₂, or <i>Fd</i>3̅<i>m</i>. The obtained particles were characterized by small-angle X-ray scattering (SAXS), dynamic light scattering (DLS), and cryogenic transmission electron microscopy (cryo-TEM). Our method enables fine-tuning of the final particle size through the volatile-to-nonvolatile volume ratio and processing conditions

Direct protein crystallization on ultrathin membranes for diffraction measurements at X-ray free-electron lasers (vol 50, pg 909, 2017)

Errors in the article by Opara, Martiel, Arnold, Braun, Stahlberg, Makita, David & Padeste [J. Appl. Cryst. (2017), 50, 909-918] are corrected

Direct protein crystallization on ultrathin membranes for diffraction measurements at X-ray free-electron lasers

A new era of protein crystallography started when X-ray free-electron lasers (XFELs) came into operation, as these provide an intense source of X-rays that facilitates data collection in the 'iffract-before-destroy' regime. In typical experiments, crystals sequentially delivered to the beam are exposed to X-rays and destroyed. Therefore, the novel approach of serial crystallography requires thousands of nearly identical samples. Currently applied sample-delivery methods, in particular liquid jets or drop-on-demand systems, suffer from significant sample consumption of the precious crystalline material. Direct protein microcrystal growth by the vapour diffusion technique inside arrays of nanolitre-sized wells is a method specifically tailored to crystallography at XFELs. The wells, with X-ray transparent Si3N4 windows as bottoms, are fabricated in silicon chips. Their reduced dimensions can significantly decrease protein specimen consumption. Arrays provide crystalline samples positioned in an ordered way without the need to handle fragile crystals. The nucleation process inside these microfabricated cavities was optimized to provide high membrane coverage and a quasi-random crystal distribution. Tight sealing of the chips and protection of the crystals from dehydration were achieved, as confirmed by diffraction experiments at a protein crystallography beamline. Finally, the test samples were shown to be suitable for time-resolved measurements at an XFEL at femtosecond resolution

Oil Transfer Converts Phosphatidylcholine Vesicles into Nonlamellar Lyotropic Liquid Crystalline Particles

There is a need for the development of low-energy dispersion methods tailored to the formation of phospholipid-based nonlamellar lyotropic liquid crystalline (LLC) particles for delivery system applications. Here, facile formation of nonlamellar LLC particles was obtained by simple mixing of a phosphatidylcholine (PC) liposome solution and an oil-in-water emulsion, with limonene or isooctane as an oil. The internal structure of the particles was controlled by the PC-to-oil ratio, consistently with the sequence observed in bulk phase. For the first time, reverse micellar cubosomes with <i>Fm</i>3̅<i>m</i> inner structure were produced. The size, morphology, and inner structure of the particles were characterized by small-angle X-ray scattering (SAXS), dynamic light scattering (DLS), and freeze-fracture cryo scanning electron microscopy (cryo-SEM). These findings pave the way to new strategies in low-energy formulation of LLC delivery systems

MAP65/Ase1 promote microtubule flexibility.

International audienceMicrotubules (MTs) are dynamic cytoskeletal elements involved in numerous cellular processes. Although they are highly rigid polymers with a persistence length of 1-8 mm, they may exhibit a curved shape at a scale of few micrometers within cells, depending on their biological functions. However, how MT flexural rigidity in cells is regulated remains poorly understood. Here we ask whether MT-associated proteins (MAPs) could locally control the mechanical properties of MTs. We show that two major cross-linkers of the conserved MAP65/PRC1/Ase1 family drastically decrease MT rigidity. Their MT-binding domain mediates this effect. Remarkably, the softening effect of MAP65 observed on single MTs is maintained when MTs are cross-linked. By reconstituting physical collisions between growing MTs/MT bundles, we further show that the decrease in MT stiffness induced by MAP65 proteins is responsible for the sharp bending deformations observed in cells when they coalign at a steep angle to create bundles. Taken together, these data provide new insights into how MAP65, by modifying MT mechanical properties, may regulate the formation of complex MT arrays