Vesicle adhesion and fusion studied by small-angle x-ray scattering.

We have studied the adhesion state (also denoted by docking state) of lipid vesicles as induced by the divalent ions Ca2+ or Mg2+ at well-controlled ion concentration, lipid composition, and charge density. The bilayer structure and the interbilayer distance in the docking state were analyzed by small-angle x-ray scattering. A strong adhesion state was observed for DOPC:DOPS vesicles, indicating like-charge attraction resulting from ion correlations. The observed interbilayer separations of ∼1.6 nm agree quantitatively with the predictions of electrostatics in the strong coupling regime. Although this phenomenon was observed when mixing anionic and zwitterionic (or neutral) lipids, pure anionic membranes (DOPS) with highest charge density σ resulted in a direct phase transition to a multilamellar state, which must be accompanied by rupture and fusion of vesicles. To extend the structural assay toward protein-controlled docking and fusion, we have characterized reconstituted N-ethylmaleimide-sensitive factor attachment protein receptors in controlled proteoliposome suspensions by small-angle x-ray scattering

Combined small angle X-ray solution scattering with atomic force microscopy for characterizing radiation damage on biological macromolecules

Background Synchrotron radiation facilities are pillars of modern structural biology. Small-Angle X-ray scattering performed at synchrotron sources is often used to characterize the shape of biological macromolecules. A major challenge with high-energy X-ray beam on such macromolecules is the perturbation of sample due to radiation damage. Results By employing atomic force microscopy, another common technique to determine the shape of biological macromolecules when deposited on flat substrates, we present a protocol to evaluate and characterize consequences of radiation damage. It requires the acquisition of images of irradiated samples at the single molecule level in a timely manner while using minimal amounts of protein. The protocol has been tested on two different molecular systems: a large globular tetremeric enzyme (β-Amylase) and a rod-shape plant virus (tobacco mosaic virus). Radiation damage on the globular enzyme leads to an apparent increase in molecular sizes whereas the effect on the long virus is a breakage into smaller pieces resulting in a decrease of the average long-axis radius. Conclusions These results show that radiation damage can appear in different forms and strongly support the need to check the effect of radiation damage at synchrotron sources using the presented protocol

Reconstitution of SNARE proteins into solid-supported lipid bilayer stacks and X-ray structure analysis.

SNAREs are known as an important family of proteins mediating vesicle fusion. For various biophysical studies, they have been reconstituted into supported single bilayers via proteoliposome adsorption and rupture. In this study we extended this method to the reconstitution of SNAREs into supported multilamellar lipid membranes, i.e. oriented multibilayer stacks, as an ideal model system for X-ray structure analysis (X-ray reflectivity and diffraction). The reconstitution was implemented through a pathway of proteomicelle, proteoliposome and multibilayer. To monitor the structural evolution in each step, we used small-angle X-ray scattering for the proteomicelles and proteoliposomes, followed by X-ray reflectivity and grazing-incidence small-angle scattering for the multibilayers. Results show that SNAREs can be successfully reconstituted into supported multibilayers, with high enough orientational alignment for the application of surface sensitive X-ray characterizations. Based on this protocol, we then investigated the effect of SNAREs on the structure and phase diagram of the lipid membranes. Beyond this application, this reconstitution protocol could also be useful for X-ray analysis of many further membrane proteins

Evolution of the Plant Reproduction Master Regulators LFY and the MADS Transcription Factors: The Role of Protein Structure in the Evolutionary Development of the Flower.

International audienceUnderstanding the evolutionary leap from non-flowering (gymnosperms) to flowering (angiosperms) plants and the origin and vast diversification of the floral form has been one of the focuses of plant evolutionary developmental biology. The evolving diversity and increasing complexity of organisms is often due to relatively small changes in genes that direct development. These "developmental control genes" and the transcription factors (TFs) they encode, are at the origin of most morphological changes. TFs such as LEAFY (LFY) and the MADS-domain TFs act as central regulators in key developmental processes of plant reproduction including the floral transition in angiosperms and the specification of the male and female organs in both gymnosperms and angiosperms. In addition to advances in genome wide profiling and forward and reverse genetic screening, structural techniques are becoming important tools in unraveling TF function by providing atomic and molecular level information that was lacking in purely genetic approaches. Here, we summarize previous structural work and present additional biophysical and biochemical studies of the key master regulators of plant reproduction - LEAFY and the MADS-domain TFs SEPALLATA3 and AGAMOUS. We discuss the impact of structural biology on our understanding of the complex evolutionary process leading to the development of the bisexual flower

Structural Basis of Membrane Protein Chaperoning through the Mitochondrial Intermembrane Space

International audienceThe exchange of metabolites between the mitochon- drial matrix and the cytosol depends on b-barrel channels in the outer membrane and a-helical carrier proteins in the inner membrane. The essential trans- locase of the inner membrane (TIM) chaperones escort these proteins through the intermembrane space, but the structural and mechanistic details remain elusive. We have used an integrated struc- tural biology approach to reveal the functional princi- ple of TIM chaperones. Multiple clamp-like binding sites hold the mitochondrial membrane proteins in a translocation-competent elongated form, thus mimicking characteristics of co-translational mem- brane insertion. The bound preprotein undergoes conformational dynamics within the chaperone bind- ing clefts, pointing to a multitude of dynamic local binding events. Mutations in these binding sites cause cell death or growth defects associated with impairment of carrier and b-barrel protein biogen- esis. Our work reveals how a single mitochondrial ‘‘transfer-chaperone’’ system is able to guide a-heli- cal and b-barrel membrane proteins in a ‘‘nascent chain-like’’ conformation through a ribosome-free compartment

Coupling high throughput microfluidics and small-angle x-ray scattering to study protein crystallization from solution

In this work, we propose the combination of small-angle X-ray scattering (SAXS) and high throughput, droplet based microfluidics as a powerful tool to investigate macromolecular interactions, directly related to protein solubility. For this purpose, a robust and low cost microfluidic platform was fabricated for achieving the mixing of proteins, crystallization reagents, and buffer in nanoliter volumes and the subsequent generation of nanodroplets by means of a two phase flow. The protein samples are compartmentalized inside droplets, each one acting as an isolated microreactor. Hence their physicochemical conditions (concentration, pH, etc.) can be finely tuned without cross-contamination, allowing the screening of a huge number of saturation conditions with a small amount of biological material. The droplet flow is synchronized with synchrotron radiation SAXS measurements to probe protein interactions while minimizing radiation damage. To this end, the experimental setup was tested with rasburicase (known to be very sensitive to denaturation), proving the structural stability of the protein in the droplets and the absence of radiation damage. Subsequently weak interaction variations as a function of protein saturation was studied for the model protein lysozime. The second virial coefficients (A2) were determined from the X-ray structure factors extrapolated to the origin. A2 obtained values were found to be in good agreement with data previously reported in literature but using only a few milligrams of protein. The experimental results presented here highlight the interest and convenience of using this methodology as a promising and potential candidate for studying protein interactions for the construction of phase diagrams

Innovative high-throughput SAXS methodologies based on photonic lab-on-a-chip sensors: application to macromolecular studies

The relevance of coupling droplet-based Photonic Lab-on-a-Chip (PhLoC) platforms and Small-Angle X-Ray Scattering (SAXS) technique is here highlighted for the performance of high throughput investigations, related to the study of protein macromolecular interactions. With this configuration, minute amounts of sample are required to obtain reliable statistical data. The PhLoC platforms presented in this work are designed to allow and control an effective mixing of precise amounts of proteins, crystallization reagents and buffer in nanoliter volumes, and the subsequent generation of nanodroplets by means of a two-phase flow. Spectrophotometric sensing permits a fine control on droplet generation frequency and stability as well as on concentration conditions, and finally the droplet flow is synchronized to perform synchrotron radiation SAXS measurements in individual droplets (each one acting as an isolated microreactor) to probe protein interactions. With this configuration, droplet physic-chemical conditions can be reproducibly and finely tuned, and monitored without cross-contamination, allowing for the screening of a substantial number of saturation conditions with a small amount of biological material. The setup was tested and validated using lysozyme as a model of study. By means of SAXS experiments, the proteins gyration radius and structure envelope were calculated as a function of protein concentration. The obtained values were found to be in good agreement with previously reported data, but with a dramatic reduction of sample volume requirements compared to studies reported in the literature

Microfluidic Assisted Self-Assembly of pH-Sensitive Low-Molecular Weight Hydrogelators Close to the Minimum Gelation Concentration

\u3cp\u3eThe fibrillation and subsequent gelation of low molecular hydrogelators is usually triggered by external stimuli. Generally, strong acids are employed to trigger the self-assembly mechanism in pH-responsive supramolecular systems. However, the generation and design of novel stable gels with performing mechanical properties is a challenging task as a result of the uneven self-assembled networks formed. Here, we report the study of the self-assembly process of a low molecular weight hydrogelator (LMWG) in the proximity of its minimum gelation concentration (MGC = 0.3 mg/ml). At such high dilution, the generation of homogeneous gels with good mechanical properties by turning pH by strong acids is a demanding task as a result of the lack of monodisperse 1D self-assembled rod-like aggregates. A microfluidic device is employed here to gradually and homogeneously change the pH. We show that self-assembly of LMWG in well-defined structures can be enhanced by using the diffusive mixing occurring in the microfluidic reactor channel. For very short mixing times, aggregates with 2 nm cross-section are found in the region adjacent to the focused LMWG solution in contact with the low pH buffer solution streams, where the pH reaches values below the pKa of the LMWG and triggers the supramolecular self-assembly. For longer mixing times, aggregates grow in size and occupy homogeneously the micro channel. The results presented here show that better controlled self-assembly can be achieved using microfluidic mixing devices and early stages of self-assembly can be efficiently studied by coupling synchrotron SAXS with microfluidics.\u3c/p\u3

Binding of myomesin to obscurin-like-1 to the muscle M-band provides a strategy for isoform-specific mechanical protection

The sarcomeric cytoskeleton is a network of modular proteins that integrate mechanical and signalling roles. Obscurin, or its homolog obscurin-like-1, bridges the giant ruler titin and the myosin crosslinker myomesin at the M-band. Yet, the molecular mechanisms underlying the physical obscurin(-like-1):myomesin connection, important for mechanical integrity of the M-band, remained elusive. Here, using a combination of structural, cellular, and single-molecule force spectroscopy techniques, we decode the architectural and functional determinants defining the obscurin(-like-1): myomesin complex. The crystal structure reveals a trans-complementation mechanism whereby an incomplete immunoglobulin-like domain assimilates an isoform-specific myomesin interdomain sequence. Crucially, this unconventional architecture provides mechanical stability up to forces of 135 pN. A cellular competition assay in neonatal rat cardiomyocytes validates the complex and provides the rationale for the isoform specificity of the interaction. Altogether, our results reveal a novel binding strategy in sarcomere assembly, which might have implications on muscle nanomechanics and overall M-band organization.We thank the Diamond Light Source and the European Synchrotron Radiation Laboratory for access to MX and SAXS beamlines, respectively. This work was supported by a British Heart Foundation grant (PG/10/67/28527) awarded to R.A.S. and M.G. as well as MRC grant MR/J010456/1 to M.G. and a British Heart Foundation grant (PG/13/50/30426) and EPSRC Fellowship (K00641X/1) to S.G.-M