Concentration dependent pathways in spontaneous self-assembly of unilamellar vesicles

Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG geförderten) Allianz- bzw. Nationallizenz frei zugänglich.This publication is with permission of the rights owner freely accessible due to an Alliance licence and a national licence (funded by the DFG, German Research Foundation) respectively.We report on the structural dynamics underlying the formation of unilamellar vesicles upon mixing dilute solutions of anionic and zwitterionic surfactant solutions. The spontaneous self-assembly was initiated by rapid mixing of the surfactant solutions using a stopped-flow device and the transient intermediate structures were probed by time-resolved small-angle X-ray scattering. The initial surfactant solutions comprised of anionic lithium perfluorooctanoate and zwitterionic tetradecyldimethylamine oxide, where the mixtures form unilamellar vesicles over a wide range of concentrations and mixing ratios. We found that disk-like transient intermediate structures are formed at higher concentrations while more elongated forms such as cylinder-like and torus-like micelles are involved at lower concentrations. These differences are attributed to monomer addition mechanism dominating the self-assembly process when the initial concentration is well below the critical micellar concentration of the anionic surfactant, while at higher concentrations the process is governed by fusion of disk-like mixed micelles. This means that the pathway of vesicle formation is determined by the proximity to the critical micellar concentration of the more soluble component

Interaction strength between proteins and polyelectrolyte brushes : a small angle X-ray scattering study

We present an investigation of amp; 946; lactoglobulin adsorption onto spherical polyelectrolyte brushes SPBs by small angle X ray scattering SAXS . The SPB consists of a polystyrene core onto which long chains of poly styrene sulfonate are grafted. The amount and the distribution of proteins adsorbed in the brush layer at low ionic strength can be derived from SAXS. The analysis of the SAXS data reveals additionally that some of the protein molecules form aggregates of about six monomers in the adsorbed state. Furthermore, the position and the amount of slightly bound protein can be detected by the combination of the SAXS results and the SPB loading after extensive ultrafiltration. The total amount of adsorbed protein is compared to data derived from isothermal titration calorimetry. The comparison of both sets of data demonstrates that the protein molecules in the inner layers of the spherical polyelectrolyte brush are firmly bound. Proteins located in the outer layers are only weakly bound and can be washed out by prolonged ultrafiltratio

pHLIP Peptide Interaction with a Membrane Monitored by SAXS

The pH (Low) Insertion Peptides (pHLIP® peptides) find application in studies of membrane-associated folding, since spontaneous insertion of these peptides is conveniently triggered by varying pH. Here we employed small angle X-ray scattering (SAXS) to investigate WT pHLIP® peptide oligomeric state in solution at high concentrations and monitor changes in liposome structure upon peptide insertion into the bilayer. We established that even at high concentrations (up to 300 μM) WT pHLIP® peptide at pH 8.0 does not form oligomers higher than tetramers (which exhibit concentration-dependent transfer to monomeric state as it was shown previously). This finding has significance for medical applications, when high concentration of the peptide is injected into blood and diluted in blood circulation. The interaction of WT pHLIP® peptide with liposomes does not alter the unilamellar vesicle structure upon peptide adsorption by lipid bilayer at high pH or upon insertion across the bilayer at low pH. At the same time, SAXS data clearly reflect the insertion of the peptide into the membrane at low pH, which opens the possibility to investigate kinetic process of a polypeptide insertion and exit from the membrane in real time by time-resolved SAXS

The Complex Systems and Biomedical Sciences group at the ESRF: current status and new opportunities after Extremely Brilliant Source upgrade

The Complex System and Biomedical Sciences (CBS) group at the European Synchrotron Radiation Facility (ESRF) in Grenoble is dedicated to the study of a broad family of materials and systems, including soft and hard condensed matter, nanomaterials, and biological materials. The main experimental methods used for this purpose are X-ray diffraction, reflectivity, scattering, photon correlation spectroscopy, and time-resolved X-ray scattering/diffraction. After a recent and successful Extremely Brilliant Source (EBS) upgrade, the Grenoble synchrotron has become the first of the 4th generation high energy facilities, which offers unprecedented beam parameters for its user community, bringing new experimental opportunities for the exploration of the nanoscale structure, kinetics, and dynamics of a myriad of systems. In this contribution, we present the impact of the recent upgrade on the selected beamlines in the CBS group and a summary of recent scientific activities after the facility reopening

Time-Resolved Small-Angle X-Ray Scattering Study of the Early Formation of Amyloid Protofibrils on a Apomyoglobin Mutant

The description of the fibrillogenesis pathway and the identification of “on-pathwayâ or â off-pathwayâ intermediates are key issues in amyloid research as they are concerned with the mechanism for onset of certain diseases and with therapeutic treatments. Recent results on the fibril formation process revealed an unexpected complexity both in the number and in the types of species involved, but the early aggregation events are still largely unknown, mainly because of their experimental inaccessibility. To provide information on the early stage events of self-assembly of an amyloidogenic protein, during the so-called lag phase, stopped-flow time-resolved small angle x-ray scattering (SAXS) experiments were performed. Using a global fitting analysis, the structural and aggregation properties of the apomyoglobin W7FW14F mutant, which is monomeric and partly folded at acidic pH but forms amyloid fibrils after neutralization, were derived from the first few milliseconds onward. SAXS data indicated that the first aggregates appear in less than 20 ms after the pH jump to neutrality and further revealed the simultaneous presence of diverse species. In particular, worm-like unstructured monomers, very large assemblies, and elongated particles were detected, and their structural features and relative concentrations were derived as a function of time on the basis of our model. The final results show that, during the lag phase, early assembling occurs due to the presence of transient monomeric species very prone to association and through successive competing aggregation and rearrangement processes leading to coexisting on-pathway and off-pathway transient species

The “Roll and Lock” Mechanism of Force Generation in Muscle

SummaryMuscle force results from the interaction of the globular heads of myosin-II with actin filaments. We studied the structure-function relationship in the myosin motor in contracting muscle fibers by using temperature jumps or length steps combined with time-resolved, low-angle X-ray diffraction. Both perturbations induced simultaneous changes in the active muscle force and in the extent of labeling of the actin helix by stereo-specifically bound myosin heads at a constant total number of attached heads. The generally accepted hypothesis assumes that muscle force is generated solely by tilting of the lever arm, or the light chain domain of the myosin head, about its catalytic domain firmly bound to actin. Data obtained suggest an additional force-generating step: the “roll and lock” transition of catalytic domains of non-stereo-specifically attached heads to a stereo-specifically bound state. A model based on this scheme is described to quantitatively explain the data