Wim P. de Boeij

Reconstitution of Membrane Proteins into Giant Unilamellar Vesicles via Peptide-Induced Fusion

In this work, we present a protocol to reconstitute membrane proteins into giant unilamellar vesicles (GUV) via peptide-induced fusion. In principle, GUV provide a well-defined lipid matrix, resembling a close-to-native state for biophysical studies, including optical microspectroscopy, of transmembrane proteins at the molecular level. Furthermore, reconstitution in this manner would also eliminate potential artifacts arising from secondary interactions of proteins, when reconstituted in planar membranes supported on solid surfaces. However, assembly procedures of GUV preclude direct reconstitution. Here, for the first time, a method is described that allows the controlled incorporation of membrane proteins into GUV. We demonstrate that large unilamellar vesicles (LUV, diameter 0.1 microm), to which the small fusogenic peptide WAE has been covalently attached, readily fuse with GUV, as revealed by monitoring lipid and contents mixing by fluorescence microscopy. To monitor contents mixing, a new fluorescence-based enzymatic assay was devised. Fusion does not introduce changes in the membrane morphology, as shown by fluorescence correlation spectroscopy. Analysis of fluorescence confocal imaging intensity revealed that approximately 6 to 10 LUV fused per microm(2) of GUV surface. As a model protein, bacteriorhodopsin (BR) was reconstituted into GUV, using LUV into which BR was incorporated via detergent dialysis. BR did not affect GUV-LUV fusion and the protein was stably inserted into the GUV and functionally active. Fluorescence correlation spectroscopy experiments show that BR inserted into GUV undergoes unrestricted Brownian motion with a diffusion coefficient of 1.2 microm(2)/s. The current procedure offers new opportunities to address issues related to membrane-protein structure and dynamics in a close-to-native state.

On the relation between the echo-peak shift and Brownian-oscillator correlation function

We show that for systems that exhibit bimodal dynamics in their system-bath correlation function the shift of the stimulated photon-echo maximum as a function of waiting time reflects fairly well the long time part of the correlation function. For early times this correspondence breaks down due to a fundamentally different behaviour of the echo-peak shift in this time domain and because of the effect of finite pulse duration on the echo-peak shift. The method is used to characterize the solvation dynamics in various dye solutions.

Reduced Protein Diffusion Rate by Cytoskeleton in Vegetative and Polarized Dictyostelium Cells

Fluorescence recovery after photobleaching measurements with high spatial resolution are performed to elucidate the impact of the actin cytoskeleton on translational mobility of green fluorescent protein (GFP) in aqueous domains of Dictyostelium discoideum amoebae. In vegetative Dictyostelium cells, GFP molecules experience a 3.6-fold reduction of their translational mobility relative to dilute aqueous solutions. In disrupting the actin filamentous network using latrunculin-A, the intact actin cytoskeletal network is shown to contribute an effective viscosity of 1.36 cP, which accounts for 53% of the restrained molecular diffusion of GFP. The remaining 47% of hindered protein motions is ascribed to other mechanical barriers and the viscosity of the cell liquid. A direct correlation between the density of the actin network and its limiting action on protein diffusion is furthermore established from measurements under different osmotic conditions. In highly locomotive polarized cells, the obstructing effect of the actin filamentous network is seen to decline to 0.46 cP in the non-cortical regions of the cell. Our results indicate that the meshwork of actin filaments constitutes the primary mechanical barrier for protein diffusion and that any noticeable reorganization of the network is accompanied by altered intracellular protein mobility.

Ultrafast optical dynamics of HITCI in ethylene glycol. A non-Markovian Brownian oscillator description

Abstract Femtosecond photon echo, chirped four-wave mixing and pump-probe experiments are reported, using a 13 fs cavity-dumped Ti:sapphire laser for excitation. It is shown that the optical dynamics of HITCI in ethylene glycol occurs on distinctly different time scales. The ultrafast solvent response is modelled by a non-Markovian solvent oscillator, while the slow diffusional solvent motions are represented by an overdamped Markovian oscillator. With this model an oscillator-bath time correlation function is obtained that exhibits a bimodal behaviour: an initial fast Gaussian decay is followed by a slower, long exponential tail.

Intracellular water diffusion probed by femtosecond nonlinear CARS microscopy

We report on a nonlinear coherent anti-Stokes Raman microscope system based on a high repetition rate femtosecond cavity-dumped visible optical parametric oscillator. This microscope enables real-time mapping of water concentration gradients in single living cells at high spatial resolution.