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.

System-bath correlation function probed by conventional and time-gated stimulated photon echo

We show how in the framework of the multimode Brownian oscillator model the system-bath correlation function can be derived from conventional and time-gated stimulated photon echo experiments and consideration of the linear optical spectra. Experiments are performed on the infrared dye DTTCI in room temperature solutions of ethylene glycol, methanol, and acetonitrile. The obtained correlation function is the sum of several Brownian oscillators, of which four are attributed to intrachromophore vibrational dynamics and the other three to solute-solvent dynamics. The ultrafast part of the correlation function on the time scale of the excitation pulses is interpreted as a free induction decay-like effect due to impulsive excitation of spectrally broad underlying vibrational structure in the dye's electronic transition. The slower parts are assigned to multiple time scale solute-solvent dynamics. The effect of vibrational coherences on the echo measurements is also analyzed; this analysis permits the dissection of the correlation function into a part due to intrachromophore dynamics and a part due to solvation dynamics. The spectral densities associated with these latter oscillators are located in the far infrared, in the same spectral region as probed by the optical Kerr effect. The measurements, however, provide no definite answer to the question of whether these spectral densities are the same

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 µm), 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 ~6 to 10 LUV fused per µm2 of GUV surface. As a model protein, bacteriorhodopsin (BR) was reconstituted into GUV, using LUV into which BR was incorporated via detergent dyalisis. 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 µm2/s. The current procedure offers new opportunities to address issues related to membrane-protein structure and dynamics in a close-to-native state