The lateral diffusion coefficients of a BODIPY tail-labeled lipid in two model systems, namely, free-standing giant unilamellar vesicles (GUVs) and supported phospholipid bilayers (SPBs), were determined by fluorescence correlation spectroscopy (FCS) using the Z-scan approach. For the first time, the performed measurements on 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bilayers maintain exactly the same experimental conditions for both systems, which allows for a quantitative comparison of lipid diffusion in these two commonly used model membranes. The results obtained revealed that the lipid mobility in free-standing bilayers (D=7.8+/-0.8 microm2 s-1) is significantly higher than in the bilayer created on the solid support (mica) (D=3.1+/-0.3 microm2 s-1).

The ReI(CO)3(4,7-dimethyl-1,10-phenanthroline)(histidine-124)(tryptophan-122) complex, denoted ReI(dmp)(W122), of Pseudomonas aeruginosa azurin behaves as a single photoactive unit that triggers very fast electron transfer (ET) from a distant (2 nm) CuI center in the protein. Analysis of time-resolved (ps-μs) IR spectroscopic and kinetics data collected on ReI(dmp)(W122)AzM (M = ZnII CuII, CuI; Az = azurin) and position-122 tyrosine (Y), phenylalanine (F), and lysine (K) mutants together with excited-state DFT/TDDFT calculations and X-ray structural characterization reveal the character, energetics, and dynamics of the relevant electronic states of the ReI(dmp)(W122) unit and a cascade of photoinduced ET and relaxation steps in the corresponding Re-azurins. Optical population of ReI(imidazole-H124)(CO)3→dmp 1CT states is followed by ~110 fs intersystem crossing and ~600 ps structural relaxation to a 3CT state whose IR spectrum indicates a mixed ReI(CO)3,A→dmp/π→π*(dmp) character for aromatic amino acids A122 (A = W, Y, F) and ReI(CO)3→dmp MLCT for ReI(dmp)(K122)AzCuII. In a few ns, the 3CT state of ReI(dmp)(W122)AzM establishes an equilibrium with the ReI(dmp•−)(W122•+)AzM charge-separated state, 3CS, whereas the 3CT state of the other Y, F, and K122 proteins decays to the ground state. In addition to this main pathway, 3CS is populated by fs and ps W(indole)→ReII ET from 1CT and the initially “hot” 3CT states, respectively. The 3CS state undergoes a tens-of-ns dmp•−→W122•+ ET recombination leading to the ground state or, in the case of the CuI azurin, competitively fast (~30 ns over 1.12 nm) CuI→W•+ ET producing ReI(dmp•−)(W122)AzCuII. The overall photoinduced CuI→Re(dmp) ET through ReI(dmp)(W122)AzCuI occurs over a 2 nm distance in <50 ns after excitation, the intervening fast 3CT-3CS equilibrium being the principal accelerating factor. No reaction was observed for the three Y, F, and K122 analogues. Although the presence of Re(dmp)(W122)AzCuII oligomers in solution was documented by mass spectrometry and phosphorescence anisotropy, kinetics data do not indicate any significant interference from intermolecular ET steps. The ground-state dmp-indole ππ interaction together with well-matched W/W•+ and excited-state ReII(CO)3(dmp•−)/ReI(CO)3(dmp•−) potentials, that result in very rapid electron interchange and 3CT - 3CS energetic proximity, are the main factors responsible for the unique ET behavior of ReI(dmp)(W122)-containing azurins.

No abstract

Although there exist a number of methods, such as NMR, X-ray, e.g., which explore the hydration of phospholipid bilayers, the solvent relaxation (SR) method has the advantage of simple instrumentation, easy data treatment and possibility of measuring fully hydrated samples. The main information gained from SR by the analysis of recorded "time-resolved emission spectra" (TRES) is micro-viscosity and micro-polarity of the dye microenvironment. Based on these parameters, one can draw conclusions about water structure in the bilayer. In this review, we focus on physical background of this method, on all the procedures that are needed in order to obtain relevant parameters, and on the requirements on the fluorescence dyes. Furthermore, a few recent applications (the effect of curvature, binding of antibacterial peptides and phase transition) illustrating the versatility of this method are mentioned. Moreover, limitations and potential problems are discussed.