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We have performed time-resolved fluorescence measurements on photosystem II (PSII) containing membranes (BBY particles) from spinach with open reaction centers. The decay kinetics can be fitted with two main decay components with an average decay time of 150 ps. Comparison with recent kinetic exciton annihilation data on the major light-harvesting complex of PSII (LHCII) suggests that excitation diffusion within the antenna contributes significantly to the overall charge separation time in PSII, which disagrees with previously proposed trap-limited models. To establish to which extent excitation diffusion contributes to the overall charge separation time, we propose a simple coarse-grained method, based on the supramolecular organization of PSII and LHCII in grana membranes, to model the energy migration and charge separation processes in PSII simultaneously in a transparent way. All simulations have in common that the charge separation is fast and nearly irreversible, corresponding to a significant drop in free energy upon primary charge separation, and that in PSII membranes energy migration imposes a larger kinetic barrier for the overall process than primary charge separation.

The photophysical properties of the lowest excited singlet states, S1(π,π*), of two porphyrin diacids have been investigated. The diacids are H4TPP2+ and H4OEP2+, the diprotonated forms of free base tetraphenylporphyrin (H2TPP) and octaethylporphyrin (H2OEP), respectively. Both diacids exhibit perturbed static and dynamic characteristics relative to the parent neutral complexes in solution at room temperature. These properties include enhanced yields of S1 → S0 radiationless deactivation (internal conversion), which increase from ∼0.1 for H2TPP and H2OEP to 0.4 for H4OEP2+ and 0.6 for H4TPP2+. The fluorescence lifetimes of both diacids are strongly temperature dependent, with an activation enthalpy of ∼1400 cm-1 for S1-state deactivation. The enhanced nonradiative decays and many other photophysical consequences of diacid formation are attributed primarily to nonplanar macrocycle distortions. Both H4TPP2+ and H4OEP2+ have been shown previously by X-ray crystallography to adopt saddle-shaped conformations, and the magnitudes of the perturbed properties for the two diacids in solution correlate with the extent of the deviations from planarity in the crystals. A model is proposed to explain the nonradiative decay behavior of the porphyrin diacids that is relevant to nonplanar porphyrins in general. The model includes the existence of decay funnels on the S1(π,π*)-state energy surface that are separated from the equilibrium conformation and other minima by activation barriers. It is suggested that these funnels involve configurations at which the potential-energy surfaces of the ground and excited states approach more closely than at the equilibrium excited-state structure(s) from which steady-state fluorescence occurs. Possible contributions to the relevant nuclear coordinates are discussed.

Structural information on intracellular fusions of the green fluorescent protein (GFP) of the jellyfish Aequorea victoria with endogenous proteins is required as they are increasingly used in cell biology and biochemistry. We have investigated the dynamic properties of GFP alone and fused to a single chain antibody raised against lipopolysaccharide of the outer cell wall of Gram-negative bacteria (abbreviated as scFv-GFP). The scFv moiety was functional as was proven in binding assays, which involved the use of both fluorescence correlation spectroscopy observing the binding of scFv-GFP to Gram-negative bacteria and a surface plasmon resonance cell containing adsorbed lipopolysaccharide antigen. The rotational motion of scFv-GFP has been investigated with time-resolved fluorescence anisotropy. However, the rotational correlation time of scFv-GFP is too short to account for globular rotation of the whole protein. This result can only be explained by assuming a fast hinge motion between the two fused proteins. A modeled structure of scFv-GFP supports this observation.The green fluorescent protein (GFP) 1 from the jellyfish Aequorea victoria has received widespread utilization as a natural fluorescent marker for gene expression, localization of gene products (1-5), and identification of protein interaction and function. GFP is a protein consisting of 238 amino acids with a molecular mass of 27 kDa and has the shape of a cylinder with a length of 4.2 nm and diameter of 2.4 nm. The chemical structure of the hexapeptide chromophore has been elucidated (6). The intrinsic fluorophore is a p-hydroxybenzylidene-imidazolidine derivative formed by a covalent modification of the sequence Ser 65 (or Thr 65 in enhanced GFP), Tyr 66 , and Gly 67 in the hexapeptide. A comprehensive review on GFP has been published (7). The crystal structure of GFP and enhanced GFP has been solved and showed the hexapeptide to be part of a central helix inside a 11-stranded ␤-barrel (8 -11).Genetic fusions of a variety of proteins with GFP have been used in numerous studies on gene or protein function. In a sense it is miraculous that in most fusion proteins GFP is functional. In many other fusion proteins, the protein used as a reporter does often not fold well, resulting in aggregates or inclusion bodies of the entire fusion protein. Sometimes the causes of aggregation can be attributed to certain (clusters of) amino acids such as hydrophobic clusters of amino acids that become solvent exposed (12). To obtain a better picture why these phenomena do not occur in GFP fusion proteins, we have investigated the behavior of the GFP moiety in fusion proteins and emphasized the motional properties. Thereto, we fused enhanced GFP with a single chain Fv fragment raised against the lipopolysaccharide (LPS) of a Gram-negative bacterium. Because the single chain antibody was linked to the N-terminal residue of GFP, the fusion protein is abbreviated as scFv-GFP. Here we report information relevant for the dynamics of GFP fusion proteins used to monitor pro...

The fluorescence lifetime strongly depends on the immediate environment of the fluorophore. Timeresolved fluorescence measurements of the enhanced forms of ECFP and EYFP in water-glycerol mixtures were performed to quantify the effects of the refractive index and viscosity on the fluorescence lifetimes of these proteins. The experimental data show for ECFP and EYFP two fluorescence lifetime components: one short lifetime of about 1 ns and a longer lifetime of about 3.7 ns of ECFP and for EYFP 3.4. The fluorescence of ECFP is very heterogeneous, which can be explained by the presence of two populations: a conformation (67% present) where the fluorophore is less quenched than in the other conformation (33% present). The fluorescence decay of EYFP is much more homogeneous and the amplitude of the short fluorescence lifetime is about 5%. The fluorescence anisotropy decays show that the rotational correlation time of both proteins scales with increasing viscosity of the solvent similarly as shown earlier for GFP. The rotational correlation times are identical for ECFP and EYFP, which can be expected since both proteins have the same shape and size. The only difference observed is the slightly lower initial anisotropy for ECFP as compared to the one of EYFP.

The fluorescence decay kinetics of the reduced nicotinamides NMNH, NADH and NADPH in aqueous solution were investigated using an Ar ion laser, mode locked in the UV, as source of excitation and single photon counting electronics in the detection system allowing for a time resolution in the picosecond range. Analysis of the experimental fluorescence decay showed that the dinucleotides did not follow a single exponential decay law. Good fitting was accomplished with a sum of two exponentials. The mononucleotide fluorescence decay was a single exponential for at least 95% of its amplitude.The heterogeneity in lifetimes of the fluorescence of the dinucleotides was interpreted in terms of an exciplex mechanism. INTRODUaIONThe fluorescence decay kinetics of the reduced nicotinamide nucleotides NMNH (reduced b-nicotinamide mononucleotide), NADH (reduced b-nicotinamide adenine dinucleotide) and NADPH (reduced p-nicotinamide adenine dinucleotide phosphate) have received

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