The green fluorescent protein (GFP) of the bioluminescent jellyfish Aequorea and its mutants have gained widespread usage as an indicator of structure and function within cells. Proton transfer has been implicated in the complex photophysics of the wild-type molecule, exhibiting a protonated A species excited at 400 nm, and two deprotonated excited-state species I* and B* with red-shifted excitation ∼475 nm. Photochromicity between the protonated and deprotonated species has been reported upon 400 nm excitation. Using precise time-resolved spectroscopy, we have been able to distinguish the fluorescence lifetimes of the I and B species (∼3.3 and ∼2.8 ns, respectively) and show that the irreversible photochromicity which we observe is due to formation in the excited state of the B species, which cannot return to other species in the ground state. The ground state A and I species are in thermal equilibrium. Anisotropy measurements indicate that the chromophore lies rigidly in the molecule with a rotational correlation time of ∼15.5 ns, as is to be expected for a molecule of this size. Time-resolved measurements of enhanced yellow fluorescent protein (EYFP) and red-shifted green fluorescent protein (RSGFP) were also analyzed.

Using a confocal epi-illuminated microscope with a polarizing beam splitter and dual-channel detection of single-molecule fluorescence induced by pulsed laser excitation, a new application of the three-dimensional, real-time spectroscopic technique BIFL (burst integrated fluorescence lifetime) is introduced. BIFL allows simultaneous registration of fluorescence intensity, lifetime, and anisotropy. It is shown to be well-suited to identify the freely diffusing fluorescent molecule Rhodamine 123 and the Enhanced Yellow Fluorescent Protein via their characteristic fluorescence anisotropy using a time-resolved analysis. Furthermore, data analysis is discussed and rotational correlation times of single molecules are determined. Applications for multidimensional single-molecule identification are outlined.

A statistical analysis of a set of genomic human Alu elements is based on a published alignment and a recent classification of these sequences. After separation of the Alu sequences into families, the consensus sequences of these families are determined, using the correct weighting of the unidirectional decay of CG-dinucleotides. For, the tenfold greater mutation rate at CG's requires separate consideration of an independent clock at every stage of analysis. The distributions of the substitutions with respect to the new consensus sequences, taking the CG and the non-CG-nucleotide positions separately, lie far closer to the expected distributions than the total diversity. Computer analysis of the folding of RNAs derived from these sequences indicates that RNA secondary structure is conserved among Alu families, suggesting its importance for Alu proliferation and/or function. The folding pattern, further substantiated by a number of compensatory mutations, includes secondary structure domains which are homologous to those observed in 7SL RNA and a defined region of interaction between the two Alu subunits. These results are consistent with a model in which a small number of conserved Alu master genes give rise via retroposition to the numerous copies of Alu pseudogenes, that then diversify by random substitution. The master genes appeared at different periods during evolution giving rise to different families of Alu sequences.

Monomer and excimer fluorescence decays of 1,3-di( 1 -pyrenyl)propane (!Py(3)lPy) in «-heptane are measured at different temperatures by using time-correlated single-photon counting (SPC). The decays at 61 °C are triple-exponential with decay times and amplitudes independent of the number of total counts, excitation wavelength, and width of the excitation pulse. At -43 °C and lower temperatures, single-exponential monomer decays are obtained, next to triple-exponential excimer records. Aggregates of lPy(3)lPy are not detected. These results can be described by the three-state model, DMD, comprising one group of rapidly interconverting excited-state monomers (M*) and two excimers (D*). No evidence for distributed decay times is found down to a time resolution of 0.1 ns/channel and 2000 channels. In support of the DMD model, molecular mechanics calculations backed by *H NMR measurements are presented, showing that only two monomer conformen of lPy(3)lPy are populated in the ground state. The interconversion between these two monomers in the excited state is too fast to be resolved by the present SPC measurements.