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A systematic study of the translational diffusion of the phospholipid derivative N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine (NBD-PE) has been undertaken in liquid crystalline phase phosphatidylcholine bilayers by using the fluorescence recovery after photobleaching technique. This work was done with the intention of comparing the experimental results with the predictions of theoretical models for diffusion in membranes. The following is shown. For NBD-PE, the dependence of the translational diffusion coefficient (Dt) upon the acyl chain length of the diffusant is not that predicted by continuum fluid hydrodynamic models for diffusion in membranes [Saffman, P.G., & Delbrueck, M. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 3111-3113; Hughes, B. D., Pailthorpe, B. A., & White, L. R. (1981) J. Fluid Mech. 110, 349-372]. Plots of Dt vs. 1/T (Arrhenius plots) are nonlinear in dilauroyl-phosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) bilayers where the acyl chain composition of the NBD-PE is matched with that of the host bilayer lipid. This suggests that a "free volume" model may be appropriate for the description of lipid diffusion in lipid bilayers. In bilayers of phosphatidylcholines with saturated acyl chains at the same "reduced temperature", the magnitude of Dt follows the order distearoylphosphatidylcholine greater than DPPC greater than DMPC greater than DLPC. This is the inverse of what may be expected from the hydrodynamic model but is in agreement with the free volume in these bilayers.(ABSTRACT TRUNCATED AT 250 WORDS)

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We present applications of polar plots for analyzing fluorescence lifetime data acquired in the frequency domain. This graphical, analytical method is especially useful for rapid FLIM measurements. The usual method for sorting out and determining the underlying lifetime components from a complex fluorescence signal is to carry out the measurement at multiple frequencies. When it is not possible to measure at more than one frequency, such as rapid lifetime imaging, specific features of the polar plot analysis yield valuable information, and provide a diagnostic visualization of the participating fluorescent species underlying a complex lifetime distributions. Data are presented where this polar plot presentation is useful to derive valuable, unique information about the underlying component distributions. We also discuss artifacts of photolysis and how this method can also be applied to samples where each fluorescence species shows a continuous distribution of lifetimes. Polar plots of frequency-domain data are commonly used for analysis of dielectric relaxation experiments (Cole-Cole plots), which have proved to be exceptionally useful in that field for decades. We compare this analytical tool that is well developed and extensively used in dielectric relaxation and chemical kinetics to fluorescence measurements.

The efficiency of fluorescence resonance energy transfer (FRET) between fluorescein and rhodamine covalently attached to both 5' termini of a series of doublestranded DNA species (ranging from 8 to 20 bp) was measured. FRET efficiency varied with a dependence compatible with dye-to-dye distances (R) calculated on the basis of doublestranded B-DNA structure; the helical geometry of doublestranded DNA in solution is clearly evident. The experimental data were consistent with a 1/[1 + (RIRo)6] dependence of FRET efficiency characteristic for the Forster dipole-dipole mechanism. The thermal dissociation of the strands of the duplex DNA species can be followed by using FRET, and from these data we have been able to obtain enthalpies of duplex formation in good agreement with earlier measurements using alternative techniques. FRET measurements at very different salt concentrations can be accurately compared. We conclude that FRET is a reliable and valuable method for studying structure and conformational transitions in nucleic acids.Nucleic acids may adopt specific and sometimes complex folded structures that are critical for their biological function. Full determination of these structures requires the measurement of distances up to 80 A or more, but there are few techniques that allow such distances to be determined in solution. This is particularly important for nucleic acids in view of the extended helical structures involved, which can lead to underdetermination of structures by methods that can only yield short distances. Fluorescence resonance energy transfer (FRET) is sensitive to distances in the longer size range and has recently proved to be very useful in the study of nucleic acid structures, such as the solution structure of the four-way DNA junction (1, 2). FRET-derived distance information in DNA and RNA could be complementary to the shorter distances determined by NMR. However, the application of FRET to fluorescence probes covalently linked to nucleic acid structures has been relatively infrequent (1,(3)(4)(5)(6)(7)(8)(9)(10)(11).The rate of nonradiative energy transfer from an excited donor molecule (D) to a nearby acceptor molecule (A) depends in a characteristic manner on the distance between the two chromophores and their relative angular disposition (12)(13)(14). Depending on the D-A molecular pairs, the efficiency of transfer responds sensitively to relatively small changes of distances in the range of 10 to 80 A. Careful evaluations of FRET experiments can yield quantitative estimates of distances between labeled positions in macromolecules, or in molecular aggregates, provided that certain spectroscopic parameters are known. Even if sufficient information is not available to calculate exact distances, relative dimensions of molecular structures can often be deduced (1, 2, 11), and in principle FRET is applicable to very complex molecular structures. The method has been applied successfully to estimate intra(inter)molecular distances between donor and acceptor molecules in biological mo...

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We have carried out fluorescence resonance energy transfer (FRET) measurements on four-way DNA junctions in order to analyze the global structure and its dependence on the concentration of several types of ions. A knowledge of the structure and its sensitivity to the solution environment is important for a full understanding of recombination events in DNA. The stereochemical arrangement of the four DNA helices that make up the four-way junction was established by a global comparison of the efficiency of FRET between donor and acceptor molecules attached pairwise in all possible permutations to the 5' termini of the duplex arms of the four-way structure. The conclusions are based upon a comparison between a series of many identical DNA molecules which have been labeled on different positions, rather than a determination of a few absolute distances. Details of the FRET analysis are presented; features of the analysis with particular relevance to DNA structures are emphasized. Three methods were employed to determine the efficiency of FRET: (1) enhancement of the acceptor fluorescence, (2) decrease of the donor quantum yield, and (3) shortening of the donor fluorescence lifetime. The FRET results indicate that the arms of the four-way junction are arranged in an antiparallel stacked X-structure when salt is added to the solution. The ion-related conformational change upon addition of salt to a solution originally at low ionic strength progresses in a continuous noncooperative manner as the ionic strength of the solution increases. The mode of ion interaction at the strand exchange site of the junction is discussed.