Collisional quenching of fluorescence is known to result in transient terms in the intensity decays. The origin of these effects is the rapid decay of closely spaced fluorophore-quenchers pairs, followed by slower diffusion-limited quenching of the remaining fluorophores. Historically, it has been difficult to detect these effects, because of their rapid timescale in non- viscous solution and the needed for high signal-to-noise ratio for a reliable analysis. We recently found that the effects can be readily detected by freqency-domain fluorometry. An important feature of the analysis is the ability to independently recover diffusion coefficients, interaction radii and specific rate constants for quenching. The diffusion coefficients are of particular interest for fluorophores in proteins and membranes. The interaction radii and specific rate constants are of interest regarding the mechanisms of quenching, electrstatic effects and symmetry and/or orientation effects in quenching. The objective of this proposal is to use state-of-the-art time and frequency- domain measurements to recover the complex decay laws which result from collisional quenching. To better understand the transient effects we will examine short and longer-lived probes in viscous and non-viscous solvents, quenched by oxygen, acrylamide, and halogenated quenchers. The effects of electrostatic interactions and modest spectral overlap will also be studied. A clear emphasis of the project will be quenching of intrinsic protein fluorescence by oxygen, acrylamide, iodide, succinimide and picolinium. Single tryptophan proteins will be chosen with variable extents of exposure to the aqueous phase. Examples include ribonuclease T1, S. Nuclease, monellin, melittin and ACTH, in order of increased exposure. To facilitate the development of theory we will examine labeled proteins, such as ANS, TNS or DENS-apomyoglobin, as the data may clarify the role of quencher partitioning into the proteins. The effects of participating will also be examined using probes like 6-In-11 bound to micelles. The role of steric effects will be examined using probes bound to cyclodextrins. Measurements will also be performed for two-dimensional diffusion of quenchers and labeled lipids in bilayers. The membrane systems will include labeled membranes in which the fluorophore is quenched by small polar and non-polar molecules (iodide, hexachlorocyclohexane) and by larger quenchers such as dibromo fatty acids or ubiquinone.
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