Quantitating the Dynamics of NBD Hexanoic Acid in Homogeneous Solution and in Solutions Containing Unilamellar Vesicles

Abstract

We report here on the motional and fluorescence lifetime dynamics of the chromophore NBDHA (6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoic acid) in neat solvents and in aqueous solutions containing unilamellar vesicles of varying composition. We measure the transient response of this chromophore by time-correlated single-photon counting, using one- and two-photon excitation to resolve the Cartesian components of the rotational diffusion constant, D. Our experimental data for NBDHA in selected solvents of varying viscosity demonstrate that one- and two-photon excitation probe different components of the rotational diffusion constant and that this chromophore reorients as a prolate rotor with an aspect ratio of approximately 2. For NBDHA in aqueous solutions containing unilamellar vesicles of varying composition, we recover the same reorientation behavior regardless of vesicle composition. Fluorescence lifetime and steady-state fluorescence data show the chromophore to reside in a polar environment that is different from neat water. We understand these data in the context of the chromophore residing in close proximity to the unilamellar vesicle polar headgroups in all cases.

Conformational Dynamics of the Plug Domain of the SecYEG Protein-conducting Channel

“…The fluorescence lifetime of NBD depends not only on the polarity of the environment but also on the hydrogen-bonding donor capacity of the solvent. Previous studies have shown that when a NBD fluorophore is exposed to different protic and aprotic solvents, the fluorescence lifetime ranges from ϳ1 ns for purely water to Ͼ10 ns in ethyl acetate (33,34). A fluorescent lifetime of around 7 ns is observed when NBD is dissolved in butanol, a value that corresponds to the fluorescence lifetimes observed for positions within or close to the hydrophobic constriction ring, which consists mainly of isoleucines.…”

Increase of Fluorescence Anisotropy Upon Self‐Assembly in Headgroup‐Labeled Surfactants

“…[7] This is the case for NBD, which is treated as a prolate rotor with a mono-exponential anisotropy decay. [10] For tryptophan, however, the highest observed anisotropy is 0.3. [7] …”

“…Fluorescence anisotropy ( r ) can be affected by a number of parameters as shown in Equation (2) where τ is the fluorescence lifetime, η is solvent viscosity, ${\cal R}$ is the universal gas constant, T is temperature, V is the effective hydrodynamic volume of the fluorophore (related to its rotational and translational diffusion coefficients) and r 0 is the maximum possible anisotropy for the molecule (usually close to 0.4 for randomly distributed geometries) 7. This is the case for NBD, which is treated as a prolate rotor with a mono‐exponential anisotropy decay 10. For tryptophan, however, the highest observed anisotropy is 0.3 7…”

Solvent-Dependent Changes in Molecular Reorientation Dynamics: The Role of Solvent−Solvent Interactions

“…It is the functional form and the time constant(s) associated with the decay of R ( t ) that are related to the local environment of the reorienting molecule. Chuang and Eisenthal developed a model for fluorescence anisotropy decay that provides a means of interpreting anisotropy decay functions containing up to five exponential components. ,,− Despite this potential for complexity, one or two decays are the functional forms observed most commonly. For the excitation and emission transition moments oriented parallel to one another, along the long axis in the chromophore π-system plane, reorientation of the chromophore as a prolate rotor ( D x > D y = D z ) gives rise to a single exponential decay of R ( t ) (eq ), and reorientation as an oblate rotor ( D z > D x = D y ) produces a two-component R ( t ) function, as described by eq .…”