Excitation wavelength dependent fluorescence anisotropy of eosin-myosin adducts

Vandermeulen, David L.; Nealon, D.G.; Gratton, Enrico; Jameson, David M.

doi:10.1016/0301-4622(90)85021-w

Cited by 13 publications

(8 citation statements)

References 24 publications

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“…(1 -100 MHz, or higher) frequency range (VanderMeulen et al, 1990;Brunet et al, 1993). Third, global analysis enables data sets obtained under different conditions to be analyzed for common factors or parameters (Beechem et al, 1985;Beechem & Gratton, 1988;Beechem, 1992).…”

Section: Resultsmentioning

confidence: 99%

Molecular Dynamics of the Anti-Fluorescein 4-4-20 Antigen-Binding Fragment. 2. Time-Resolved Fluorescence Spectroscopy

Lim

Jameson²,

Gentry³

et al. 1995

Biochemistry

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Time-resolved fluorescence experiments were performed to investigate the dynamic aspects of the antigen-binding fragment (Fab) of a high-affinity monoclonal antibody (4-4-20) which binds the fluorescent hapten fluorescein. Both the unliganded Fab and a complex of the Fab with a nonfluorescent analog of fluorescein (fluoresceinamine, FLM) were examined. A fluorescence polarization probe [5-[[2-[(iodoacetyl)amino]ethyl]amino]naphthalene-1-sulfonic acid, AEDANS] was covalently attached to the C-terminus of the Fab. Experiments were performed at three different temperatures (10, 25, and 35 degrees C), and phase-modulation data sets were collected for five different molar ratios of FLM to Fab at each temperature. Global analyses were then used to extract values for fluorescence lifetime and rotational correlation time from these data. In the lifetime analysis the best fit was obtained when the emission of AEDANS was described by a Lorentzian distribution of lifetimes (tau = 15.6 ns, distribution width = 3.4 ns, both at 25 degrees C), which suggested that the probe experienced a heterogeneous environment. Anisotropy analyses suggested that two different rotational components were present. The first was attributed to the global motion of the Fab and exhibited a rotational correlation time (theta 1) of ca. 33 ns at 25 degrees C. This component was relatively unaffected by antigen binding. The second rotational component was attributed to the local or segmental motion within the Fab and exhibited a rotational correlation time (theta 2) of 1.1 ns at 25 degrees C. This value increased by more than 50% upon antigen binding, a result which was consistent with molecular dynamics simulations of the same Fab--fluorescein system [Lim & Herron (1995) Biochemistry 34, 6962-6974]. Furthermore, statistical analysis showed that this increase was significant at the 95% confidence level.

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Section: Resultsmentioning

confidence: 99%

Molecular Dynamics of the Anti-Fluorescein 4-4-20 Antigen-Binding Fragment. 2. Time-Resolved Fluorescence Spectroscopy

Lim

Jameson²,

Gentry³

et al. 1995

Biochemistry

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“…In more viscous solvents, such as propylene glycol/water mixtures, the apparent Debye rotational relaxation times of eosin differ upon excitation in the regions of positive and negative anisotropy. 32 In aqueous glycerol solutions, the rotational correlation time is proportional to viscosity/temperature in the microsecond time range. 33 As shown in Supplementary Figure 6, we confirmed the fluorescence emission level of eosin changes with the pH and the solvent's viscosity.…”

Section: Analytical Chemistrymentioning

confidence: 99%

Enhanced Visualization of Hematoxylin and Eosin Stained Pathological Characteristics by Phasor Approach

Luo

Liu

et al. 2017

Anal. Chem.

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The phasor approach to fluorescence lifetime imaging microscopy (FLIM) is used to identify different types of tissues from hematoxylin and eosin (H&E) stained basal cell carcinoma (BCC) sections. The results suggest that working directly on the phasor space with the clustering assignment achieves immunofluorescence like simultaneous five or six-color imaging by using multiplexed fluorescence lifetimes of H&E. The phase approach is of particular effectiveness for enhanced visualization of the abnormal morphology of a suspected nidus. Moreover, the phasor approach to H&E FLIM data can determine the actual paths or the infiltrating trajectories of basophils and immune cells associated with the preneoplastic or neoplastic skin lesions. The integration of the phasor approach with routine histology proved its available value for skin cancer prevention and early detection. We therefore believe that the phasor analysis of H&E tissue sections is an enhanced visualization tool with the potential to simplify the preparation process of special staining and serve as color contrast aided imaging in clinical pathological examination.

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“…A fluorescent probe molecule, with colinear absorption and emission dipole moments, is attached at a known orientation relative to the protein. In xanthene derivative fluorophores, such as rhodamine and eosin, the absorption and emission dipoles lie nearly parallel (Chen and Bowman, 1965;VanderMeulen et al, 1990;Van der Heide et al, 1992) and the approximation of colinearity is justified. In a fluorescence polarization experiment, intensity ratios are obtained that provide information on the orientational order and rotational dynamics of the transition dipole moments in the system (Lakowicz, 1983;Zannoni et al, 1983;Kooyman et al, 1983).…”

Section: Fluorescence Polarizationmentioning

confidence: 99%

A Maximum Entropy Analysis of Protein Orientations Using Fluorescence Polarization Data from Multiple Probes

Heide

Hopkins

Goldman

2000

Biophysical Journal

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Techniques have recently become available to label protein subunits with fluorescent probes at predetermined orientation relative to the protein coordinates. The known local orientation enables quantitative interpretation of fluorescence polarization experiments in terms of orientation and motions of the protein within a larger macromolecular assembly. Combining data obtained from probes placed at several distinct orientations relative to the protein structure reveals functionally relevant information about the axial and azimuthal orientation of the labeled protein segment relative to its surroundings. Here we present an analytical method to determine the protein orientational distribution from such data. The method produces the broadest distribution compatible with the data by maximizing its informational entropy. The key advantages of this approach are that no a priori assumptions are required about the shape of the distribution and that a unique, exact fit to the data is obtained. The relative orientations of the probes used for the experiments have great influence on information content of the maximum entropy distribution. Therefore, the choice of probe orientations is crucial. In particular, the probes must access independent aspects of the protein orientation, and two-fold rotational symmetries must be avoided. For a set of probes, a "figure of merit" is proposed, based on the independence among the probe orientations. With simulated fluorescence polarization data, we tested the capacity of maximum entropy analysis to recover specific protein orientational distributions and found that it is capable of recovering orientational distributions with one and two peaks. The similarity between the maximum entropy distribution and the test distribution improves gradually as the number of independent probe orientations increases. As a practical example, ME distributions were determined with experimental data from muscle fibers labeled with bifunctional rhodamine at known orientations with respect to the myosin regulatory light chain (RLC). These distributions show a complex relationship between the axial orientation of the RLC relative to the fiber axis and the azimuthal orientation of the RLC about its own axis. Maximum entropy analysis reveals limitations in available experimental data and supports the design of further probe angles to resolve details of the orientational distribution.

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Excitation wavelength dependent fluorescence anisotropy of eosin-myosin adducts

Cited by 13 publications

References 24 publications

Molecular Dynamics of the Anti-Fluorescein 4-4-20 Antigen-Binding Fragment. 2. Time-Resolved Fluorescence Spectroscopy

Molecular Dynamics of the Anti-Fluorescein 4-4-20 Antigen-Binding Fragment. 2. Time-Resolved Fluorescence Spectroscopy

Enhanced Visualization of Hematoxylin and Eosin Stained Pathological Characteristics by Phasor Approach

A Maximum Entropy Analysis of Protein Orientations Using Fluorescence Polarization Data from Multiple Probes

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