A fluorescence depolarization study of the orientational distribution of crossbridges in muscle fibres

Abstract

A fluorescence depolarization study of the orientational distribution of crossbridges in dye-labelled muscle fibres is presented. The characterization of this distribution is important since the rotation of crossbridges is a key element in the theory of muscle contraction. In this study we exploited the advantages of angle-resolved experiments to characterize the principal features of the orientational distribution of the crossbridges in the muscle fibre. The directions of the transition dipole moments in the frame of the dye and the orientation and motion of the dye relative to the crossbridge determined previously were explicitly incorporated into the analysis of the experimental data. This afforded the unequivocal determination of all the second and fourth rank order parameters. Moreover, this additional information provided discrimination between different models for the orientational behaviour of the crossbridges. Our results indicate that no change of orientation takes place upon a transition from rigor to relaxation. The experiments, however, do no rule out a conformational change of the myosin S1 during the transition.

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

“…Another possibility for combining probe data is to describe the protein orientational distribution by directly determining its order parameters (Ajtai et al, 1992;Van der Heide et al, 1994). To unambiguously define the orientation at second-rank order would require five probe axes, and to define it to fourth-rank order would require a total of nine axes (Zannoni et al, 1983).…”

“…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, 1983;Kooyman et al, 1983). The orientation distribution of the probe with respect to the sample director f can be expressed in terms of the order parameters ͗P 2 (cos ␤ fd )͘ and ͗P 4 (cos ␤ fd )͘, which represent the second-and fourth-rank Legendre polynomials averaged over the distribution of all dipole orientations ␤ fd (Van Gurp et al, 1988;Van der Heide et al, 1994;Dale et al, 1999;cf., Eq. 1 in the text here).…”

The orientation of eosin-5-maleimide on human erythrocyte band 3 measured by fluorescence polarization microscopy

“…Whereas microscopic polar-ized fluorescence images of single cells have been used previously to determine chromophore orientation (Axelrod, 1979;Florine-Casteel, 1990), the models used in those studies make specific assumptions about the form of the nanosecond time scale motion that do not apply to eosinlabeled band 3. Other previous work provides examples of determination of transition dipole orientations by angledependent fluorescence measurements (e.g., Van der Meer et al, 1982;Van der Heide et al, 1994;Burghardt and Ajtai, 1994). However, the experimental geometries used to derive the previous models are not compatible with our instrument.…”

“…The chromophore orientation is represented by two order parameters ((P2(cOs 6a)) and (P2(cos Oe))) and three correlation functions, CO, C1, and C2, which correspond in the URD model to the residual anisotropy and the amplitudes of the e-Dt and e4Dt exponential decay terms, respectively. There is a large body of theoretical modeling and experimental data describing fluorescence in oriented systems, using different experimental geometries (e.g., Van der Meer et al, 1982;Zannoni et al, 1983;Van der Heide et al, 1994;Burghardt and Ajtai, 1994). Whereas some similar approaches have expanded C0, Cl, and C2 in terms of orientational order parameters (e.g., Van der Meer et al, 1982), we are primarily interested in these correlation functions because of their appearance in the URD model.…”

Probing the Orientational Distribution of Dyes in Membranes through Multiphoton Microscopy

“…In most cases, the long axis of the dye molecule can be assumed to be coincident with its transition dipole moment (TDM). Previously, studies that have quantified tilt angle have employed solid-state NMR (6)(7)(8), electron paramagnetic resonance (9,10), polarization coherent anti-Stokes Raman scattering (11), infrared dichroism (12)(13)(14)(15), polarization anisotropy fluorescence (16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26), and secondharmonic generation (SHG) microscopy (11,(27)(28)(29)(30)(31)(32)(33)(34). Experimental data from these investigations have been complemented by computational methods for modeling molecules in membranes (8,(35)(36)(37)(38).…”