Effect of orientational order on the decay of the fluorescence anisotropy in membrane suspensions. A new approximate solution of the rotational diffusion equation

We discussed the time-dependence of fluorescent emission anisotropy of a cylindrical probe in membrane vesicles. We showed that, if the motion of the probe were described as diffusion in an anisotropic environment, it would be possible to determine not only the second-rank but also the fourth-rank orientational order parameter from the decay of the fluorescence anisotropy. The approximations involved were based on an interpolation of short-time and long-time behavior of the relevant correlation functions. A general expression was derived for the time dependence of the fluorescence anisotropy in closed form, which applies to any particular distribution model. It was shown to be in good agreement with previously reported results for the cone model and the Gaussian model. Finally, the applicability of the theory to time-resolved and differential phase fluorescence depolarization experiments was discussed.     

Microsecond rotational motions of eosin-labeled myosin measured by time-resolved anisotropy of absorption and phosphorescence

We have studied submicrosecond and microsecond rotational motions within the contractile protein myosin by observing the time-resolved anisotropy of both absorption and emission from the long-lived triplet state of eosin-5-iodoacetamide covalently bound to a specific site on the myosin head. These results, reporting anisotropy data up to 50 microseconds after excitation, extend by two orders of magnitude the time range of data on time-resolved site-specific probe motion in myosin. Optical and enzymatic analyses of the labeled myosin and its chymotryptic digests show that more than 95% of the probe is specifically attached to sulfhydryl-1 (SH1) on the myosin head. In a solution of labeled subfragment-1 (S-1) at 4 degrees C, absorption anisotropy at 0.1 microseconds after a laser pulse is about 0.27. This anisotropy decays exponentially with a rotational correlation time of 210 ns, in good agreement with the theoretical prediction for end-over-end tumbling of S-1, and with times determined previously by fluorescence and electron paramagnetic resonance. In aqueous glycerol solutions, this correlation time is proportional to viscosity/temperature in the microsecond time range. Furthermore, binding to actin greatly restricts probe motion. Thus the bound eosin is a reliable probe of myosin-head rotational motion in the submicrosecond and microsecond time ranges. Our submicrosecond data for myosin monomers (correlation time 400 ns) also agree with previous results using other techniques, but we also detect a previously unresolvable slower decay component (correlation time 2.6 microseconds), indicating that the faster motions are restricted in amplitude. This restriction is not consistent with the commonly accepted free-swivel model of S-1 attachment in myosin. In synthetic thick filaments of myosin, both fast (700 ns) and slow (5 microseconds) components of anisotropy decay are observed. In contrast to the data for monomers, the anisotropy of filaments has a substantial residual component (26% of the initial anisotropy) that does not decay to zero even at times as long as 50 microseconds, implying significant restriction in overall rotational amplitude. This result is consistent with motion restricted to a cone half-angle of about 50 degrees. The combined results are consistent with a model in which myosin has two principal sites of segmental flexibility, one giving rise to submicrosecond motions (possibly corresponding to the junction between S-1 and S-2) and the other giving rise to microsecond motions (possibly corresponding to the junction between S-2 and light meromyosin).(ABSTRACT TRUNCATED AT 400 WORDS)     

Rotational motions of myosin heads in myofibril studied by phosphorescence anisotropy decay measurements

We studied the rotational Brownian motions of myosin heads, of which the sulfhydryl group was selectively labeled with the triplet probe 5-eosinylmaleimide, in myofibril by using flash-induced phosphorescence anisotropy decay measurements. The anisotropy decay curve under relaxing conditions consisted of a fast (submicrosecond) and a slow (a few microseconds) component and a small constant part as in the synthetic myosin filaments in solution. The decay curves could be analyzed by assuming that a head part, i.e. subfragment 1 (S1), wobbles in the first cone and a part connecting S1 and the tail of a myosin molecule of which the length is shorter than subfragment 2 (S2) wobbles in the second cone (a double-cone model); the semiangles of the former and the latter cones were about 30 degrees and 50 degrees, respectively. The rotational freedom of myosin heads was only slightly restricted by the limited space of the filament lattice in myofibrils. Under rigor conditions, no motion of myosin heads was observed in the 10-microseconds time scale.     

Dynamic structure of lipid bilayers studied by nanosecond fluorescence techniques

Molecular motions in liposomes of dipalmitoyl-phosphatidylcholine (DPPC) were studied by nanosecond fluorescence techniques. As a fluorescent probe for the hydrocarbon region, 1,6-diphenyl-1,3,5-hexatriene (DPH) was used. Time courses of fluorescence intensity IT(t) and emission anisotropy r(t) of DPH embedded in DPPC liposomes were measured at various temperatures. The value of the fluorescence lifetime tau obtained froma single exponential decay of IT(t) was somewhat higher than that in liquid paraffin below the transition temperature Tt and decreased above Tt. Higher values of tau below Tt indicate the almost complete hydrophobic environment. The decay curves of r(t) were separated into two phases: an initial fast decreasing phase of the order of one nanosecond and a second almost constant phase. This indicates that the orientational motion of DPH in the hydrocarbon region is described by a wobbling diffusion restricted by a certain anisotropic potential. The results were analyzed on the model that the wobbling diffusion is confined in a cone with a uniform diffusion constant. Though temperature dependence of the cone angle was sigmoidal, that of the wobbling diffusion constant was like the exponential function. The change in the cone angle at Tt was sharper than that in the wobbling diffusion constant at Tt. Estimated values of the viscosity in the cone were an order of magnitude smaller than the values of "microviscosity" which were estimated from the steady-state emission anisotropy without considering the restrictions on the rotational motion.     

Effect of librational motion on fluorescence depolarization and nuclear magnetic resonance relaxation in macromolecules and membranes.

The theory of fluorescent emission anisotropy [r(t)] of a cylindrical probe in a membrane suspension is developed. It is shown, independent of any model, that the limiting anisotropy [r(infinity)] is proportional to the square to the order parameter of the probe. The order parameter determines the first nontrivial term in the expansion of the equilibrium orientational distribution function of the probe in a series of Legendre polynomials. Following Kinosita, Kawato, and Ikegami, the motion of the probe is described as diffusion ("wobbling") within a cone of semiangle theta 0. Within the framework of this model, an accurate single-exponential approximation for r(t) is considered. An analytic expression relating the effective relaxation time, which appears in the above approximation, to theta 0 and the diffusion coefficient for wobbling is derived. The model is generalized to the situation where the probe is attached to a macromolecule whose motion cannot be neglected on the time scale of the fluorescence experiment. Finally, by exploiting the formal similarity between the theory of fluorescence depolarization and 13C-NMR dipolar relaxation, expressions for T1, T2, and the nuclear Overhauser enhancement are derived for a protonated carbon which is nonrigidly attached to a macromolecule and undergoes librational motion described as diffusion on a spherical "cap" of semiangle theta 0.     

Membrane dynamic alterations associated with viral transformation and reversion. Decay of fluorescence emission and anisotropy studies of 3T3 cells.

Fluorescence lifetimes, anisotropies and rotational correlation time values of 1,6-diphenyl-1,3,5-hexatriene (DPH) in membranes of normal, transformed, and revertant 3T3 cells were determined by nanosecond (nsec), photon counting spectrofluorimetry. No change in lifetime values with transformation or reversion is observed. Fluorescence anisotropy decay curves show at least two components; an initial relatively fast decay and a non-zero “plateau” level component. The observed changes in the average anisotropy values, which qualitatively follow steady-state fluorescence polarization values, is due primarily to changes in the non-zero “plateau” level component. The anisotropy decay curves suggest that the rotational motion of the probe is restricted to a limited angular range. The present results are compared with model membrane systems.     

A new analysis method for the membrane viscosity from steady-state fluorescence depolarization

The absolute value of the viscosity in membrane lipid bilayers, which is different from the microviscosity advocated by Shinitzky, could be calculated from steady-state fluorescence depolarization of a hydrocarbon fluorophore, 1,6-diphenyl-1,3,5-hexatriene (DPH). This method was based on the theory of time-resolved fluorescence anisotropy and empirical relationships between fluorescence life time and the anisotropy parameters such as half cone angle in wobbling motion and wobbling diffusion rate of the fluorescent probe. Obtained viscosity values of various membranes from this method were consistent with those from time resolved method within experimental error.     

Internal motion of F-actin in 10(-6)-10(-3) s time range studied by transient absorption anisotropy: detection of torsional motion

By means of laser flash photolysis, the transient absorption anisotropy (TAA) of the triplet probe, 5-iodoacetamide-Eosin, labeling rabbit skeletal F-actin was measured in the 10(-6)-10(-3) s time range. The TAA curve at 20 degrees C showed a relatively slow decay phase covering several hundred microseconds and a large residual anisotropy (approximately 0.1 at 2 ms). After analysis with Barkley & Zimm's formula, it was concluded that the TAA of Eosin-F-actin can be approximated by the anisotropy decay due to torsional motion of F-actin.     

The rotational diffusion of cytochrome b5 in lipid bilayer membranes. Influence of the lipid physical state.

A derivative of the integral membranes protein, cytochrome b5, has been prepared in which the native heme group has been replaced by the structurally similar rhodium(III)-protoporphyrin IX. This metalloporphyrin has a finite triplet yield with a single exponential decay time of 22 microsecond in water. After insertion of the metalloporphyrin into the protein, its triplet-state decay becomes strongly nonexponential with at least three equal amplitude components with time constants varying over a range of 100. The derivatized protein has been incorporated into unilamellar liposomes prepared from dimyristoyllecithin, and the rotational diffusion of the protein in the lipid bilayer has been studied at temperatures above and below the lipid phase transition temperature via triplet absorbance anisotropy decay. The anisotropy decay curves are biphasic both above and below the lipid phase transition. The rotational diffusion constant is found to be 2.4 X 10(5) s-1 at 35 degrees C, and 1.1 X 10(4) s-1 at 10 degrees C, both being calculated from the fast decay component. The ratio of the limiting anisotropy to the initial anisotropy is 0.6 at both temperatures. This implies a cone of restricted motion of 34 degrees for the protein in the bilayer.     

Submicrosecond phospholipid dynamics using a long-lived fluorescence emission anisotropy probe.

The use of the long-lived fluorescence probe coronene (mean value of tau(FL) approximately 200 ns) is described for investigating submicrosecond lipid dynamics in DPPC model bilayer systems occurring below the lipid phase transition. Time-resolved fluorescence emission anisotropy decay profiles, measures as a function of increasing temperature toward the lipid-phase transition temperature (T(C)), for coronene-labeled DPPC small unilamellar vesicles (SUVs), are best described in most cases by three rotational decay components (phi(i = 3)). We have interpreted these data using two dynamic lipid bilayer models. In the first, a compartmental model, the long correlation time (phi(N)) is assigned to immobilized coronene molecules located in "gel-like" or highly ordered lipid phases (S-->1) of the bilayer, whereas a second fast rotational time (phi(F) approximately 2-5 ns) is associated with probes residing in more "fluid-like" regions (with corresponding lower ordering, S-->0). Interests here have focused on the origins of an intermediate correlation time (50-100 ns), the associated amplitude (beta(G)) of which increases with increasing temperature. Such behavior suggests a changing rotational environment surrounding the coronene molecules, arising from fluidization of gel lipid. The observed effective correlation time (phi(EFF)) thus reflects a discrete gel-fluid lipid exchange rate (k(FG)). A refinement of the compartmental model invokes a distribution of gel-fluid exchange rates (d(S,T)) corresponding to a distribution of lipid order parameters and is based on an adapted Landau expression for describing "gated" packing fluctuations. A total of seven parameters (five thermodynamic quantities, defined by the free energy versus temperature expansion; one gating parameter (gamma) defining a cooperative "melting" requirement; one limiting diffusion rate (or frequency factor: d(infinity))) suffice to predict complete anisotropy decay curves measured for coronene at several temperatures below the phospholipid T(C). The thermodynamic quantities are associated with the particular lipid of interest (in this case DPPC) and have been determined previously from ultrasound studies, thus representing fixed constants. Hence resolved variables are r(O), temperature-dependent gate parameters (gamma), and limiting diffusion rates (d(infinity)). This alternative distribution model is attractive because it provides a general probe-independent expression for distributed lipid fluctuation-induced probe rotational rates occurring within bilayer membranes below the phospholipid phase transition on the submicrosecond time scale.     

Rotational dynamics of actin

The rotational diffusion of actin was studied with the technique of time-resolved phosphorescence anisotropy using actin labeled at Cys-374 with erythrosin iodoacetamide. Immediately after the polymerization of actin was initiated, the correlation time increased sharply, passing through a maximum at 5 min and then declined to low values. F-Actin at equilibrium showed no anisotropy decay. The results were interpreted as indicating the initial formation of short mobile filaments which became increasingly immobile as elongation proceeded, leaving a decay which was dominated by shorter filaments. Some of these short filaments could have arisen by fragmentation of longer filaments. Eventually, the shorter filaments themselves became immobilized by entanglement within the gel matrix. The infinite-time anisotropy increased during polymerization, reflecting a smaller range of angular motion of the probe brought about by restricted torsional motion on the submicrosecond time scale. The results were compared with the length distribution of actin filaments revealed by electron microscopy [Kawamura, M., & Maruyama, K. (1970) J. Biochem. (Tokyo) 67, 437-457]. Polymerization in the presence of 1 microM cytochalasin B abolished the maximum in the correlation time profile and tended to prevent the immobilization of filaments by favoring shorter capped filaments which retained considerable rotational freedom. Addition of spectrin dimer to F-actin caused an increase in the time-invariant anisotropy. Subsequent additions of spectrin-binding proteins (erythrocyte bands 2.1 and 4.1) caused further increases in the anisotropy in a concentration-dependent manner, suggesting additional restriction of submicrosecond torsional motions. The results suggest that actin filaments within nonmuscle cells are rotationally immobile particularly if they are cross-linked by actin-binding proteins.     

The effect of chloroplast coupling factor ATP synthetase (CF1 · CF0) reconstitution on fluidity properties of isolated thylakoid lipid vesicles

The reconstitution of chloroplast coupling factor ATP synthetase (CF₁ · CF₀) with thylakoid lipids by cholate dialysis produced vesicles that displayed higher steady-state anisotropy (r_s) values for both 1,6-diphenyl-1,3,5-hexatriene (DPH) and trimethylammonium-diphenyl hexatriene fluorescence than the pure lipid alone. This is interpreted as meaning that the insertion of protein into the lipid bilayer brings about an increase in the ordering of acyl chains. This ordering effect became more obvious as the protein-to-lipid ratio was increased. Time-resolved decay analyses of DPH fluorescence anisotropy confirmed the conclusion drawn from the steady-state measurements, but further indicated that the dynamic motion of the probe was also slightly restricted after CF₁ · CF₀ incorporation. The restriction of DPH motion and the change in the half-angle for its cone of rotation was observed at relatively low protein-to-lipid ratios as compared with other reconstituted or biological membranes, suggesting that perhaps lipid-protein interactions occur with the inserted CF₁ · CF₀ complex.     

Dynamic depolarization of interacting fluorophores. Effect of internal rotation and energy transfer.

Effects of internal rotation on the fluorescence decay functions and time-dependent anisotropies of fluorophores bound to a spherical macromolecule are theoretically investigated in the presence of the intramolecular energy transfer interaction by solving relevant rotational diffusion equations. The model system examined is one in which the energy donor is internally rotating around an axis fixed at the macromolecule and the acceptor is fixed at a definite position in the macromolecule. The effect of internal rotation in the system is described by Hill's functions with two cosine terms. The fluorescence decay function and anisotropy decay are functions of the ratio of energy-transfer probability averaged over the internal rotation angle to the rotary diffusion co-efficient. When the internal rotation is much faster than energy transfer, the decay function of the donor is predicted to be a single exponential, and the anisotropy decay is essentially described by the expression derived by Gotlieb and Wahl (1963. J. Chim. Phys. 60:849-856). However, deviation from it becomes pronounced as the rotation becomes slower. Methods of numerical analysis are presented for decay function and anisotropy decay, as well as relative quantum yield and polarization anisotropy under steady-state excitation, and examined for a simplified system under the variation of the diffusion coefficient.     

Rotational modes of Ca2+-liganded calmodulin, as determined by time-domain fluorescence

The time decay of fluorescence anisotropy was monitored as a function of pH and temperature for complexes of 2,6-toluidinylnaphthalenesulfonate with calmodulin, with its proteolytic fragments, and with the 1:1 complex of calmodulin and melittin. For all the conditions examined the anisotropy decay of native calmodulin involved at least two rotational modes. These corresponded to a short correlation time of 2-3 ns, reflecting a localized motion in the vicinity of the binding site and a longer correlation time which arises from the rotation of a major portion of the molecule. The relative amplitudes of the two rotational modes were dependent upon temperature in the range 11-40 degrees C, the contribution of the more rapid mode increasing with temperature. The maximum immobilization of the probe occurred at pH 5.0 and 12 degrees C. While these results indicate the presence of internal rotations in Ca2+-liganded calmodulin, the magnitude of the longer correlation time is consistent with the crystallographic structure.     

Environmental modulation of M13 coat protein tryptophan fluorescence dynamics

The effects of detergent [deoxycholate (DOC) and phospholipid [dimyristoylphosphatidylcholine (DMPC)] environments on the rotational dynamics of the single tryptophan residue 26 of bacteriophage M13 coat protein have been investigated by using time-resolved single photon counting measurements of the fluorescence intensity and anisotropy decay. The total fluorescence decay of tryptophan-26 is complex but rather similar in DOC as compared to DMPC when analyzed in terms of a lifetime distribution (exponential series method). This similarity, in conjunction with the almost identical steady-state fluorescence spectra, indicates only minor differences between the tryptophan environments in DOC and DMPC. The reorientational dynamics of tryptophan-26 are dominated by slow rotation of the entire protein in both detergent and phospholipid environments. The resolved anisotropy decay in DOC can be approximated by a simple hydrodynamic model of protein/detergent micelle rotational diffusion, although the data indicative slightly greater complexity in the rotational motion. The tryptophan fluorescence anisotropy is not sensitive to protein conformational changes in DOC detected by nuclear magnetic resonance on the basis of pH independence in the range 7.5-9.1. In DMPC bilayers, restricted tryptophan motion with a correlation time of approximately 2 ns is observed together with a second very slow reorientational component. Resolution of the time constant for this slow rotation is obscured by the tryptophan fluorescence time window being too short to clearly locate its anisotropic limit. The possible contribution made by axial rotational diffusion of the protein to this slow rotational process is discussed.(ABSTRACT TRUNCATED AT 250 WORDS)     

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

The dominant motional mode for membrane proteins is uniaxial rotational diffusion about the membrane normal axis, and investigations of their rotational dynamics can yield insight into both the oligomeric state of the protein and its interactions with other proteins such as the cytoskeleton. However, results from the spectroscopic methods used to study these dynamics are dependent on the orientation of the probe relative to the axis of motion. We have employed polarized fluorescence confocal microscopy to measure the orientation of eosin-5-maleimide covalently reacted with Lys-430 of human erythrocyte band 3. Steady-state polarized fluorescence images showed distinct intensity patterns, which were fit to an orientation distribution of the eosin absorption and emission dipoles relative to the membrane normal axis. This orientation was found to be unchanged by trypsin treatment, which cleaves band 3 between the integral membrane domain and the cytoskeleton-attached domain. this result suggests that phosphorescence anisotropy changes observed after trypsin treatment are due to a rotational constraint change rather than a reorientation of eosin. By coupling time-resolved prompt fluorescence anisotropy with confocal microscopy, we calculated the expected amplitudes of the e-Dt and e-4Dt terms from the uniaxial rotational diffusion model and found that the e-4Dt term should dominate the anisotropy decay. Delayed fluorescence and phosphorescence anisotropy decays of control and trypsin-treated band 3 in ghosts, analyzed as multiple uniaxially rotating populations using the amplitudes predicted by confocal microscopy, were consistent with three motional species with uniaxial correlation times ranging from 7 microseconds to 1.4 ms.     

Ligand interactions in the ArsA protein, the catalytic component of an anion-translocating adenosinetriphosphatase.

The ars operon of the conjugative R-factor R773 produces resistance to arsenicals in cells of Escherichia coli. The operon encodes an oxyanion pump which is composed of a membrane subunit, the 45.5-kDa ArsB protein, and a catalytic subunit, the 63-kDa ArsA protein. Purified ArsA protein is an arsenite(antimonite)-stimulated ATPase. From its amino acid sequence, as deduced from the nucleotide sequence, the ArsA protein has four tryptophanyl residues which could serve as intrinsic fluorescent probes for the study of substrate-induced conformational changes. Both static and dynamic measurements of tryptophan fluorescence were performed with the ArsA protein. Results from static anisotropy measurements indicated differences in molecular motion with addition of ATP, SbO2-, or Mg2+. These results were supported by time decay measurements of fluorescence anisotropy. The results of time decay measurements indicated a shorter correlation time, reflecting localized motion in the vicinity of the probe, and a longer correlation time, which could have arisen from rotation of the major portion of the molecule. The longer correlation time changed with addition of the various effectors, especially MgCl2, suggesting that binding of Mg2+ decreases probe mobility.     

Liquid-ordered microdomains in lipid rafts and plasma membrane of U-87 MG cells: a time-resolved fluorescence study

Lipid rafts, the functional microdomains in the cell membrane, are believed to exist as liquid-ordered (Lo) phase domains along with the liquid-disordered (Ld) phase of the bulk of the cell membranes. We have examined the lipid order in model and natural membranes by time-resolved fluorescence of trimethylammonium-1,6-diphenylhexatriene incorporated into the membranes. The lipid phases were discerned by the limiting anisotropy, rotational diffusion rate and distribution of the fluorescence lifetime. In dipalmitoylphosphatidylcholine (DPPC)-cholesterol mixtures the gel phase exhibited higher anisotropy and a two-fold slower rotational diffusion rate of the probe as compared to the Ld phase. On the other hand, the Lo phase exhibited higher limiting anisotropy but a rotational diffusion rate comparable to the Ld phase. The Ld and Lo phases elicited unimodal distribution of lifetimes with distinct mean values and their co-existence in phospholipid-cholesterol mixtures was reflected as a biphasic change in the width of the lifetime distribution. Global analysis of the lifetimes yielded a best fit with two lifetimes which were identical to those observed in single Lo or Ld phases, but their fractional contribution varied with cholesterol concentration. Attributing the shorter and longer lifetime components to the Ld and Lo phases, respectively, the extent of the Lo/Ld phase domains in the membranes was estimated by their fractional contribution to the fluorescence decay. In ternary mixtures of egg PC-gangliosides-cholesterol, the gangliosides induced heterogeneity in the membrane but the Ld phase prevailed. The Lo phase properties were observed only in the presence of cholesterol. Results obtained in the plasma membrane and detergent-resistant membrane fractions (DRMs) isolated from U-87 MG cells revealed that DRMs mainly possess the Lo phase; however, a substantially large proportion of plasma membrane also exists in the Lo phase. Our data show that, besides cholesterol, the membrane proteins play a significant role in the organization of lipid rafts and, furthermore, a considerable amount of heterogeneity is present among the lipid rafts.