Time-resolved Phosphorescence Anisotropy Research Articles

Structural Dynamics of SERCA and Phospholamban by Fluorescence and Phosphorescence

We used fluorescence and phosphorescence to investigate the structure and dynamics of phospholamban (PLB), and its regulation of its inhibited target, sarcoplasmic reticulum calcium ATPase (SERCA). Polarized TIRF of PLB, labeled in the cytoplasmic domain helix with bifunctional rhodamine (BFR), shows that this domain lies parallel to the membrane surface. The structural and functional effects of PLB phosphorylation and mutation are under investigation. Single molecular TIRF microscopy was used to measure the translational diffusional coefficient of Alexa488 labeled monomeric PLB reconstituted into a supported lipid bilayer. The diffusional coefficient of monomeric PLB is 0.7 μm2/s, which is consistent with its molecular weight. Time-resolved phosphorescence anisotropy of erythrosin iodoacetamide (ErIA) labeled SERCA in cardiac sarcoplasmic reticulum (SR) was measured with and without phosphorylation of PLB in presence of high and low Ca concentrations. Phosphorylation of PLB decreased the final anisotropy of ErIA labeled SERCA at low Ca, indicating decreased SERCA self-association. This supports the proposal that PLB inhibits SERCA by inducing SERCA-SERCA association, which is relieved by phosphorylation.

Compartmentalization of the Type I Fcε receptor and MAFA on mast cell membranes

The Mast cell Function-associated Antigen (MAFA) is a membrane glycoprotein on rat mast cells (RBL-2H3) expressed at a ratio of ∼ 1:30 with respect to the Type I Fcε receptor (FcεRI). Despite this stoichiometry, clustering MAFA by its specific mAb G63 substantially inhibits secretion of both granular and de novo synthesized mediators induced upon FcεRI aggregation. Since the FcεRIs apparently signal from within raft micro-environments, we investigated possible co-localization of MAFA within these membrane compartments containing aggregated FcεRI. We used cholera toxin B subunit (CTB) to cluster the raft component ganglioside GM1 and studied the effects of this perturbation on rotation of FcεRI and MAFA by time-resolved phosphorescence anisotropy of erythrosin-conjugated probes. CTB treatment would be expected to substantially inhibit rotation of raft-associated molecules. Experimentally, CTB has no effect on rotational parameters such as the long-time anisotropy ( r ∞) of unperturbed FcεRI or MAFA. However, on cells where FcεRI-IgE has previously been clustered by antigen (DNP 14-BSA), CTB treatment increases the FcεRI-IgE's r ∞ by 0.010 and MAFA's by 0.014. Similarly, CTB treatment of cells where MAFA had been clustered by mAb G63 increases MAFA's r ∞ by 0.010 but leaves FcεRI's unaffected. Evaluation of raft localization of FcεRI and MAFA using sucrose gradient ultracentrifugation of Triton X-100 treated membrane fragments demonstrates that a significant fraction of MAFA molecules sediments with rafts when FcεRI is clustered by antigen or when MAFA itself is clustered by mAb G63. The large excess of FcεRI over MAFA explains why clustering MAFA does not substantively affect FcεRI dynamics. Moreover, in single-particle tracking studies of individual FcεRI-IgE or MAFA molecules, these proteins, upon clustering by antigen, move into small membrane compartments of reduced, but similar, dimensions. This provides additional indication of constitutive interactions between FcεRI and MAFA. Taken together, these results of distinct methodologies suggest that MAFA functions within raft microdomains of the RBL-2H3 cell membrane and thus in close proximity to the FcεRI which themselves signal from within the raft environment.

Cofilin Increases the Torsional Flexibility and Dynamics of Actin Filaments

We have measured the effects of cofilin on the conformation and dynamics of actin filaments labeled at Cys374 with erythrosin-iodoacetemide (ErIA), using time-resolved phosphorescence anisotropy (TPA). Cofilin quenches the phosphorescence intensity of actin-bound ErIA, indicating that binding changes the local environment of the probe. The cofilin concentration-dependence of the phosphorescence intensity is sigmoidal, consistent with cooperative actin filament binding. Model-independent analysis of the anisotropies indicates that cofilin increases the rates of the microsecond rotational motions of actin. In contrast to the reduction in phosphorescence intensity, the changes in the rates of rotational motions display non-nearest-neighbor cooperative interactions and saturate at substoichiometric cofilin binding densities. Detailed analysis of the TPA decays indicates that cofilin decreases the torsional rigidity (C) of actin, increasing the thermally driven root-mean-square torsional angle between adjacent filament subunits from approximately 4 degrees (C = 2.30 x 10(-27) Nm2 radian(-1)) to approximately 17 degrees (C = 0.13 x 10(-27) Nm2 radian(-1)) at 25 degrees C. We favor a mechanism in which cofilin binding shifts the equilibrium between thermal ErIA-actin filament conformers, and facilitates two distinct structural changes in actin. One is local in nature, which affects the structure of actin's C terminus and is likely to mediate nearest-neighbor cooperative binding and filament severing. The second is a change in the internal dynamics of actin, which displays non-nearest-neighbor cooperativity and increases the torsional flexibility of filaments. The long-range effects of cofilin on the torsional dynamics of actin may accelerate P(i) release from filaments and modulate interactions with other regulatory actin filament binding proteins.

SERCA structural dynamics induced by ATP and calcium.

We have used time-resolved phosphorescence anisotropy (TPA) to probe rotational dynamics of the rabbit skeletal sarcoplasmic reticulum Ca-ATPase (SERCA), to test the hypothesis, generated from X-ray crystallography, that large-scale structural changes are induced by Ca in this system. Previous TPA studies on SERCA used primarily erythrosin 5'-isothiocyanate (ErITC), which binds to the nucleotide-binding domain and inactivates the enzyme. To investigate rotational dynamics of the active enzyme, we labeled SERCA with erythrosin 5'-iodoacetamide, which binds to the phosphorylation domain and has a minimal effect on the calcium-dependent ATPase activity. In the absence of nucleotide and the presence of calcium, TPA results were similar to those observed previously with ErITC, consistent with the global uniaxial rotation of SERCA monomers and oligomers and small amplitude internal protein dynamics. The removal of Ca had only a slight effect, while the addition of adenosine 5'-triphosphate (ATP) increased the amplitude of internal dynamics and changed the probe's orientation, corresponding to tilting of the phosphorylation domain by at least 20 degrees . Ca partially reversed the ATP effects. A nonhydrolyzable ATP analogue had the same effects as ATP, showing that the observed changes were not dependent on active ion transport. Computational analysis indicates that these ligands affect primarily the internal dynamics of the enzyme, with negligible effects on global dynamics and enzyme association. Melittin, which has been shown to aggregate and inhibit SERCA, eliminated the nucleotide-induced internal dynamics and increased the final anisotropy. We propose that (i) the large Ca-dependent structural changes suggested by SERCA crystallography are more dependent on ATP than on Ca and (ii) aggregation-induced inhibition of SERCA is due to the functional coupling between global and internal protein dynamics.

Membrane Organization of Luteinizing Hormone Receptors Differs between Actively Signaling and Desensitized Receptors

Signaling by the luteinizing hormone/choriogonadotropin receptor (LHR) is of considerable interest because of its requirement for successful reproduction. Time-resolved phosphorescence anisotropy and fluorescence resonance energy transfer were used to investigate the organization of endogenous LHRs in porcine follicular membranes in two distinct signaling states, active and desensitized. Desensitized LHRs exhibited approximately 3-fold slower rotational correlation times compared with active LHRs (59 +/- 4 and 21 +/- 9 mus, respectively), suggesting that with agonist-dependent desensitization the receptors are organized into larger protein complexes. Incubation of membranes with inhibitors of LHR desensitization, such as neutralizing anti-arrestin antibodies, a synthetic peptide corresponding to the third intracellular loop of the LHR but not the corresponding scrambled peptide, or catalytically inactive ARNO, resulted in faster rotational diffusion times equivalent to those of actively signaling LHRs. Furthermore, desensitized LHRs exhibited a 2.4-fold increase in fluorescence resonance energy transfer between LHRs suggesting that the larger protein aggregates formed during desensitization contain more self-associated LHRs. These results indicate that agonist-dependent LHR desensitization precedes organization of LHRs at the cells surface into larger protein aggregates.

Rotational dynamics of colloidal tracer spheres in suspensions of charged rigid rods

The short-time rotational dynamics of colloidal silica tracer spheres in suspensions of rigid silica rods is investigated, using time-resolved phosphorescence anisotropy, as a function of tracer radius aT, rod volume fraction φ, and the range κ−1 of the double-layer repulsions between the like-charged rods and tracer spheres. A large tracer size aT and a small screening length κ−1 appear to maximize hydrodynamic hindrance of tracer diffusion for given φ. The marked φ-dependence of the rotational dynamics is primarily determined by the large excluded volumes of the high-aspect ratio rods. Stokes–Einstein–Debye (SED) scaling of the rotational diffusion coefficients with the inverse viscosity of the rod suspensions holds fairly well, expect for small aT and large κ−1. The ionic strength dependence of deviations from SED scaling is rationalized in terms of an effective hard-rod model with the bare length L replaced by an effective length L+4κ−1.

Rotational dynamics of charged colloidal spheres: Role of particle interactions

Time-resolved phosphorescence anisotropy (TPA) is used to measure the short-time rotational diffusion coefficient Dsr(φ) of charged tracer spheres as a function of the volume fraction φ of like-charged colloidal host spheres in nonaqueous solvents. Sphere interactions are varied from long-range repulsive to short-range attractive by changing the ionic strength and the solvent composition. It is shown that Dsr(φ) is very sensitive to details of the interaction near contact, in agreement with theory. In contrast, the low-shear viscosity ηL(φ) of the host dispersions is mostly controlled by the tail of the interaction potential. We discuss the applicability of Stokes–Einstein–Debye scaling Dsr(φ)∝1/ηL(φ), and Dsr(φ)∝1/η∞(φ), where η∞ is the high-frequency-limiting viscosity. Scaling with ηL(φ) fails at high particle and low salt concentrations, while scaling with η∞ is fairly good, in particular when an apparent nonstick boundary condition is imposed on the friction factor. We conclude that TPA is well suited for use as a microrheological technique.

Interactions of the mast cell function-associated antigen with the type I Fcε receptor

Clustering the mast cell function-associated antigen (MAFA), a membrane glycoprotein expressed on 2H3 cells, by its specific monoclonal antibody G63 substantially inhibits secretion normally triggered by aggregating these cells’ Type I Fcε receptor (FcεRI). To explore possible MAFA-FcεRI interactions giving rise to this inhibition, we have studied by time-resolved phosphorescence anisotropy the rotational behavior of both MAFA and FcεRI as ligated by various reagents involved in FcεRI-induced degranulation and MAFA-mediated inhibition thereof. From 4 to 37 °C the rotational correlation times (mean±S.D.) of FcεRI-bound, erythrosin-conjugated IgE resemble those observed for MAFA-bound erythrosin-conjugated G63 Fab, 82±17 μs and 79±31 μs at 4 °C, respectively. Clustering the FcεRI-IgE complex by antigen or by anti-IgE increases the phosphorescence anisotropy of G63 Fab and slows its rotational relaxation. Lateral diffusion of G63 Fab is also slowed by antigen clustering of the receptor. Taken together, these results suggest that unperturbed MAFA associates with clustered FcεRI. They are also consistent with its interaction with the isolated receptor, a situation also suggested by FRET measurements on the system.

The mast cell function-associated antigen and its interactions with the type I Fcepsilon receptor.

Rat mucosal-type mast cells of the RBL-2H3 line express a glycoprotein termed the MAst cell Function-associated Antigen (MAFA). When MAFA is clustered by its specific monoclonal antibody G63, secretion normally triggered by aggregating these cells' type I Fcepsilon receptor (FcepsilonRI) is substantially inhibited. The nature of MAFA-FcepsilonRI interactions giving rise to this inhibition remains unclear. Rotational diffusion of a membrane protein is a sensitive probe of its involvement in intermolecular interactions. We have therefore studied by time-resolved phosphorescence anisotropy the rotational behavior of both MAFA and FcepsilonRI as ligated by various reagents involved in FcepsilonRI-induced degranulation and MAFA-mediated inhibition thereof. From 4 to 37 degrees C, the rotational correlation times (mean +/- SD) of FcepsilonRI-bound, erythrosin-conjugated IgE resemble those observed for MAFA-bound, erythrosin-conjugated G63 Fab, 82 +/- 17 and 79 +/- 31 micros at 4 degrees C, respectively. Clustering the FcepsilonRI-IgE complex by antigen or by anti-IgE increases the phosphorescence anisotropy of G63 Fab and slows its rotational relaxation. Lateral diffusion of G63 Fab is also slowed by antigen clustering of the receptor. Taken together, these results indicate that unperturbed MAFA associates with clustered FcepsilonRI. They are also consistent with its interaction with the isolated receptor.

Rotational tracer diffusion in binary colloidal sphere mixtures.

We demonstrate that tracer/host size asymmetry and electrostatic interactions strongly affect rotational self-diffusion in binary mixtures of charged colloidal tracer and host spheres. Tracer diffusion coefficients, measured with time-resolved phosphorescence anisotropy, are compared with calculations of rotational diffusion including two- and three-particle hydrodynamic interactions. We also show that the inverse dependence of the rotational diffusion coefficient on the suspension viscosity is approached only at large size ratios.

Rotational Diffusion of Tracer Spheres in Packings and Dispersions of Colloidal Spheres Studied with Time-Resolved Phosphorescence Anisotropy

We introduce the time-resolved phosphorescence anisotropy (TPA) method to study rotational diffusion of phosphorescent colloidal silica spheres in dispersions and in random close packings of host spheres. The tracer diffusion coefficients in sphere dispersions agree with hard-sphere predictions, demonstrating that TPA is a reliable technique for studying rotational dynamics. We also assess the rotational diffusion of phosphorescent tracers in sphere packings, exploiting the fact that the TPA technique can be used for slightly turbid samples. We find that the fraction of immobilized tracers directly reflects the pore size distribution as extracted from the Voronoi construction of a simulated random close packing. A calculation of the average distance between tracer and neighboring particle shows that the dependence of the rotational diffusion coefficient on the average distance between tracer and medium spheres is stronger for packings than for dispersions.

Phosphorescent Colloidal Silica Spheres as Tracers for Rotational Diffusion Studies

We introduce a novel, phosphorescent, colloidal silica sphere tagged with eosin-5-isothiocyanate as a tracer for rotational diffusion on the millisecond time scale measured in situ with time-resolved phosphorescence depolarization. The suitability of the tracer was investigated by a combination of spectral and time-resolved fluorescence and phosphorescence measurements. The phosphorescence quantum yield of dye molecules is high, because they are effectively shielded for quenching by oxygen or solute molecules, though some oxygen and water may penetrate the silica sphere. Both energy transfer between eosin molecules and internal rotational mobility of eosin cause a limited decay of the initial phosphorescence anisotropy. Finally, the hydrodynamic radius of the tracer found from the time-resolved phosphorescence anisotropy experiment coincides with the value found by dynamic light scattering experiments. Our results show that silica tracer spheres tagged with eosin are suitable tracers for rotational diffusion measurements.

Biological function of the LH receptor is associated with slow receptor rotational diffusion

The biological activity of luteinizing hormone (LH) receptors can be affected by modifications to the receptor’s amino acid sequence or by binding of hormone antagonists such as deglycosylated hCG. Here we have compared rotational diffusion of LH receptors capable of activating adenylate cyclase with that of non-functional hormone-occupied receptors at 4°C and 37°C using time-resolved phosphorescence anisotropy techniques. Binding of hCG to the rat wild-type receptor expressed on 293 cells (LHR-wt cells) or to the LH receptor on MA-10 cells produces functional receptors which exhibit rotational correlation times longer than 1000 μs. However, modification of the LH receptor by substitution of Lys583→Arg (LHR-K583R) results in a receptor that is non-functional and which has a significantly shorter rotational correlation time of 130±12 μs following binding of hCG. When these receptors are treated with deglycosylated hCG, an inactive form of hCG, the rotational correlation times for the LH receptors on LHR-wt and MA-10 cells are also shorter, namely 64±8 and 76±14 μs, respectively. Finally, a biologically active truncated form of the rat LH receptor expressed in 293 cells (LHR-t631) has slow rotational diffusion, greater than 1000 μs, when occupied by hCG and a significantly shorter rotational correlation time of 103±12 μs when occupied by deglycosylated hCG. The effects of rat LH binding to LH receptors on these various cell lines were similar to those of hCG although the magnitude of the changes in receptor rotational diffusion were less pronounced. We suggest that functional LH receptors are present in membrane complexes that exhibit slow rotational diffusion or are rotationally immobile. Shorter rotational correlation times for non-functional hormone–receptor complexes may reflect the absence of essential interactions between these complexes and other membrane proteins.

Phosphorescence and fluorescence characterization of fluorescein derivatives immobilized in various polymer matrices

The phosphorescence quantum yield, zero-time anisotropy, and lifetime are the three factors determining the suitability of a phosphorescent dye for rotational diffusion experiments. Furthermore, knowledge of the absolute orientation of the phosphorescence dipole moment in the molecular frame is a prerequisite for a quantitative analysis of time-resolved phosphorescence anisotropy (TPA) measurements in orientationally anisotropic systems. These factors have been characterized for four dye molecules, consisting of a fluorescein core with two or four substituted heavy-atoms (iodine and bromine). The characterization was performed by a combination of spectral and time-resolved phosphorescence and fluorescence measurements on dye molecules embedded in immobilizing polymer matrices. A quantitative criterion was found for the time-region where to use which dye, as well as for the dye concentrations to be used. For the latter criterion the effect of Forster radiationless energy transfer on the phosphorescence anisotropy was treated explicitly. The phosphorescence dipole moment was found to be tilted somewhat out of the molecular plane, and aligned more towards the in-plane fluorescence dipole moment than to the in-plane S1←S0 absorption dipole moment. The results for the four fluorescein derivatives indicate that the effect of the spin-orbital coupling is to pull the in-plane component of the phosphorescence dipole moment towards the fluorescence dipole moment, causing an increase of the phosphorescence quantum yield. This effect is primarily influenced by the Z-number of the substituted heavy atoms. The number of substituted heavy atoms plays a less important role.

Differences in structural dynamics of muscle and yeast actin accompany differences in functional interactions with myosin.

We have used spectroscopic probes ErIA and IAEDANS attached to Cys374 to compare the structural dynamics of yeast actin filaments with that of muscle actin, to understand the structural basis of the less productive interaction of yeast actin with myosin. Time-resolved phosphorescence anisotropy (TPA) of ErIA and steady-state fluorescence of IAEDANS were measured. TPA indicated more rapid rotational motion and more restricted angular amplitude in yeast actin. The fluorescence spectrum was less intense and more red-shifted in yeast actin, suggesting more exposure of the probe to solvent. These results indicate that the two actins differ substantially in the conformational dynamics of the C-terminal region. Binding of myosin S1 induced significantly different spectroscopic changes in TPA and fluorescence of muscle and yeast actin. As a result, the spectroscopic differences between the two actins were decreased by the addition of S1. These results suggest that yeast actin is less effective at activating myosin because of larger changes required in the structure of actin upon strong myosin binding. These results provide insight into the relationship between actomyosin dynamics and function, and they provide a useful framework for structure-function analysis of mutant yeast actin.

Dynamics of molecules involved in antigen presentation: effects of fixation

Antigen presentation by MHC class II molecules can be enhanced by paraformaldehyde fixation of antigen-presenting cells prior to assay. This treatment might be expected to aggregate membrane proteins and thus stabilize and strengthen transient protein-protein interactions involved in intercellular cooperation. Lateral and rotational dynamics of the MHC class II antigen I-A d on A20 cells fixed with various concentrations of paraformaldehyde were examined by fluorescence photobleaching recovery and time-resolved phosphorescence anisotropy, respectively. Probes were tetramethylrhodamine and erythrosin conjugates of MKD6 Fab fragments. Increasing concentrations of paraformaldehyde led to a progressive increase in the limiting anisotropy of I-A d at 4°C from the value of 0.042 for untreated cells, indicative of large aggregate formation, while leaving the rotational correlation time of 29 μs unchanged, a measure of the unperturbed molecule. On the other hand, the translational diffusion constants decreased from ∼2×10 −10 cm 2 s −1, while the fractional recovery remained unchanged at about 40–50%. Taken together, these results suggest that fixation crosslinks class II molecules to each other or to other membrane proteins into structures large enough (>500,000 kDa) to diffuse translationally with perceptibly size-dependent rates. The fixation effects on both class II rotation and lateral diffusion were half-maximal at paraformaldehyde concentrations of ∼0.2%. Possible relations between the biological effector functions of class II and the physical sizes of fixation-induced aggregates are discussed.

Rotational Dynamics of the Regulatory Light Chain in Scallop Muscle Detected by Time-Resolved Phosphorescence Anisotropy

We have used time-resolved phosphorescence anisotropy (TPA) to study the rotational dynamics of chicken gizzard regulatory light chain (RLC) bound to scallop adductor muscle myofibrils in key physiological states. Native RLC from scallop myofibrils was extracted and replaced completely with gizzard RLC labeled specifically at Cys 108 with erythrosin iodoacetamide (ErIA). The calcium sensitivity of the ATPase activity of the labeled myofibril preparation was quite similar to that of the native sample, indicating that the ErIA-labeled RLC is functionally bound to the myosin head. In rigor (in the absence of ATP, when all the myosin heads are rigidly bound to the thin filament), a slight decay was observed in the first few microseconds, followed by no change in the anisotropy. This indicates small-amplitude restricted motions of the RLC or the entire LC domain of myosin. Addition of calcium to rigor restricts these motions further. Relaxation with ATP (no Ca) causes a large decay in the anisotropy, indicating large-amplitude rotational motion with correlation times of 5-50 micros. Further addition of calcium, to induce contraction, resulted in a decrease in the rate and amplitude of anisotropy decay. In particular, there is clear evidence for a slow rotational motion with a correlation time of approximately 300 micros, which is not present either in rigor or relaxation. This indicates rotational motion that specifically correlates with force generation. The changes in the rotational dynamics of the light-chain domain in rigor, relaxation, and contraction support earlier work based on probes of the catalytic domain that muscle contraction is accompanied by a disorder-to-order transition of the myosin head. However, the motions of the LC domain are different from those of the catalytic domain, which indicates rotation of the two domains relative to each other.

An Autoinhibitory Peptide from the Erythrocyte Ca-ATPase Aggregates and Inhibits Both Muscle Ca-ATPase Isoforms

We have studied the effects of C28R2, a basic peptide derived from the autoinhibitory domain of the plasma membrane Ca-ATPase, on enzyme activity, oligomeric state, and E1-E2 conformational equilibrium of the Ca-ATPase from skeletal and cardiac sarcoplasmic reticulum (SR). Time-resolved phosphorescence anisotropy (TPA) was used to determine changes in the distribution of Ca-ATPase among its different oligomeric species in SR. C28R2, at a concentration of 1–10 μM, inhibits the Ca-ATPase activity of both skeletal and cardiac SR (CSR). In skeletal SR, this inhibition by C28R2 is much greater at low (0.15 μM) than at high (10 μM) Ca 2+, whereas in CSR the inhibition is the same at low and high Ca 2+. The effects of the peptide on the rotational mobility of the Ca-ATPase correlated well with function, indicating that C28R2-induced protein aggregation and Ca-ATPase inhibition are much more Ca-dependent in skeletal than in CSR. In CSR at low Ca 2+, phospholamban (PLB) antibody (functionally equivalent to PLB phosphorylation) increased the inhibitory effect of C28R2 slightly. Fluorescence of fluorescein 5-isothiocyanate-labeled SR suggests that C28R2 stabilizes the E1 conformation of the Ca-ATPase in skeletal SR, whereas in CSR it stabilizes E2. After the addition of PLB antibody, C28R2 still stabilizes the E2 conformational state of CSR. Therefore, we conclude that C28R2 affects Ca-ATPase activity, conformation, and self-association differently in cardiac and skeletal SR and that PLB is probably not responsible for the differences.

Differential effects of general anesthetics on the quaternary structure of the Ca-ATPases of cardiac and skeletal sarcoplasmic reticulum.

The effects of the general anesthetics hexanol, halothane, and diethyl ether on Ca-ATPase activity and on the oligomeric state of the Ca-ATPase of sarcoplasmic reticulum (SR) from cardiac and skeletal muscle were investigated. The effects of these general anesthetics on Ca-ATPase activity were similar in cardiac and skeletal SR and were characterized by stimulation of Ca-ATPase activity at lower concentrations of anesthetics and inhibition at higher concentrations. The distribution of the Ca-ATPase among its oligomeric states was estimated from the time-resolved phosphorescence anisotropy (TPA) decay of SR in which Ca-ATPase was covalently labeled with erythrosin isothiocyanate (ERITC) or with erythrosin iodoacetamide (ERIA). In contrast to the similar responses of Ca-ATPase activity, there were marked differences in the responses to general anesthetics of the TPA decay between cardiac and skeletal SR. In cardiac SR hexanol, halothane, and diethyl ether caused pronounced increases in the limiting anisotropy at very long times (r infinity), which indicate increases in the fraction of oligomers too large to rotate on the millisecond time scale of the experiments. In skeletal SR, by contrast, there were no significant changes in r infinity in response to the three general anesthetics. This difference between cardiac and skeletal SR in response to general anesthetics is not due to the presence of phospholamban in cardiac SR, since SR from AT-1 cells, which have the SERCA2a isoform of Ca-ATPase, but only trace levels of phospholamban, have increases in r infinity in response to the general anesthetics that resemble those in cardiac SR. Experiments with cardiac SR labeled with ERIA give similar results, showing that the results with ERITC are not an artifact of the labeling procedure. Increasing the ionic strength with LiCl diminished the proportion of large immobile oligomers of cardiac Ca-ATPase under control conditions but enhanced the formation of large oligomers in response to hexanol.

Perturbations of functional interactions with myosin induce long-range allosteric and cooperative structural changes in actin.

The role of the rotational dynamics of actin filaments in their interaction with myosin was studied by comparing the effect of myosin subfragment 1 (S1) with two other structural perturbations, which have substantial inhibitory effects on activation of myosin ATPase and in vitro motility of F-actin: (1) binding of the antibody fragment Fab(1-7) against the first seven N-terminal residues and (2) copolymerization with monomers treated with the zero-length cross-linker 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC), referred to as EDC-actin. The rotational motion of actin was measured by time-resolved phosphorescence anisotropy (TPA) of erythrosin iodoacetamide (ErIA) attached to Cys 374 on actin. The binding of S1 in a rigor complex (no nucleotide) induced intramonomer (allosteric) and intermonomer (cooperative) structural changes that increased the residual anisotropy of labeled F-actin, indicating a conformational change in the region of the C terminus. Similar allosteric and cooperative changes were induced by binding of Fab(1-7) and by copolymerization of the ErIA-labeled actin monomers with EDC-actin. This suggests that the functional perturbations transform actin to a form resembling the rigor actomyosin complex. The correlation of the perturbation-induced changes in TPA of actin with the functional effects suggests that the actomyosin interaction can be inhibited by stabilization of actin in one of its structural intermediates.