Micro-viscosity of liquid oil confined in colloidal fat crystal networks.

Molecular rotors may be utilized as non-invasive, non-disruptive and highly sensitive alternatives to conventional measures of bulk viscosity when the oil is entrained in a colloidal fat crystal network. Oil viscosity changes based on the molecular confinement of the oil, which is dependent on its molecular volume. Changes in micro-viscosity were not dependent on the solids content, but instead were strongly dependent on the box-counting fractal dimension in high-space filling colloidal fat crystal networks (i.e., D > 1.89). A bulk oil viscosity is often an overestimation of the actual viscosity of the entrained oil and may not be appropriate when predicting diffusion in multi-phase materials.

Mean DNA bend angle and distribution of DNA bend angles in the CAP-DNA complex in solution.

In order to define the mean DNA bend angle and distribution of DNA bend angles in the catabolite activator protein (CAP)-DNA complex in solution under standard transcription initiation conditions, we have performed nanosecond time-resolved fluorescence measurements quantifying energy transfer between a probe incorporated at a specific site in CAP, and a complementary probe incorporated at each of five specific sites in DNA. The results indicate that the mean DNA bend angle is 77(+/-3) degrees - consistent with the mean DNA bend angle observed in crystallographic structures (80(+/-12) degrees ). Lifetime-distribution analysis indicates that the distribution of DNA bend angles is relatively narrow, with <10 % of DNA bend angles exceeding 100 degrees. Millisecond time-resolved luminescence measurements using lanthanide-chelate probes provide independent evidence that the upper limit of the distribution of DNA bend angles is approximately 100 degrees. The methods used here will permit mutational analysis of CAP-induced DNA bending and the role of CAP-induced DNA bending in transcriptional activation.

Differential dynamic behavior of actin filaments containing tightly-bound Ca2+ or Mg2+ in the presence of myosin heads actively hydrolyzing ATP.

We have investigated how Ca2+ or Mg2+ bound at the high-affinity cation binding site in F-actin modulates the dynamic response of these filaments to ATP hydrolysis by attached myosin head fragments (S1). Rotational motions of the filaments were monitored using steady-state phosphorescence emission anisotropy of the triplet probe erythrosin-5-iodoacetamide covalently attached to cysteine 374 of actin. The anisotropy of filaments containing only Ca2+ increased from 0.080 to 0.137 upon binding S1 in a rigor complex and decreased to 0.065 in the presence of ATP, indicating that S1 induced additional rotational motions in the filament during ATP hydrolysis. The comparable anisotropy values for Mg(2+)-containing filaments were 0.067, 0.137, and 0.065, indicating that S1 hydrolysis did not induce measurable rotational motions in these filaments. Phalloidin, a fungal toxin which stabilizes F-actin and increases its rigidity, increased the anisotropy of F-actin containing either Ca2+ or Mg2+ but not the anisotropy of the 1:1 S1-actin complexes of these filaments. Mg(2+)-containing filaments with phalloidin bound also displayed increased rotational motions during S1 ATP hydrolysis. A strong positive correlation between the phosphorescence anisotropy of F-actin under specific conditions and the extent of the rotational motions induced by S1 during ATP hydrolysis suggested that the long axis torsional rigidity of F-actin plays a crucial role in modulating the dynamic response of the filaments to ATP hydrolysis by S1. Cooperative responses of F-actin to dynamic perturbations induced by S1 during ATP hydrolysis may thus be physically mediated by the torsional rigidity of the filament.

Differential mobility of skeletal and cardiac tropomyosin on the surface of F-actin.

Polarized phosphorescence from the triplet probe erythrosin-5-iodoacetamide attached to sulfhydryls in rabbit skeletal and cardiac muscle tropomyosin (Tm) was used to measure the microsecond rotational dynamics of these tropomyosins in a complex with F-actin. The steady-state phosphorescence anisotropy of skeletal tropomyosin on F-actin was 0.025 +/- 0.005 at 20 degrees C; the comparable anisotropy for cardiac tropomyosin was 0.010 +/- 0. 003. Measurements of the anisotropy as a function of temperature and solution viscosity (modulated by addition of glycerol) indicated that both skeletal and cardiac tropomyosin undergo complex rotational motions on the surface of F-actin. Models assuming either long axis rotation of a rigid rod or torsional twisting of a flexible rod adequately fit these data; both analyses indicated that cardiac Tm is more mobile than skeletal Tm and that the increased mobility on the surface of F-actin reflected either the rotational motion of a smaller physical unit or the torsional twisting of a less rigid molecule. The binding of myosin heads (S1) to the Tm-F-actin complexes increased the anisotropy to 0.049 +/- 0.004 for skeletal and 0.054 +/- 0.007 for cardiac tropomyosin. The titration of the skeletal tropomyosin-F-actin complex by S1 showed a break at an S1/actin ratio of 0.14; this complex had an anisotropy of 0.040 +/- 0.007, suggesting that one bound head effectively restricted the motion of each skeletal tropomyosin. A similar titration with cardiac tropomyosin reached a plateau at an S1/actin ratio of 0.4, suggesting that 2-3 myosin heads are required to immobilize cardiac Tm. Surface mobility is predicted by structural models of the interaction of tropomyosin with the actin filament while the decrease in tropomyosin mobility upon S1 binding is consistent with current theories for the proposed role of myosin binding in the mechanism of tropomyosin-based regulation of muscle contraction.

Influence of tightly bound Mg2+ and Ca2+, nucleotides, and phalloidin on the microsecond torsional flexibility of F-actin.

To better understand the relationship between structure and molecular dynamics in F-actin, we have monitored the torsional flexibility of actin filaments as a function of the type of tightly bound divalent cation (Ca2+ or Mg2+) or nucleotide (ATP or ADP), the level of inorganic phosphate and analogues, KCl concentration, and the level of phalloidin. Torsional flexibility on the microsecond time scale was monitored by measuring the steady-state phosphorescence emission anisotropy (rFA) of the triplet probe erythrosin-5-iodoacetamide covalently bound to Cys-374 of skeletal muscle actin; extrapolations to an infinite actin concentration corrected the measured anisotropy values for the influence of variable amounts of rotationally mobile G-actin in solution. The type of tightly bound divalent cation modulated the torsional flexibility of F-actin polymerized in the presence of ATP; filaments with Mg2+ bound (rFA = 0.066) at the active site cleft were more flexible than those with Ca2+ bound (rFA = 0.083). Filaments prepared from G-actin in the presence of MgADP were more flexible (rFA = 0.051) than those polymerized with MgATP; the addition of exogenous inorganic phosphate or beryllium trifluoride to ADP filaments, however, decreased the filament flexibility (increased the anisotropy) to that seen in the presence of MgATP. While variations in KCl concentration from 0 to 150 mM did not modulate the torsional flexibility of the filament, the binding of phalloidin decreased the torsional flexibility of all filaments regardless of the type of cation or nucleotide bound at the active site. These results emphasize the dynamic malleability of the actin filament, the role of the cation-nucleotide complex in modulating the torsional flexibility, and suggest that the structural differences that have previously been seen in electron micrographs of actin filaments manifest themselves as differences in torsional flexibility of the filament.

Influence of lipid composition on pediocin PA-1 binding to phospholipid vesicles.

Pediocin PA-1 bound to anionic lipid vesicles with saturated or unsaturated fatty acid chains in a lipid concentration-dependent fashion. Little change in binding parameters was observed for zwitterionic lipid vesicles. Decreasing the anionic lipid content of the vesicles gave a higher relative dissociation constant for the peptide-lipid interactions and further supports the electrostatic interaction model of binding.

Analysis of the conformational stability of the active domain of recombinant mouse TIMP-1 by intrinsic fluorescence.

Intrinsic fluorescence was used to examine the stability of an active, N-terminal domain of mouse tissue inhibitor of metalloproteinase (TIMP-1) fused with an N-terminal polyhistidine tag. Emission and quenching studies suggested that the single tryptophan is on the protein surface partially exposed to solvent. The TIMP-1 recombinant unfolded reversibly in the presence of guanidinium chloride with the transition midpoint at 2.35M; extrapolation gave a stabilization free energy of 5.1 kcal mol-1 at 25 degrees C. Analysis of the temperature dependence of the fluorescence intensity gave a melting transition with midpoint at 51 degrees C and an enthalpy and heat capacity change on unfolding of 32 kcal mol-1 and 0.45 kcal K-1 mol-1, respectively, values comparable to other single domain proteins. Comparison with literature data indicated that the stability of mouse recombinant TIMP-1 more closely resembled that of human metalloproteinase inhibitor TIMP-2 than TIMP-1 despite closer homology to the human TIMP-1 protein.

Electrostatic interactions, but not the YGNGV consensus motif, govern the binding of pediocin PA-1 and its fragments to phospholipid vesicles.

The purpose of this study was to characterize in detail the binding of pediocin PA-1 and its fragments to target membranes by using tryptophan fluorescence as a probe. Based on a three-dimensional model (Y. Chen, R. Shapira, M. Eisenstein, and T. J. Montville, Appl. Environ. Microbiol. 63:524-531, 1997), four synthetic N-terminal pediocin fragments were selected to study the mechanism of the initial step by which the bacteriocin associates with membranes. Binding of pediocin PA-1 to vesicles of phosphatidylglycerol, the major component of Listeria membranes, caused an increase in the intrinsic tryptophan fluorescence intensity with a blue shift of the emission maximum. The Stern-Volmer constants for acrylamide quenching of the fluorescence of pediocin PA-1 in buffer and in the lipid vesicles were 8.83 +/- 0.42 and 3.53 +/- 0.67 M-1, respectively, suggesting that the tryptophan residues inserted into the hydrophobic core of the lipid bilayer. The synthetic pediocin fragments bound strongly to the lipid vesicles when a patch of positively charged amino acid residues (K-11 and H-12) was present but bound weakly when this patch was mutated out. Quantitative comparison of changes in tryptophan fluorescence parameters, as well as the dissociation constants for pediocin PA-1 and its fragments, revealed that the relative affinity to the lipid vesicles paralleled the net positive charge in the peptide. The relative affinity for the fragment containing the YGNGV consensus motif was 10-fold lower than that for the fragment containing the positive patch. Furthermore, changing the pH from 6.0 to 8.0 decreased binding of the fragments containing the positive patch, probably due to deprotonation of His residues. These results demonstrate that electrostatic interactions, but not the YGNGV motif, govern pediocin binding to the target membrane.

Physiochemical characterization of the nisin-membrane interaction with liposomes derived from Listeria monocytogenes.

Mechanistic information about the bacteriocin nisin was obtained by examining the efflux of 5(6)-carboxy-fluorescein from Listeria monocytogenes-derived liposomes. The initial leakage rate (percentage of efflux per minute) of the entrapped dye was dependent on both nisin and lipid concentrations. At all nisin concentrations tested, 5(6)-carboxyfluorescein efflux plateaued before all of the 5(6)-carboxyfluorescein was released (suggesting that pore formation was transient), but efflux resumed when more nisin was added. Isotherms for the binding of nisin to liposomes constructed on the basis of the Langmuir isotherm gave an apparent binding constant of 6.2 x 10(5)M(-1) at pH 6.0. The critical number of nisin molecules required to induce efflux from liposomes at pH 6.0 was approximately 7,000 molecules per liposome. The pH affected the 5(6)-carboxyfluorescein leakage rates, with higher pH values resulting in higher leakage rates. The increased leakage rate observed at higher pH values was not due to an increase in the binding affinity of the nisin molecules towards the liposomal membrane. Rather, the critical number of nisin molecules required to induce activity was decreased (approximately 1,000 nisin molecules per liposome at pH 7.0). These data are consistent with a poration mechanism in which the ionization state of histidine residues in nisin plays an important role in membrane permeabilization.

Microsecond rotational dynamics of F-actin in ActoS1 filaments during ATP hydrolysis.

Rabbit skeletal muscle F-actin labeled at Cys374 with the triplet probe erythrosin-5-iodoacetamide had a steady-state phosphorescence anisotropy (rp) of 0.090 +/- 0.005 at 20 degrees C in 100 mM KCl, pH 7.0, buffer. Titration with skeletal muscle S1 fragment increased rp to 0.138 +/- 0.006 at a mole ratio of 1:1. In the presence of ATP, the anisotropy of the actoS1 complex initially decreased to 0.050 +/- 0.005, a value significantly smaller than the anisotropy of pure F-actin; rp subsequently increased to 0.126 +/- 0.002. The time course of the increase matched that expected from the measured actin-activated ATPase of S1. The plateau value at long time, 0.126, was identical to that of actoS1 in the presence of exogenous ADP or ADP plus phosphate. Characterization of the spectroscopic properties of the erythrosin probe indicated that the changes in rp were not due to changes in fast probe motions on the surface of the filament or the phosphorescence emission lifetime, or in the orientation of the probe on the surface of F-actin, suggesting that they reflect large-scale changes in the microsecond rotational dynamics of actin. ATP hydrolysis by actoS1 thus appeared to induce rotational motions of or within F-actin on the phosphorescence time scale (approximately 300 microseconds). Although the precise physical origin of the induced rotational motions is unknown, this study provides direct evidence that large-scale conformational fluctuations of the actin filament are associated with the force-generating event in actomyosin.

Inhibition of yeast (1,3)-beta-glucan synthase by phospholipase A2 and its reaction products.

Fungal (1,3)-beta-glucan synthases are sensitive to a wide range of lipophilic inhibitors and it has been proposed that enzyme activity is highly sensitive to perturbations of the membrane environment. Yeast membranes were exposed to phospholipases and various lipophilic compounds, and the resultant effects on glucan synthase activity were ascertained. Glucan synthase from Saccharomyces cerevisiae was rapidly inactivated by phospholipase A2 (PLA2), and to a lesser extent by phospholipase C. Inactivation was time and dose-dependent and was protected against by EDTA and fatty-acid binding proteins (bovine and human serum albumins). Albumins also partially protected against inhibition by papulacandin B. PLA2 reaction products were structurally characterized and it was shown that fatty acids and lysophospholipids were the inhibitory moieties, with no novel inhibitory compounds apparent. Glucan synthase was inhibited by a range of fatty acids, monoglycerides and lysophospholipids. Inhibition by fatty acids was non-competitive, and progressive binding of [14C]oleic acid correlated with activity loss. Fluorescence anisotropy studies using diphenylhexatriene (DPH) confirm that fatty acids increase membrane fluidity. These results are consistent with proposals suggesting that glucan synthase inhibition is due in part to non-specific detergent-like disruption of the membrane environment, in addition to direct interactions of lipophilic inhibitors with specific target sites on the enzyme complex.

Use of cilofungin as direct fluorescent probe for monitoring antifungal drug-membrane interaction.

Cilofungin is an antifungal cyclopeptide which inhibits cell wall (1,3)-beta-glucan biosynthesis in fungal organisms, and its action against Candida albicans (1,3)-beta-glucan synthase has been widely studied. Since glucan synthase inactivation is thought to partially result from perturbations of the membrane lipid environment, the interaction of cilofungin with fungal membranes and phosphatidylcholine membrane vesicles was studied. Cilofungin, which contains two independent aromatic groups, has an excitation maximum of 270 nm and an emission maximum of 317 nm in aqueous solution. Comparison of the fluorescence properties of cilofungin with those of the analogs pneumocandin B0, N-acetyl-tyrosinamide, and 4-hydroxybenzamide indicated that the emission of cilofungin largely derived from the p-octyloxybenzamide side chain. Microsomal membranes from Saccharomyces cerevisiae, C. albicans, and phosphatidylcholine membrane vesicles induced a blue shift in the cilofungin emission spectrum and increased the cilofungin steady-state emission anisotropy, providing direct evidence for a cilofungin-membrane interaction. Cilofungin interacted more strongly with membranes of C. albicans than with those of S. cerevisiae, correlating with previous findings that C. albicans is far more susceptible than S. cerevisiae to the action of cilofungin. These findings support the hypothesis that drug-induced inhibition of the (1,3)-beta-glucan synthesis results from the perturbation of the membrane environment and the interaction with the glucan synthase complex combined. The study demonstrated ways in which the fluorescence properties of drugs can be used to directly evaluate drug-membrane interactions and structure-activity relationships.

Local conformation of rabbit skeletal myosin rod filaments probed by intrinsic tryptophan fluorescence.

Rabbit skeletal myosin rod contains two tryptophan residues per chain (four per coiled-coil) that are located about 50 and 175 A from the N-terminus of the light meromyosin (LMM) region of rod. We have characterized the local polarity, excited-state photophysics, solvent accessibility, and rotational dynamics of these tryptophans in myosin rod filaments at 125 mM KCl using steady-state and time-resolved fluorescence techniques. The fluorescence decays were described using a complex bimodal distribution with a discrete long-lifetime component of 5.44 ns (amplitude of 0.51) and a Gaussian distribution of short lifetimes with mean of 0.105 ns and width of 2.15 ns (amplitude 0.49). The discrete long-lifetime species was efficiently quenched by the neutral quencher acrylamide with a bimolecular collision constant (kq) of 0.85 x 10(9) M-1 s-1. The emission spectrum, lifetime distribution, and quenching behavior of the tryptophans in myosin rod monomers (at 0.5 M KCl) were quite similar. Time-resolved anisotropy decays of the rod monomers and filaments exhibited nearly identical double-exponential decays to a constant. Each had a fast, subnanosecond component (amplitude 0.07), probably corresponding to fast wobble of the tryptophans on the coiled-coil surface, a slower, approximately 6-ns component (amplitude approximately 0.04), corresponding to an unidentified internal, segmental mode of motion of the coiled-coil, and a constant (r infinity) of 0.15.(ABSTRACT TRUNCATED AT 250 WORDS)

Tryptophan photophysics in rabbit skeletal myosin rod.

The fibrous region of myosin (myosin rod) is an alpha-helical, two-stranded coiled-coil made up of identical hydrophobic d sites in the heptad repeat that forms the basis for hydrophobic dimerization. The fluorescence excitation and emission spectra of rod in high salt buffer (where the rod exists as a coiled-coil monomer) at 20 degrees C are red- and blue-shifted, respectively, from the comparable spectra of N-acetyl-tryptophanamide or L-tryptophan. These spectral shifts, as well as red-shifts in the emission spectra induced by excitation on the red edge of the absorption or by increases in temperature, indicate that (on average) the tryptophans are partially exposed to aqueous solvent yet in contact with the protein matrix. The tryptophan intensity decays show an unusual bimodal distribution; the major species has a discrete lifetime of about 5.2 ns while the minor species exhibits a complex decay with a broad (3.4 ns full width at half maximum) Gaussian distribution of lifetimes centered around 1.3 ns. The long lifetime species has a blue-shifted excitation and red-shifted emission characteristic of the indole chromophore in a polar (probably aqueous) environment while the short lifetime species has the spectral parameters characteristic of indole in a non-polar environment. Although assignment of these lifetime species to particular tryptophans in the rod is problematic, this study indicates that the coiled-coil interface presents a complex heterogeneous environment that may undergo rapid conformational mobility.

Characterization of skeletal muscle actin labeled with the triplet probe erythrosin-5-iodoacetamide.

We have labeled rabbit skeletal muscle actin with the triplet probe erythrosin-5-iodoacetamide and characterized the labeled protein. Labeling decreased the critical concentration and lowered the intrinsic viscosity of F-actin filaments; labeled filaments were motile in an in vitro motility assay but were less effective than unlabeled F-actin in activating myosin S1 ATPase activity. In unpolymerized globular actin (G-actin), both the prompt and delayed luminescence were red-shifted from the spectra of the free dye in solution and the fluorescence anisotropy of the label was high (0.356); filament formation red shifted all excitation and emission spectra and increased the fluorescence anisotropy to 0.370. The erythrosin phosphorescence decay was at least biexponential in G-actin with an average lifetime of 99 microseconds while in F-actin the decay was approximately monoexponential with a lifetime of 278 microseconds. These results suggest that the erythrosin dye was bound at the interface between two actin monomers along the two-start helix. The steady-state phosphorescence anisotropy of F-actin was 0.087 at 20 degrees C and the anisotropy increased to approximately 0.16 in immobilized filaments. The phosphorescence anisotropy was also sensitive to binding the physiological ligands phalloidin, cytochalasin B and tropomyosin. This study lays a firm foundation for the use of this triplet probe to study the large-scale molecular dynamics of F-actin.

Tryptophan fluorescence quenching in rabbit skeletal myosin rod.

The solvent accessibility of the four tryptophans of rabbit skeletal muscle myosin rod was investigated using steady-state and time-resolved fluorescence quenching by iodide, acrylamide, and cesium. The quenching by iodide and acrylamide was biphasic; the discrete, long lifetime component was quenched with bimolecular collision constants (kq) of 1 x 10(9) M-1 s-1 and 1.6 x 10(9) M-1 s-1, respectively, while the Gaussian distributed, short lifetime component was quenched with a kq value of 0.3 x 10(9) M-1 s-1 and 0.04 x 10(9) M-1 s-1, respectively. Comparison with kq values for N-acetyl-tryptophanamide indicated that the fractional solvent accessibility was about 25% for the long and less than 10% for the short lifetime component. Cesium quenching was monophasic and provided evidence of an excess of positive charge around these tryptophans. Our findings cast doubt on the general application of the simple coiled-coil model to describe coiled-coil interactions in this protein in solution.

Influence of hydration on the internal dynamics of hen egg white lysozyme in the dry state.

Proteins exist in a predominantly aqueous solvent environment. Hydration of the protein surface significantly affects many aspects of the protein's structure and function; these effects may be related to the molecular dynamics of the protein. We have examined the influence of hydration on the internal dynamics of hen egg white lysozyme using room-temperature phosphorescence from the intrinsic tryptophan residues. Powders of lyophilized lysozyme were hydrated in a phosphorimeter using a flow system that allowed for continuous manipulation of relative humidity over the range 0-92%; this system allowed us to directly compare intensity differences that result from changes in hydration. Lysozyme phosphorescence intensity decreased as a function of hydration over the entire relative humidity range; the decrease was not linear but appeared to occur in distinct phases. The phosphorescence intensity decays were multiexponential over the hydration range studied, and hydration had the largest influence on the long lifetime component. These data suggest that the protein exists in multiple, static conformations in the dry state and that water binding to polar (as opposed to charged) sites on the protein surface induces local and/or global softening of the protein structure.

N-(iodoacetyl)-p-phenylenediamine-EDTA: a reagent for high-efficiency incorporation of an EDTA-metal complex at a rationally selected site within a protein.

We have developed a highly efficient procedure to incorporate an EDTA:metal complex at a rationally selected site within a full-length protein. Our procedure has two steps: In step one, we use site-directed mutagenesis to introduce a unique solvent-accessible cysteine residue at the site of interest. In step two, we derivatized the resulting protein with N-(iodoacetyl)-p-phenylenediamine-EDTA:metal, a novel haloacetyl derivative of EDTA:metal. We have used this procedure to incorporate each of three EDTA:metal complexes at amino acid 2 of the helix-turn-helix motif of the sequence-specific DNA binding protein Cro: a radioactive and nucleolytic EDTA:metal complex (EDTA:55Fe), a radioactive EDTA:metal complex (EDTA:63Ni), and a fluorescent and heavy-atom EDTA:metal complex (EDTA:Eu). Incorporation of EDTA:metal was highly efficient (> 80% for EDTA:55Fe and EDTA:63Ni; 60% for EDTA:Eu) and highly site-specific (> 99%). We have analyzed DNA affinity cleaving by the Cro derivative having EDTA:55Fe at amino acid 2 of the helix-turn-helix motif. The Cro derivative cleaves DNA at base pairs -4 to 6 of the DNA half site in the protein-DNA complex, indicating that amino acid 2 of the helix-turn-helix motif of Cro is close to base pairs -4 to 6 of the DNA half site in the Cro-DNA complex in solution.(ABSTRACT TRUNCATED AT 250 WORDS)

Time-resolved rotational dynamics of phosphorescent-labeled myosin heads in contracting muscle fibers.

We have measured the microsecond rotational motions of myosin heads in contracting rabbit psoas muscle fibers by detecting the transient phosphorescence anisotropy of eosin-5-maleimide attached specifically to the myosin head. Experiments were performed on small bundles (10-20 fibers) of glycerinated rabbit psoas muscle fibers at 4 degrees C. The isometric tension and physiological ATPase activity of activated fibers were unaffected by labeling 60-80% of the heads. Following excitation of the probes by a 10-ns laser pulse polarized parallel to the fiber axis, the time-resolved emission anisotropy of muscle fibers in rigor (no ATP) showed no decay from 1 microsecond to 1 ms (r infinity = 0.095), indicating that all heads are rigidly attached to actin on this time scale. In relaxation (5 mM MgATP but no Ca2+), the anisotropy decayed substantially over the microsecond time range, from an initial anisotropy (r0) of 0.066 to a final anisotropy (r infinity) of 0.034, indicating large-amplitude rotational motions with correlation times of about 10 and 150 microseconds and an overall angular range of 40-50 degrees. In isometric contraction (MgATP plus saturating Ca2+), the amplitude of the anisotropy decay (and thus the amplitude of the microsecond motion) is slightly less than in relaxation, and the rotational correlation times are about twice as long, indicating slower motions than those observed in relaxation. While the residual anisotropy (at 1 ms) in contraction is much closer to that in relaxation than in rigor, the initial anisotropy (at 1 microsecond) is approximately equidistant between those of rigor and relaxation.(ABSTRACT TRUNCATED AT 250 WORDS)

Rotational dynamics of the single tryptophan of porcine pancreatic phospholipase A2, its zymogen, and an enzyme/micelle complex. A steady-state and time-resolved anisotropy study.

The rotational dynamics of the single tryptophan of porcine pancreatic phospholipase A2 and its zymogen (prophospholipase A2) have been studied by polarized fluorescence using steady-state and time-resolved single-photon counting techniques. The motion of Trp-3 in phospholipase A2 consists of a rapid subnanosecond wobble of the indole ring with an amplitude of about +/- 20 degrees accompanied by slower isotropic rotation of the entire protein. The rotational correlation times for overall particle rotational diffusion are consistent with conventional hydrodynamic theory. When phospholipase A2 binds to micelles of n-hexadecylphosphocholine, the amplitude of the fast ring rotation decreases. The whole particle rotational correlation time of the enzyme/micelle complex is smaller than the minimum value calculated from hydrodynamic theory. A similar result is obtained for the micelle itself by using the lipophilic probe transparinaric acid. These low values for the particle correlation times can be understood by postulating that an isotropic motion of the fluorophore in the small detergent particles contributes to the angular reorientation of the fluorophore. The internal reorientational motion of the tryptophan in the zymogen, prophospholipase A2, is of larger amplitude than that observed for the enzyme; specifically, the proenzyme exhibits a motion with a significant amplitude on the nanosecond time scale. This additional freedom of motion is attributed to segmental mobility of the N-terminal residues of prophospholipase A2. This demonstrates that this region of the protein is flexible in the zymogen but not in the processed enzyme. The implications of these findings for the mechanism of surface activation of phospholipase A2 are discussed by analogy with a trypsinogen-trypsin activation model.