Katalin Ajtai

Fluorescence emission and anisotropy from rhodamine dimers

The absorption, fluorescence emission, and excitation fluorescence anisotropy spectra of a rhodamine dye in a water-glycerol solution at high concentration were investigated to determine spectroscopic properties of the ground state dimer. The combination of data from these spectra measured at several dye concentrations contained sufficient constraints on the model for dimer association to permit an estimate of: the association constant, the extinction coefficients, the relative fluorescence quantum yield, and the emission spectra of the monomeric and dimeric species. The rhodamine dimer is an efficient fluorescence emitter with fluorescence anisotropy equivalent to that of the pure monomeric species over the range of excitation wavelengths covering its two lowest energy transitions.

Inhibition of myosin ATPase by metal fluoride complexes

Magnesium (Mg2+) is the physiological divalent cation stabilizing nucleotide or nucleotide analog in the active site of myosin subfragment 1 (S1). In the presence of fluoride, Mg2+ and MgADP form a complex that traps the active site of S1 and inhibits myosin ATPase. The ATPase inactivation rate of the magnesium trapped S1 is comparable but smaller than the other known gamma-phosphate analogs at 1.2 M-1 s-1 with 1 mM MgCl2. The observed molar ratio of Mg/S1 in this complex of 1.58 suggests that magnesium occupies the gamma-phosphate position in the ATP binding site of S1 (S1-MgADP-MgFx). The stability of S1-MgADP-MgFx at 4 degrees C was studied by EDTA chase experiments but decomposition was not observed. However, removal of excess fluoride causes full recovery of the K+-EDTA ATPase activity indicating that free fluoride is necessary for maintaining a stable trap and suggesting that the magnesium fluoride complex is bonded to the bridging oxygen of beta-phosphate more loosely than the other known phosphate analogs. The structure of S1 in S1-MgADP-MgFx was studied with near ultraviolet circular dichroism, total tryptophan fluorescence, and tryptophan residue 510 quenching measurements. These data suggest that S1-MgADP-MgFx resembles the M**.ADP.Pi steady-state intermediate of myosin ATPase. Gallium fluoride was found to compete with MgFx for the gamma-phosphate site in S1-MgADP-MgFx. The ionic radius and coordination geometry of magnesium, gallium and other known gamma-phosphate analogs were compared and identified as important in determining which myosin ATPase intermediate the analog mimics.

Ventricular myosin modifies in vitro step-size when phosphorylated

Cardiac and skeletal muscle myosins have the central role in contraction transducing ATP free energy into the mechanical work of moving actin. Myosin has a motor domain containing ATP and actin binding sites and a lever-arm that undergoes rotation impelling bound actin. The lever-arm converts torque generated in the motor into the linear displacement known as step-size. The myosin lever-arm is stabilized by bound essential and regulatory light chains (ELC and RLC). RLC phosphorylation at S15 is linked to modified lever-arm mechanical characteristics contributing to myosin filament based contraction regulation and to the response of the muscle to disease. Myosin step-size was measured using a novel quantum dot (Qdot) assay that previously confirmed a 5nm step-size for fast skeletal myosin and multiple unitary steps, most frequently 5 and 8nm, and a rare 3nm displacement for β cardiac myosin (βMys). S15 phosphorylation in βMys is now shown to change step-size distribution by advancing the 8nm step frequency. After phosphorylation, the 8nm step is the dominant myosin step-size resulting in significant gain in the average step-size. An increase in myosin step-size will increase the amount of work produced per ATPase cycle. The results indicate that RLC phosphorylation modulates work production per ATPase cycle suggesting the mechanism for contraction regulation by the myosin filament.

Effect of Ionic Strength on the Conformation of Myosin Subfragment1–Nucleotide Complexes

The effect of ionic strength on the conformation and stability of S1 and S1-nucleotide-phosphate analog complexes in solution was studied. It was found that increasing concentration of KCl enhances the reactivity of Cys(707) (SH1 thiol) and Lys(84) (reactive lysyl residue) and the nucleotide-induced tryptophan fluorescence increment. In contrast, high KCl concentration lowers the structural differences between the intermediate states of ATP hydrolysis in the vicinity of Cys(707), Trp(510) and the active site, possibly by increasing the flexibility of the molecule. High concentrations of neutral salts inhibit both the formation and the dissociation of the M**.ADP.Pi analog S1.ADP.Vi complex. High ionic strength profoundly affects the structure of the stable S1.ADP.BeF(x) complex, by destabilizing the M*.ATP intermediate, which is the predominant form of the complex at low ionic strength, and shifting the equilibrium to favor the M**.ADP.Pi state. The M*.ATP intermediate is destabilized by perturbation of ionic interactions possibly by disruption of salt bridges. Two salt-bridge pairs, Glu(501)-Lys(505) in the Switch II helix and Glu(776)-Lys(84) connecting the catalytic domain to the lever arm, seem most appropriate to consider for participating in the ionic strength-induced transition of the open M*.ATP to the closed M**.ADP.Pi state of S1.

Cleft containing reactive thiol of myosin closes during ATP hydrolysis

The probe binding cleft of myosin subfragment 1 (S1) contains the reactive thiol, SH1, and tryptophan 510 (Trp-510). Solvent accessibility to Trp-510, measured using the acrylamide quenching of its fluorescence, is highest in rigor and decreases during the ATPase cycle prior to force generation. These data suggest the probe binding cleft closes during ATP hydrolysis and opens during force generation. The closing of the probe binding cleft may be the origin of the shape change of S1 during ATP hydrolysis.

Myosin cross-bridge orientation in rigor and in the presence of nucleotide studied by electron spin resonance.

The tilt series electron spin resonance (ESR) spectrum from muscle fibers decorated with spin labeled myosin subfragment 1 (S1) was measured from fibers in rigor and in the presence of MgADP. ESR spectra were measured at low amplitude modulation of the static magnetic field to insure that a minimum of spectral lineshape distortion occurs. Ten tilt series ESR data sets were fitted simultaneously by the model-independent methodology described in the accompanying paper (Burghardt, T. P., and A. R. French, 1989. Biophys. J. 56:525-534). By this method the average and standard error in the mean of order parameters for the probe angular distribution were calculated for the two states of the fiber investigated. The average order parameters were used to reconstruct the probe angular distribution in two dimensions, one angular dimension corresponding to a polar angle measured relative to the fiber axis, and the other a torsional angular degree of freedom of the probe. We find that the probe angular distributions for the rigor and MgADP states of the fiber differ such that the rigor distribution is broader and shifted relative to the distribution in the presence of MgADP. The shape of the rigor distribution suggests the presence of two probe orientations, one similar to that in the presence of MgADP, and another at a different orientation. The shape of the distribution in the presence of MgADP suggests that the binding of the nucleotide to the rigor cross-bridge shifts the spin population into a more homogeneous one by causing a cross-bridge rotation.

Chemical Decoupling of ATPase Activation and Force Production from the Contractile Cycle in Myosin by Steric Hindrance of Lever-Arm Movement

The myosin motor protein generates force in muscle by hydrolyzing Adenosine 5-triphosphate (ATP) while interacting transiently with actin. Structural evidence suggests the myosin globular head (subfragment 1 or S1) is articulated with semi-rigid catalytic and lever-arm domains joined by a flexible converter domain. According to the prevailing hypothesis for energy transduction, ATP binding and hydrolysis in the catalytic domain drives the relative movement of the lever arm. Actin binding and reversal of the lever-arm movement (power stroke) applies force to actin. These domains interface at the reactive lysine, Lys84, where trinitrophenylation (TNP-Lys84-S1) was observed in this work to block actin activation of myosin ATPase and in vitro sliding of actin over myosin. TNP-Lys84-S1s properties and interactions with actin were examined to determine how trinitrophenylation causes these effects. Weak and strong actin binding, the rate of mantADP release from actomyosin, and actomyosin dissociation by ATP were equivalent in TNP-Lys84-S1 and native S1. Molecular dynamics calculations indicate that lever-arm movement inhibition during ATP hydrolysis and the power stroke is caused by steric clashes between TNP and the converter or lever-arm domains. Together these findings suggest that TNP uncouples actin activation of myosin ATPase and the power stroke from other steps in the contraction cycle by inhibiting the converter and lever-arm domain movements.

Myosin head rotation in muscle fibers measured using polarized fluorescence photobleaching recovery.

The technique of polarized fluorescence photobleaching recovery (PFPR) has been applied for the first time to investigation of the rotational correlation time of the myosin head in muscle fibers. This is a novel application of PFPR because it is the first time PFPR has been applied to a sample which is not cylindrically symmetric about the optical axis. Therefore we present a method for analysis of PFPR results from an oriented sample such as the muscle fibers aligned perpendicularly to the optical axis used here. Control experiments performed on fluorescently labeled myosin heads in solution demonstrate that, under some conditions, our PFPR apparatus can easily measure a rotational correlation time of less than 200 μs. Validity of this application of PFPR to muscle fibers is provided by the agreement of our results with published results from a variety of other spectroscopic techniques. In particular, using glycerinated rabbit psoas muscle fibers, we find that for relaxed fibers and isometrically contracting fibers, the myosin heads undergo high-amplitude rotations on the submillisecond time domain. For fibers in rigor the myosin heads are highly oriented and nearly immobile. For fibers in ADP the myosin heads are highly ordered in a distribution quite different from that in rigor, and they are slightly more mobile than in rigor.

Optical activity of a nucleotide-sensitive tryptophan in myosin subfragment 1 during ATP hydrolysis

The xanthene probes 5-iodoacetamido-fluorescein and -tetramethylrhodamine specifically modify skeletal muscle myosin subfragment 1 (S1) at the reactive thiol residue (SH1) and fully quench the fluorescence emission from tryptophan residue 510 (Trp510) in S1 (T.P. Burghardt and K. Ajtai, Biophys. Chem., 60 (1996) 119; K. Ajtai and T.P. Burghardt, Biochemistry, 34 (1995) 15943). The difference between the fluorescence intensity obtained from S1 and probe-modified S1 comes solely from Trp510 in chymotryptic S1, a protein fragment that contains five tryptophan residues. The rotary strength and quantum efficiency of Trp510 were measured using difference signals from fluorescence detected circular dichroism (FDCD) and fluorescence emission spectroscopy. These structure-sensitive signals indicate that the binding of nucleotide or nucleotide analogs to the active site of S1 causes structural changes in S1 at Trp510 and that a one-to-one correspondence exists between Trp510 conformation and transient states of myosin during contraction. The Trp510 rotary strength and quantum efficiency were interpreted structurally in terms of the indole side-chain conformation using model structures and established computational methods.

The conformation of xanthene dyes in the myosin sulfhydryl one binding site Part I. Methods and model systems

Derivatives of the fluorescent probes fluorescein and rhodamine specifically and covalently modify the highly reactive thiol (SH1) of myosin subfragment 1 (S1). Both probes develop circular dichroism (CD) upon modification of SH1 at the visible absorption band of the chromophore. A model system of chiral complexing agents (aromatic chiral amines) interacting with fluorescein in solvent develops a CD signal that mimics that produced by S1. The model system suggests that a specific interaction of the probe with an aromatic chiral residue in the SH1 binding pocket induces the CD signal. Several other spectroscopic signals, including absorption and fluorescence intensity and anisotropy, characterize the fluorescein or rhodamine binding to SH1. A coupled dipole method is adapted to interpret these spectroscopic signals in terms of the probe-S1 complex conformation. The computation of the orientation of the principal hydrodynamic frame (PHF) of S1 from its crystallographic alpha-carbon backbone structure permits the known orientation of the probe in the PHF of S1 to further constrain the conformation of the probe-S1 complex. The coupled dipole interpretation of spectroscopic data combined with constraints relating the probe dipole orientation to the PHF of S1 determines the conformation of the probe-S1 complex. The methods developed here are applied to the spectroscopic signals from fluorescein or rhodamine in the SH1 binding site of S1 to obtain an atomic resolution model of the probe-S1 conformation [Ajtai and Burghardt, Biochemistry, 34 (1995) 15943-15952].