Structural characterization of beta-cardiac myosin subfragment 1 in solution.

beta-cardiac myosin subfragment 1 (betaS1) tertiary structure and dynamics were characterized with proteolytic digestion, nucleotide analogue trapping kinetics, and intrinsic fluorescence changes accompanying nucleotide binding. Proteolysis of betaS1 produces the 25, 50, and 20 kDa fragments and a new cut within the 50-kDa fragment at Arg369. F-actin inhibits cleavage of the 50-kDa fragment and fails to inhibit cleavage at the 50/20 kDa junction, suggesting betaS1 presents an actoS1 conformation fundamentally different from skeletal S1. Time-dependent changes in Mg(2+)-ATPase accompanying proteolysis identifies cleavage points that lie within the energy transduction pathway. The nucleotide analogue trapping kinetics reveal the presence of a reversible weakly actin attached state. Comparison of nucleotide analogue induced betaS1 structures with the transient structures occurring during ATPase indicates analogue induced and transient structures are in a one-to-one correspondence. Tryptophan fluorescence enhancement accompanies the binding or trapping of nucleotide or nucleotide analogues. Isolation of Trp508 fluorescence shows it is an ATP-sensitive tryptophan and that its vicinity changes conformation sequentially with the transient intermediates accompanying ATPase. These studies elucidate energy transduction and suggest how mutations of betaS1 implicated in disease might undermine function, stability, or efficiency.

Effect of ionic strength on the conformation of myosin subfragment 1-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.

Conformation of myosin interdomain interactions during contraction: deductions from muscle fibers using polarized fluorescence.

Myosin cross-bridge subfragment 1 (S1) is the ATP catalyzing motor protein in muscle. It consists of three domains that catalyze ATP and bind actin (catalytic), conduct energy transduction (converter), and transport the load (lever arm). Force development during contraction is thought to result from rotary lever arm movement with the cross-bridge attached to actin. To elucidate cross-bridge structure during force development, two crystal structures of S1 were extrapolated to working "in solution" or oriented "in tissue" forms, using structure-sensitive optical spectroscopic signals from two extrinsic probes. The probes were located at two interfaces containing the catalytic, converter, and lever arm domains of S1. Observed signals included circular dichroism (CD) and absorption originating from S1 in solution in the presence and absence of actin and fluorescence polarization from cross-bridges in muscle fibers. Theoretical signals were calculated from S1 crystal structure models perturbed with lever arm movement from swiveling at three conserved glycines, 699, 703, and 710 (chicken skeletal myosin numbering). Best agreement between the computed and observed signals gave structures showing that actin binding to S1 causes movement of the lever arm. A three-state model of S1 conformation during contraction consists of three actin-bound cross-bridge states observed from muscle fibers in isometric contraction, in the presence of MgADP, and in rigor. Structures best representing these states show that most of the lever arm rotation occurs between isometric contraction and the MgADP states, i.e., during phosphate release. Smaller but significant lever arm rotation occurs with ADP dissociation. Structural changes within the S1 interfaces studied are discussed in the accompanying paper [Burghardt et al. (2001) Biochemistry 40, 4834-4843].

Conformation of myosin interdomain interactions during contraction: deductions from proteins in solution.

Myosin subfragment 1 (S1) is the ATP catalyzing motor protein in muscle. It consists of three domains that catalyze ATP and bind actin (catalytic), conduct energy transduction (converter), and transport the load (lever arm). These domains interface in two places identified as interface I, containing the reactive thiol (SH1) and ATP-sensitive tryptophan (Trp510), and interface II, containing the reactive lysine residue (RLR). Two crystal structures of S1 were extrapolated to working "in solution" or oriented "in tissue" forms, using structure-sensitive optical spectroscopic signals from extrinsic probes located in the interfaces. Observed signals included circular dichroism (CD) and absorption originating from S1 in solution in the presence and absence of actin and fluorescence polarization from cross-bridges in muscle fibers. Theoretical signals were calculated from S1 crystal structure models perturbed with lever arm movement from swiveling at three conserved glycines, 699, 703, and 710 (chicken skeletal myosin numbering). Structures giving the best agreement between the computed and observed signals were selected as the representative forms. Both interfaces undergo dramatic conformational change during ATPase and force development. Changes at interface I suggest the molecular basis for the collisional quenching sensitivity of Trp510 to nucleotide binding. The probe conformation at SH1 suggests how it alters S1 ATPases. At interface II, the spatial relationship of the lever arm and the extrinsic probe at RLR suggests how the probe alters S1 ATPases and that it should inhibit lever arm movement during the power stroke. The latter possibility, if true, establishes a part of the corridor through which the lever arm swings during the power stroke. Global structural changes in actomyosin are discussed in the accompanying paper [Burghardt et al. (2001) Biochemistry 40, 4821-4833].

Trinitrophenylated reactive lysine residue in myosin detects lever arm movement during the consecutive steps of ATP hydrolysis.

Trinitrophenylation of the reactive lysine (Lys84) in skeletal myosin subfragment 1 (S1) introduces a chiral probe (TNP) into an interface of the catalytic and lever arm domains of S1 [Muhlrad (1977) Biochim. Biophys. Acta 493, 154-166]. Characteristics of the TNP absorption and circular dichroism (CD) spectra in TNP-modified S1 (TNP-Lys84-S1), and the Lys84 trinitrophenylation rate in native S1, indicate a one-to-one correspondence between ATPase transients and trapped phosphate analogues. Phosphate analogue-induced structures of TNP-Lys84-S1 were modeled using the crystallographic coordinates of S1 [Rayment et al. (1993) Science 261, 50-58] with swivels at Gly699 and Gly710 to approximate conformational changes during ATPase. The CD and absorption spectral characteristics of the model structures were compared to those observed for analogue-induced structures. The model calculations, first tested on a trinitrophenylated hexapeptide with known structure, were applied to TNP-Lys84-S1. They showed that ATP binding initiates swiveling at Gly699 and that swiveling at both Gly710 and Gly699 accompanied ATP splitting just prior to product release. The computed lever arm trajectory during ATPase suggests (i) a plausible mechanism for the nucleotide-induced inhibition of Lys84 trinitrophenylation, and (ii) trinitrophenylation-induced changes in S1 Mg2+- and K+-EDTA ATPase are from collision of the lever arm with TNP at Lys84. TNP is a site-specific structural perturbant of S1 and a chiral reporter group for the effect of Lys84 modification on dynamic S1 structure. As such, TNP-Lys84-S1 is equivalent to a genetically engineered mutant with intrinsic sensitivity to structure local to the modified residue.

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.

ATP sensitive tryptophans of hsp90.

The nature of the interaction between the nucleotide ATP and hsp90 was investigated by observing fluorescence quenching of the four tryptophan residues in hsp90 as a function of quencher type and temperature. ATP and acrylamide quench the fluorescence from tryptophan free in solution principally by static and collisional mechanisms, respectively. Acrylamide quenching of tryptophan fluorescence in hsp90 is also principally collisional and identifies two classes of residues, one readily accessible to quenching the other less accessible. ATP quenching of tryptophan fluorescence in hsp90 is more complex exhibiting no overall preferred mechanism. However, ATP competitively inhibits acrylamide quenching of the readily accessible class of tryptophan residues by static quenching with the quenching constant providing an upper limit for the ATP dissociation constant. The ATP-free tryptophan dissociation constant is more than a factor of three larger than that for ATP-hsp90 suggesting that the ATP-hsp90 interaction is specific. The static quenching of tryptophan fluorescence in hsp90 by ATP implies that the nucleotide binds in close proximity to one or more of the tryptophan residues.

Near UV circular dichroism from biomimetic model compounds define the coordination geometry of vanadate centers in MeVi- and MeADPVi-rabbit myosin subfragment 1 complexes in solution.

The circular dichroism (CD) spectrum was measured from vanadate (Vi) cyclic esters of chiral vicinal diols, hydroxycarboxylates, and cyclodextrines as a function of Vi concentration ([Vi]) and at the lowest energy transitions of the vanadium. At low [Vi] and in the presence of excess vicinal diols, hydroxycarboxylates, or cyclodextrines the CD signal intensity scales linearly with [Vi] indicating the predominance of a monomeric cyclic ester. At higher [Vi], the signal intensity in the presence of the vicinal diols and hydroxycarboxylates become nonlinear in [Vi], indicating formation of a dimeric cyclic ester. Vanadium-51 NMR (51V-NMR) indicates the coordination geometry of several of these model Vi centers in solution and identifies the CD signals characteristic to Vi trigonal bipyramidal (tbp) and octahedral (Oh) coordination geometries from monomeric and dimeric species. The CD spectra from monomeric and dimeric forms of the tbp-coordinated model compounds have two apparent transitions with amplitudes of opposite sign at wavelengths > or = 240 nm. Spectra from the monomeric and dimeric Oh coordinated species are distinct from the tbp-type spectra over the same wavelength domain because of the presence of two additional transitions with opposite sign amplitudes. These model spectra were compared to the vanadate CD spectra from Vi bound to rabbit myosin subfragment 1 (S1) in solution, in the presence of divalent metal cations (MeVi-S1) or trapped with MeADP (MeADPVi-S1). Polymeric MeVi binds to the active site of S1 and the vanadate centers in MnVi-S1 or CoVi-S1 produce a CD signal resembling that from the tbp model. The trapped ATPase transition state analog MeADPVi produces a different CD signal resembling that from the Oh model.

Tertiary structural changes in the cleft containing the ATP sensitive tryptophan and reactive thiol are consistent with pivoting of the myosin heavy chain at Gly699.

The conformation of myosin subfragment 1 (S1) in the vicinity of the ATP sensitive tryptophan (Trp510) and the highly reactive thiol (SH1), both residing in the "probe-binding" cleft at the junction of the catalytic and lever arm domains, was studied to ascertain its role in the mechanism of energy transduction and force generation. In glycerinated muscle fibers in rigor, a fluorescent probe linked to SH1 detects a strained probe-binding cleft conformation following a length transient by altering emission intensity without detectably rotating. In myosin S1 in solution, the optical activity of Trp510 senses conformation change in the probe-binding cleft caused by substrate analog trapping of S1 in various structures attainable transiently during normal energy transduction. Also in S1 in solution, the induced optical activity of a fluorescein probe linked to SH1 shows sensitivity to changing probe-binding cleft conformation caused by nucleotide binding to the S1 active site. The changes in the optical activity of Trp510 and SH1 bound fluorescein in response to nucleotide or nucleotide analog binding are interpreted structurally using the S1 crystallographic coordinates and aided by a model of energy transduction that pivots at Gly699 to change probe-binding cleft conformation and to displace the S1 lever arm as during force generation. The crystallographic structure of the probe-binding cleft in S1 resembles most the nucleotide bound conformation in the native protein. A different structure, generated by pivoting at Gly699, better resembles the native rigor conformation of the probe-binding cleft. Pivoting at Gly699 rotates probes at SH1 suggesting that length transients on fibers in rigor do not cause pivoting at Gly699 or reverse the power stroke.

Probes bound to myosin Cys-707 rotate during length transients in contraction.

It is widely conjectured that muscle shortens because portions of myosin molecules (the "cross-bridges") impel the actin filament to which they transiently attach and that the impulses result from rotation of the cross-bridges. Crystallography indicates that a cross-bridge is articulated-consisting of a globular catalytic/actin-binding domain and a long lever arm that may rotate. Conveniently, a rhodamine probe with detectable attitude can be attached between the globular domain and the lever arm, enabling the observer to tell whether the anchoring region rotates. Well-established signature effects observed in shortening are tension changes resulting from the sudden release or quick stretch of active muscle fibers. In this investigation we found that closely correlated with such tension changes are changes in the attitude of the rhodamine probes. This correlation strongly supports the conjecture about how shortening is achieved.

Effect of metal cations on the conformation of myosin subfragment-1-ADP-phosphate analog complexes: a near-UV circular dichroism study.

The interaction of myosin with actin, coupled with hydrolysis of ATP, is the molecular basis of muscle contraction. The head segment of myosin, called S1, contains the distinct binding sites for ATP and actin and is responsible for the ATPase activity. The myosin-catalyzed ATP hydrolysis consists of several intermediate steps and each step is accompanied by conformational changes in the S1 segment. The rate-limiting step of the ATP hydrolysis is the dissociation of the S1 x ADP x Pi complex which is accelerated by actin. The substitution of Pi with phosphate analogs (PA), such as vanadate, beryllium fluoride (BeFx) or aluminum fluoride (AlF4-), yields stable complexes which mimic the intermediates of the ATP hydrolysis. In this work, tertiary structure changes in S1 in the vicinity of aromatic residues was studied by comparing near-UV circular dichroism (CD) spectra from S1-nucleotide-phosphate analog complexes in the presence of Mg2+ and other cations. A significant difference between the MgATP and MgADP spectra indicated notable tertiary structural changes accompanying the M**ADP x Pi --> M*ADP transition. The spectra of the S1 x MgADP x BeFx and S1 x MgADP x AlF4- complexes resemble to those obtained upon addition of MgATPgammaS and MgATP to S1, and correspond to the M* x ATP and M** x ADP x Pi intermediates, respectively. We have found recently that the presence of divalent metal cations (Me2+) is essential for the formation of stable S1 x MeADP x PA complexes. Moreover, the nature of the metal cations strongly influences the stability of these complexes [Peyser, Y. M., et al. (1996) Biochemistry 35, 4409-4416]. In the present work we studied the effect of Mg2+, Mn2+, Ca2+, Ni2+, Co2+, and Fe2+ on the near-UV CD spectrum of the ATP, ADP, ADP x BeFx, and ADP x AlF4- containing S complexes. The CD spectra obtained with ADP, ATP ADP x BeFx and ADP x AlF4- were essentially identical in the presence of Co2+ and rather similar in the case of Ca2+, while they were partially different in other cases. An interesting correlation was found between actin activation and ATP versus ADP difference spectra in the presence of various metal ions. The distribution of the fractional concentration of the intermediates of ATP hydrolysis was estimated in the presence of each cation from the CD spectra with phosphate analogs. In the presence of Mg2+ the predominant intermediate is the M** x ADP x Pi state, which is in accordance with the kinetic studies. On the other hand with non-native cations the predominant intermediate is the M* x ADP state and the release of ADP is the rate limiting step in the myosin-catalyzed ATP hydrolysis. According to the results, the near-UV CD spectrum originating from aromatic residues in S1 not only can distinguish identifiable states in the ATP hydrolysis cycle but can also pinpoint to changes in the tertiary structure caused by complex formation with nucleotide or nucleotide analog and various divalent metal cations. These findings, that are correlative with actin activation, and thus with the power stroke, suggest new strategies for perturbing S1 structure in the continuous efforts directed toward the elucidation of the mechanism of muscle contraction.

Myosin as cofactor and substrate in fibrinolysis.

Myosin accelerates plasminogen activation by tissue-type plasminogen activator (tPA), and is degraded extensively by plasmin. Myosin binds both tPA and plasminogen, and enhances activation of des1-77-plasminogen by tPA but not by urokinase-type plasminogen activator (uPA). Myosin decreases K(M) and increases k(cat) for des1-77-plasminogen activation by tPA, to yield catalytic efficiencies in excess of 8000 M-1 s-1. The effect of myosin is attributed to its C-terminal portion, the myosin rod. With a K(M) of 3 microM, myosin is a high-affinity substrate for plasmin. The findings indicate that myosin is a cofactor for plasminogen activation and a substrate for plasmin.

Mechanism for coupling free energy in ATPase to the myosin active site.

Acrylamide quenching of tryptophan 510 (Trp510) fluorescence in rabbit skeletal myosin subfragment 1 (S1) indicates the conformation of the probe binding cleft, containing the highly reactive thiol (SH1) and Trp510, in the presence of nucleotides or nucleotide analogs trapped in the active site of S1 [Park et al. (1996) Biochim. Biophys. Acta 1296, 1-4]. The Trp510 quenching efficiency shows that the probe binding cleft closes slightly in the presence of beryllium fluoride trapped MgADP (MgADPBeFx-S1) and most tightly in the presence of vanadate trapped MgADP (MgADPVi-S1) with aluminum fluoride and scandium fluoride trapped MgADP (MgADPA1F4-S1 and MgADPScFx-S1) falling in between in the order MgADPBeFx > MgADPA1F4 > MgADPScFx > MgADPVi. These nucleotide analogs are identified with sequential structural changes in MgATP during hydrolysis in the same order with beryllium fluoride occurring earliest in the ATPase cycle. Correlation of the separation distance of the gamma-phosphate analog metal from the oxygen connecting it to the beta-phosphate in ADP, to the extent of cleft closure, suggests that this distance in the nucleotide transition state determines the conformation of the probe binding cleft. Trp510 quenching efficiency was also measured as a function of the base moiety of the vanadate trapped Mg-nucleotide diphosphate (MgNDPVi-S1). The extent of cleft closure is largest in the presence of the natural substrate NDP and follows the order MgADPVi > MgCDPVi > MgUDPVi > MgIDPVi > MgGDPVi with very little difference between MgADPVi and MgCDPVi. These data follow the order of the effectiveness of the corresponding nucleotide triphosphates to support force production in muscle fibers [Pate et al. (1993) J. Biol. Chem. 268, 10046-10053]. In both the fiber and S1, it appears that the 6-position amino group of the bases of ADP and CDP is required to properly anchor the nucleotide in the active site, possibly at tyrosine 135 as suggested by X-ray crystallographic studies [Fisher et al. (1995) Biochemistry 34, 8960-8972]. Finally, the Trp510 quenching efficiency was measured as a function of the size of the divalent cation trapped in the active site of S1 with ADPVi. These data failed to show a correlation suggesting that the divalent cation is not involved with the propagation of influence from the active site to the probe binding cleft. The forgoing experiments suggest that the changing conformation of ATP during hydrolysis, parameterized by the increasing distance between the beta- and the gamma-phosphate, stresses the active site of S1 through protein-nucleotide contacts at the gamma-phosphate and nucleotide base. The stress-induced strain in the cross-bridge may be the mechanism by which energy in ATP is transferred to the myosin structure.

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.

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.

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].

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.

Conformation of xanthene dyes in the sulfhydryl 1 binding site of myosin. 2.

The fluorescent dyes 5'-(iodoacetamido)tetramethylrhodamine (5'IATR) and 5'-(iodoacetamido)-fluorescein (5'IAF) bind covalently to the reactive sulfhydryl (SH1) of myosin subfragment 1 (S1), the 5'IATR as a dimer and the 5'IAF as a monomer. The conformation of the dimer and the dye-protein complex was investigated by comparison of several spectroscopic signals of the molecules before and after their association into a complex and interpretation of any changes using a coupled dipole oscillator model adapted for this problem [Burghardt & Ajtai (1995) Biophys. Chem. (submitted for publication)]. Absorption and fluorescence spectroscopies were performed on 5'IAF, 5'IATR, and rhodamine 6G (R6G) and rhodamine B (RB) as models of dimer conformation. Absorption, fluorescence, and circular dichroism (CD) spectroscopies were performed on 5'IATR-modified S1 (5'R-S1) and 5'IAF-modified S1 (5'F-S1). Combined spectroscopic and 2-D NMR data from rhodamines in solution determined the conformations of the dimers. Xanthene rings from dimers of identical dyes (homodimers) stacked in two structures having very different spectroscopic signatures. Xanthene rings from the heterodimer of R6G and RB stacked in one conformation. The two homodimer conformations of 5'IATR are equally likely to form in solution. The other rhodamine homodimers have one dominant, but not exclusive, structure. Both conformations of the 5'IATR dimer were coupled to a tryptophan as a model of the dye-protein interaction at SH1. The calculated CD from one dimer conformer (dimer A) coupled to tryptophan is negative for the lowest energy CD absorption band. The other dimer (dimer B) gives positive CD on the two lowest energy CD absorption bands. Both dimer structures of 5'IATR contributed to the early time-dependent CD signal from 5'IATR binding to SH1, but at equilibrium the CD signal indicated only dimer B, suggesting that the SH1 binding pocket converts dimer A into dimer B. The time-dependent CD signal from 5'IAF changes amplitude but not shape during the reaction with SH1. The model calculation accounting for the spectroscopic signals of 5'R-S1 and 5'F-S1 indicates several likely conformations of the 5'IATR dimer-tryptophan and 5'IAF-tryptophan complexes embedded in S1. These structures fit to the alpha-carbon structure of the SH1 binding pocket when the 5'IATR dimer and 5'IAF interact closely with Trp510 [Rayment et al. (1993) Science 261, 50-58].(ABSTRACT TRUNCATED AT 400 WORDS)

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.

Following the rotational trajectory of the principal hydrodynamic frame of a protein using multiple probes.

A generalized set of fluorescence polarization intensities and electron paramagnetic resonance (EPR) spectra from multiple fluorescent and EPR probes of a specific site on an oriented and immobilized protein element in a biological assembly, combined with anisotropic rotational relaxation studies of the purified and labeled protein element freely rotationally diffusing in solution, are used to determine the angular distribution of the principal hydrodynamic frame of the protein element in the biological assembly. This multiprobe analysis method removes all of the basic ambiguities in the measured principal frame angular distribution introduced by limitations or symmetries intrinsic to standard fluorescence polarization intensity ratios and EPR spectra. The angular distribution of the principal frame is also more highly resolved than that previously reported for multiprobe determinations of probe angular distributions and is more useful for determining biological mechanisms because it indicates the order and orientation of the protein element rather than that of the extrinsic probe. Application of this method to the determination of the principal hydrodynamic frame distribution of myosin cross-bridges in muscle fibers in four physiological states, including the active isometric state, demonstrates the method's practicality by indicating the path of cross-bridge orientation changes during the active cycle [Ajtai, Toft, & Burghardt (1994) Biochemistry (following paper in this issue)].