An investigation of the SH1-SH2 and SH1-ATPase distances in myosin subfragment-1 by resonance energy transfer using nanosecond fluorimetry

The separation between the two reactive thiols SH1 (Cys-704) and SH2 (Cys-694) and that between SH1 and the active site of myosin subfragment-1 were further investigated by F?rster energy transfer techniques. The SH1-SH2 distance was determined with the probe 5-[[2-[(iodoacetyl)amino]ethyl] amino]naphthalene-1-sulfonic acid (AEDANS) attached to SH1 as the energy donor and 5-(iodoacetamido)fluorescein (IAF) attached to SH2 as energy acceptor. The results derived from measurements of donor lifetimes yielded a donor-acceptor separation in the range 26-52 A, with the distance R(2/3) based on rapid and isotropic probe motions being 40 A. These parameters were not sensitive to added MgADP, in agreement with previous results obtained by using the steady-state method. The SH1-SH2 distance was also determined with AEDANS attached to SH1 and N-(4-dimethylamino-3,5-dinitrophenyl)maleimide (DDPM) attached to SH2. The range in R for the AEDANS/DDPM pair was 12-36 A, with R(2/3) equal to 27 A. The transfer efficiency between these two probes increased by an average of 38% upon addition of MgADP. These results are in agreement with those previously reported (Dalbey, R.E., Weiel, J. and Yount, R.G. (1983) Biochemistry 22, 4696-4706), but the uncertainty in choosing an appropriate value of the orientation factor to describe the AEDANS-DDPM separation does not allow a unique interpretation of the observed increase in energy transfer because it could reflect either an increase in the average orientation factor or a decrease in the donor-acceptor separation. Nevertheless, the results are consistent with the notion that nucleotide binding induces structural perturbations that can be sensed by SH1 and SH2. The distance between SH1 and the ATPase site was determined with AEDANS linked to SH1 and the nucleotide analogue 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-diphosphate (TNP-ADP) noncovalently bound to the active site as energy acceptor. The bound TNP-ADP was highly immobilized, with a depolarization factor approaching unity. The separation between AEDANS at SH1 and TNP-ADP at the active site was in the range 15-44 A. The actual minimal separation between SH1 and the active site is probably less than 15 A, which suggests that direct interaction between the two sites cannot be ruled out from energy transfer results.     

Excitation energy transfer studies on the proximity between SH1 and the adenosinetriphosphatase site in myosin subfragment 1

Excitation energy transfer studies were carried out to determine the distance between the adenosinetriphosphatase (ATPase) site and a unique "fast-reacting" sulfhydryl (referred to as SH1) in myosin subfragment 1. The fluorescent moiety of the probe N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylene-diamine was used as the donor attached at SH1. The chromophoric nucleotide analogue 2'(3')-0-(2,4,6-trinitrophenyl)adenosine 5'-diphosphate was used as the acceptor noncovalently bound at the ATPase site. The energy transfer efficiency was found to be 56% by measuring the decrease in donor fluorescence lifetime. The critical transfer distance, R0(2/3), was determined to be 40.3 A. Since both donor and acceptor are likely to be rigidly attached, a statistical interpretation of the data was applied (Hillel, Z., & Wu, C.-W. (1976) Biochemistry 15, 2105] to determine distances. The method yielded the following conclusions: most probable distance = 38.7 A; maximum possible distance = 52 A; 10% probability for the distance to be less than 20 A; 3% probability to be less than 15 A. It may be concluded that despite the great influence that the two sites exert on each other, it is not likely that SH1 interacts directly with the ATPase site in myosin subfragment 1. This conclusion is in agreement with the findings of Wiedner et al. [Wiedner, H., Wetzel, R., & Eckstein, F. (1978) J. Biol. Chem. 253, 2763] and Botts et al. [Botts, J., Ue., K., Hozumi, T., & Samet, J. (1979) Biochemistry 18, 5157].     

Fluorescence energy transfers between points in acto-subfragment-1 rigor complex

Fluorescence energy transfer was measured by time-resolved and steady-state fluorimetry in order to investigate the spatial relationships between the nucleotide binding site of actin, the Cys-373 residue of actin, and the SH1 of myosin subfragment-1 in the rigor complex of acto-subfragment-1. N-Iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (IAEDANS) bound to the Cys-373 of actin or the fluorescent ADP analogue 1-N6-ethenoadenosine-5'-diphosphate (epsilon-ADP) bound to F-actin was used as a donor and 4-(N-(iodoacetoxy)ethyl-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazo le (IANBD) or 5-iodoacetamidofluorescein (IAF) bound to SH1 of myosin subfragment-1 was used as an acceptor. Assuming the random orientation factor, K2, to be 2/3, the distance between Cys-373 residue of actin and SH1 of myosin subfragment-1 was calculated to be about 50 A, in agreement with the values previously reported, 60 A (Takashi, R. (1969) Biochemistry 18, 5164-69) and 50 A (Trayer, H.R. and Trayer, I.P. (1983) Eur. J. Biochem. 135, 47-59). The distance between the nucleotide binding site of actin and SH1 of myosin subfragment-1 was calculated to be about 70 A or greater.     

Two-state equilibria of myosin subfragment 1 and its complexes with ADP and actin

We previously reported that the nucleotide complex of myosin subfragment 1, S1.epsilon ADP, exists in two states on the basis of the temperature dependence of the fluorescence decay of bound 1,N6-ethenoadenosine diphosphate (epsilon ADP) [Aguirre, R., Lin. S.-H., Gonsoulin, F., Wang, C.-K., & Cheung, H.C. (1989) Biochemistry 28, 799-809]. We have extended the previous study of the equilibrium between the two states, S1L.ADP in equilibrium S1H.ADP, by using a fluorescently labeled myosin S1 (S1-AF). In S1 alkylated with IAF [5-(iodoacetamido)fluorescein], the decay of the label emission was biexponential both in the presence and absence of ADP and/or actin. In the presence of ADP, the two decay times were 4.30 (alpha 1 = 0.55) and 0.80 ns (alpha 2 = 0.45) at 12.4 degrees C, in a medium containing 60 mM KCl, 30 mM TES (pH 7.5), and 2 mM MgCl2. The steady-state fluorescence intensities of S1-AF, (S1-AF).ADP, acto.(S1-AF), and acto.(S1-AF).ADP were dependent on temperature over the range of 5-30 degrees C. By combining lifetime and steady-state intensity data, we obtained for the two-state transition (S1-AF)L.ADP in equilibrium (S1-AF)H.ADP the following parameters: delta H degrees = 16.1 kcal/mol (67.3 kJ/mol) and delta S degrees = 55.8 cal/(deg.mol) [233.5 J/(deg.mol)], in agreement with previous results obtained with epsilon ADP. The delta H degrees values for the two-state transition of S1-AF, acto.(S1-AF), and acto.(S1-AF).ADP are 13.0, 21.6, and 5.2 kcal/mol, respectively. The corresponding delta S degrees values are 46.9, 79.5, and 17.4 cal/(deg.mol).(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetic studies on the association and dissociation of myosin subfragment 1 and actin.

The reactions of pyrene-labeled actin with myosin subfragment 1 (S1) and S1-ligand complexes at low ionic strength are described by the schemes [formula: see text] where M refers to a myosin head; A is actin; L is ligand; the asterisk refers to a high fluorescence state of actin; and K1 and K3 are association constants. K1 is reduced approximately 10-fold for M.ADP or M.pyrophosphate versus M alone. The rate constant of the isomerization step (k2) is 150-200 s-1 for A*M, A*M.ADP, and A*M-pyrophosphate (20 degrees C). The interaction between the ligand the actin binding sites reduces K2 from 2,000 for A*M to 50-100 for A*M.ADP and to approximately unity for A*M-pyrophosphate. The A*M.ADP state is equated with the AM'.ADP state of Sleep and Hutton (Sleep, J., A., and Hutton, R. L. (1980) Biochemistry 19, 1276-1283).     

Fluorescence resonance energy transfer within the complex formed by actin and myosin subfragment 1. Comparison between weakly and strongly attached states

Fluorescence resonance energy transfer measurements have been made between Cys-374 on actin and Cys-177 on the alkali light chain of myosin subfragment 1 (S1) using several pairs of donor-acceptor chromophores. The labeled light chain was exchanged into subfragment 1 and the resulting fluorescently labeled subfragment 1 isolated by ion-exchange chromatography on SP-Trisacryl. The efficiency of energy transfer was measured by steady-state fluorescence in a strong binding complex of acto-S1 and found to represent a spatial separation between the two probes of 5.6-6.3 nm. The same measurements were then made with weak binding acto-S1 complexes generated in two ways. First, actin was complexed with p-phenylenedimaleimide-S1, a stable analogue of S1-adenosine 5'-triphosphate (ATP), obtained by cross-linking the SH1 and SH2 heavy-chain thiols of subfragment 1 [Greene, L. E., Chalovich, J. M., & Eisenberg, E. (1986) Biochemistry 25, 704-709]. Large increases in transfer efficiency indicated that the two probes had moved closer together by some 3 nm. Second, weak binding complexes were formed between subfragment 1 and actin in the presence of the regulatory proteins troponin and tropomyosin, the absence of calcium, and the presence of ATP [Chalovich, J. M., & Eisenberg, E. (1982) J. Biol. Chem. 257, 2432-2437]. The measured efficiency of energy transfer again indicated that the distance between the two labeled sites had moved closer by about 3 nm. These data support the idea that there is a considerable difference in the structure of the acto-S1 complex between the weakly and strongly bound states.     

Spatial relationship between a fast-reacting thiol and a reactive lysine residue of myosin subfragment 1

Fluorescence energy transfer was used to examine the spatial proximity between two key side chains in myosin subfragment 1 (S-1), viz., the reactive thiol (SH1) located on the C-terminal 20K tryptic fragment and the reactive lysyl (RLR) on the N-terminal 27K tryptic fragment of S-1 heavy chain. S-1 was specifically labeled at SH1 with an energy donor, N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine (AEDANS), and at RLR with an energy acceptor, 2,4,6,-trinitrobenzenesulfonate (TNBS). Prior blocking of SH1 with AEDANS increased the pK of RLR from 9.04 to 9.42. Trinitrophenylation of SH1-blocked S-1 was about 50% slower and sharply reduced the Ca2+ ATPase activity. Reciprocally, blocking of RLR with TNBS slowed the rate of reaction of SH1 and AEDANS by 40-60%. Addition of the second label does not grossly alter the conformation resulting from the first label. S-1 labeled at RLR with TNBS and at SH1 with optically inert iodoacetamide shows the same TNP difference spectrum +/- MgADP (lambda min 365 nm) as S-1 with S 1 free. Also, S-1 labeled at SH1 with AEDANS and at RLR with an optically inert methyl group shows the same AEDANS emission spectrum (lambda em max 475 nm), excited-state lifetime (tau = 20.3 ns) and rotational correlation time (phi = 106 ns) as S-1 with RLR free. When the decrease of either the quantum yield or the excited-state lifetime of the donor in the absence and presence of the acceptor was measured, the energy transfer efficiency was found to be 70%. The apparent interchromophore distance was calculated to be 2.6 nm through the use of the F?rster equation with an uncertainty of less than 12%.     

Analysis of the conformational change of myosin during ATP hydrolysis using fluorescence resonance energy transfer

In order to elucidate the molecular basis of energy transduction by myosin as a molecular motor, a fluorescent ribose-modified ATP analog 2'(3')-O-[6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl]-ATP (NBD-ATP), was utilized to study the conformational change of the myosin motor domain during ATP hydrolysis using the fluorescence resonance energy transfer (FRET) method. The FRET efficiency from the fluorescent probe, BD- or AD-labeled at the reactive cysteine residues, SH1 (Cys 707) or SH2 (Cys697), respectively, to the NBD fluorophore in the ATP binding site was measured for several transient intermediates in the ATPase cycle. The FRET efficiency was greater than that using NBD-ADP. The FRETs for the myosin.ADP.AlF4- and myosin.ADP.BeFn ternary complexes, which mimic the M*.ADP.P(i) state and M.ATP state in the ATPase cycle, respectively, were similar to that of NBD-ATP. This suggests that both the SH1 and SH2 regions change their localized conformations to move closer to the ATPase site in the M*.ATP state and M**.ADP.P(i) state than in the M*.ADP state. Furthermore, we measured energy transfer from BD in the essential light chain to NBD in the active site. Assuming the efficiency at different states, myosin adopts a conformation such that the light chain moves closer to the active site by approximately 9 A during the hydrolysis of ATP.     

Spatial proximity of ATP-sensitive tryptophanyl residue(s) and Cys-697 in myosin ATPase.

The reactive thiol Cys-697 (SH2) in myosin ATPase was labeled with a fluorescent analog of maleimide, 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS) (Hiratsuka, T. (1992) J. Biol. Chem. 267, 14941-14948). Although the tryptophan fluorescence of myosin subfragment-1 (S-1) was slightly affected by incorporation of the MIANS fluorophore, the tryptophan fluorescence of the resultant S-1 derivative (MIANS-S-1) was enhanced by ATP in a manner similar to that of unlabeled S-1. The quenching of tryptophan fluorescence of MIANS-S-1 was shown to result from a transfer of the excitation energy from tryptophanyl residue(s) to the MIANS fluorophore attached to SH2, which absorbed and fluoresced maximally at 325 and 418 nm, respectively. The energy transfer measurements were performed in the presence of acrylamide and compared to those performed in the absence of the quencher. The energy transfer efficiencies were found to be unaltered by acrylamide, indicating that the observed fluorescence energy transfer is originated exclusively from the tryptophanyl residue(s) that are not affected by acrylamide, i.e. the ATP-sensitive tryptophanyl residue(s) of S-1 (Torgerson, P. M. (1984) Biochemistry 23, 3002-3007). The distance between the tryptophanyl residue(s) and Cys-697 was calculated to be 27 A assuming a single donor-acceptor pair. Trp-510 is proposed to be one of the ATP-sensitive tryptophanyl residues.     

Movement of Cys-697 in myosin ATPase associated with ATP hydrolysis.

To detect movement of Cys-697 (SH2) in myosin subfragment-1 (S-1) associated with ATP hydrolysis, SH2 was labeled with the environmentally sensitive fluorescent analog of maleimide, 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS). Complex formation of S-1 labeled at Cys-697 with MIANS (MIANS-S-1) with adenyl-5'-yl imidodiphosphate and ADP resulted in a significant decrease in the fluorescence intensity of approximately 40 and 30%, respectively. When ATP was added to MIANS-S-1, the fluorescence intensity decreased rapidly by approximately 40%, and this fluorescence level was maintained during the steady state of ATP hydrolysis. As the substrate was used up, the fluorescence intensity increased to approximately 70% of the original value. These results together with model experiments with MIANS-N-acetylcysteine indicate that in the presence of ATP, the MIANS fluorophore attached to SH2 is located in a less hydrophobic environment than is the fluorophore in the absence of ligand and that the hydrolysis of ATP enhances hydrophobicity around the fluorophore. Acrylamide fluorescence quenching studies of MIANS-S-1 confirmed these results, indicating that addition of ATP and ADP to MIANS-S-1 results in an increase in the Stern-Volmer quenching constant of the fluorophore by factors of approximately 3 and 2.5, respectively. The present observations suggest that binding of ATP causes a movement of SH2 toward the protein surface, whereas it goes back into the protein interior after ATP hydrolysis. The results also confirmed previous observations by a chemical cross-linking approach (Hiratsuka, T. (1987) Biochemistry 26, 3168-3173).     

Fluorescence energey transfer in myosin subfragment-1

Fluorescent probes have been selectively introduced into skeletal muscle myosin subfragment-1 and the fluorescence emission characteristics of the labeled products studied. The fluorophores employed were the thiol-specific reagents N-[[(iodoacetyl)aminolethyl-5-naphthylamine-1-sulfonic acid and 5-(iodoacetamido)fluorescein, the spectral properties of which render them a particularly effective donor-acceptor pair in F?rster energy-transfer studies. Alkali 1 light chain, labeled at a single cysteine with either of these probes, was incorporated into chymotryptic subfragment-1 by the exchange procedure of Wagner & Weeds [Wagner, P.D., & Weeds, A.G. (1977) J. Mol. Biol. 109, 455-473]. The resultant, fluorescently labeled subfragment-1 was isolated by ion-exchange chromatography. Determination of the extent of incorporation by extinction and fluorescence indicated that greater than 80% of the subfragment-1 population possessed a fluorescently labeled alkali 1 light chain. The introduction of labeled alkali 1 did not perturb the K+-, Ca2+-, or actin-activated adenosine triphosphatases of subfragment-1. The addition of adenosine triphosphate (ATP), liganded by various cations, to this singly labeled subfragment-1 induced a 6-10% decrease in the fluorescence intensity of the extrinsic chromophore. An intensity decrease of approximately 4% was obtained when the hydrolysis of ATP was complete, and also upon direct addition of adenosine diphosphate. The ATP analogue adenylyl imidodiphosphate induced a decrease of approximately 7% in intensity. The addition of F-actin to the subfragment-1 in the presence of MgATP elicited no further fluorescence intensity change. A second, appropriate fluorophore was introduced into the singly labeled subfragment-1 at the SH1 thiol on the heavy chain. F?rster energy transfer was observed between this labeled site and the fluorophore previously introduced on the alkali 1 light chain. The measured efficiency of energy transfer indicated that the two fluorophores were approximately 40 A apart. The same value was obtained upon reversal of the donor and acceptor attachment sites, suggesting that the uncertainty in the calculated distance introduced by the choice of orientation factor is probably less than 20%. Steady-state observations did not reveal any obvious change in this distance upon the addition of MgATP and then F-actin to the doubly labeled subfragment-1.     

Is SH1-SH2-cross-linked myosin subfragment 1 a structural analog of the weakly-bound state of myosin?

Myosin subfragment 1 (S1) with SH1 (Cys(707)) and SH2 (Cys(697)) groups cross-linked by p-phenylenedimaleimide (pPDM-S1) is thought to be an analog of the weakly bound states of myosin bound to actin. The structural properties of pPDM-S1 were compared in this study to those of S1.ADP.BeF(x) and S1.ADP.AlF(4)(-), i.e., the established structural analogs of the myosin weakly bound states. To distinguish between the conformational effects of SH1-SH2 cross-linking and those due to their monofunctional modification, we used S1 with the SH1 and SH2 groups labeled with N-phenylmaleimide (NPM-S1) as a control in our experiments. The state of the nucleotide pocket was probed using a hydrophobic fluorescent dye, 3-[4-(3-phenyl-2-pyrazolin-1-yl)benzene-1-sulfonylamido]phen ylboronic acid (PPBA). Differential scanning calorimetry (DSC) was used to study the thermal stability of S1. By both methods the conformational state of pPDM-S1 was different from that of unmodified S1 in the S1.ADP.BeF(x) and S1.ADP.AlF(4)(-) complexes and closer to that of nucleotide-free S1. Moreover, BeF(x) and AlF(4)(-) binding failed to induce conformational changes in pPDM-S1 similar to those observed in unmodified S1. Surprisingly, when pPDM cross-linking was performed on S1.ADP.BeF(x) complex, ADP.BeF(x) protected to some extent the nucleotide pocket of S1 from the effects of pPDM modification. NPM-S1 behaved similarly to pPDM-S1 in our experiments. Overall, this work presents new evidence that the conformational state of pPDM-S1 is different from that of the weakly bound state analogs, S1.ADP.BeF(x) and S1.ADP.AlF(4)(-). The similar structural effects of pPDM cross-linking of SH1 and SH2 groups and their monofunctional labeling with NPM are ascribed to the inhibitory effects of these modifications on the flexibility/mobility of the SH1-SH2 helix.     

Thiol-specific cross-linkers of variable length reveal a similar separation of SH1 and SH2 in myosin subfragment 1 in the presence and absence of MgADP.

A series of thiol-specific cross-linking reagents were prepared for studying the kinetics of cross-linking between SH1 (Cys(707)) and SH2 (Cys(697)) in rabbit skeletal muscle myosin subfragment 1. The reagents were of the type RSS(CH(2))(n)()SSR, with R = 3-carboxy-4-nitrophenyl and n = 3, 6, 7, 8, 9, 10, and 12, spanning distances from 9 to 20 A. The reactions were monitored spectrophotometrically by measuring the release of 2-nitro-5-thiobenzoate. Reaction rates for modification of SH1 (k(1)) and for cross-linking (k(2)) were measured by the decrease of the K(+)(EDTA)-ATPase activity and the decrease of the Ca(2+)-ATPase activity, respectively, and corrected for the different reactivities of C(n). Cross-linking rates in the presence and absence of MgADP showed similar dependence on the length of the reagents: While the cross-linking rates for n = 3 or n = 6 were close to those for n = 0 (Ellman's reagent), those for n = 7 and 8 were significantly increased. Thus the distance between SH1 and SH2 appears to be equal in both states and can be estimated as >/=15 A, based on the length of the reagent with n = 8 in stretched conformation. Under rigor conditions, reactivity of SH1 differed significantly from that in the presence of MgADP, presumably because of shielding through a lipophilic domain. Similarly, the cross-linking rates k(2) for C(3), C(6), and C(7) in the absence of MgADP were ca. 15 times lower than in the presence of MgADP, suggesting a change in the structure of the SH2 region that depends on nucleotide binding. The results are discussed in terms of recent X-ray structures of S1 and S1-MgADP [Rayment et al. (1993) Science 261, 50-58; Gulick et al. (1997) Biochemistry 36, 11619-11628].     

Differing ADP release rates from myosin heavy chain isoforms define the shortening velocity of skeletal muscle fibers

To understand mammalian skeletal myosin isoform diversity, pure myosin isoforms of the four major skeletal muscle myosin types (myosin heavy chains I, IIA, IIX, and IIB) were extracted from single rat muscle fibers. The extracted myosin (1-2 microg/15-mm length) was sufficient to define the actomyosin dissociation reaction in flash photolysis using caged-ATP (Weiss, S., Chizhov, I., and Geeves, M. A. (2000) J. Muscle Res. Cell Motil. 21, 423-432). The ADP inhibition of the dissociation reaction was also studied to give the ADP affinity for actomyosin (K(AD)). The apparent second order rate constant of actomyosin dissociation gets faster (K(1)k(+2) = 0.17 -0.26 microm(-1) x s(-1)), whereas the affinity for ADP is weakened (250-930 microm) in the isoform order I, IIA, IIX, IIB. Both sets of values correlate well with the measured maximum shortening velocity (V(0)) of the parent fibers. If the value of K(AD) is controlled largely by the rate constant of ADP release (k(-AD)), then the estimated value of k(-AD) is sufficiently low to limit V(0). In contrast, [ATP]K(1)k(+2) at a physiological concentration of 5 mm ATP would be 2.5-6 times faster than k(-AD).     

Interaction of myosin LYS-553 with the C-terminus and DNase I-binding loop of actin examined by fluorescence resonance energy transfer

Fluorescence resonance energy transfer (FRET) experiments were carried out in the absence of nucleotide (rigor) or in the presence of MgADP between fluorescent donor probes (IAEDANS (5((((2-iodoacetyl)amino)ethyl)amino)-naphthalene-1-sulfonic acid) at Cys-374 or DANSYL (5-dimethylamino naphthalene-1-(N-(5-aminopentyl))sulfonamide) at Gln-41 of actin and acceptor molecules (FHS (6-[fluorescein-5(and 6)-carboxamido] hexanoic acid succinimidyl ester) at Lys-553 of skeletal muscle myosin subfragment 1. The critical F?rster distance (R(0)) was determined to be 44 and 38 A for the IAEDANS-FHS and DANSYL-FHS donor-acceptor pairs, respectively. The efficiency of energy transfer between the acceptor molecules at Lys-553 of myosin and donor probes at Cys-374 or Gln-41 of actin was calculated to be 0.78 +/- 0.01 or 0.94 +/- 0.01, respectively, corresponding to distances of 35.6 +/- 0.4 A and 24.0 +/- 1.6 A, respectively. MgADP had no significant effect on the distances observed in rigor. Thus, rearrangements in the acto-myosin interface are likely to occur elsewhere than in the lower 50-kDa subdomain of myosin as its affinity for actin is weakened by MgADP binding.     

Conformational flexibility of the Cys 697-Cys 707 segment of myosin subfragment-1. Distance distributions by frequency-domain fluorometry

The separation between Cys 697 (SH1) and Cys 707 (SH2) of the heavy chain of myosin subfragment-1 was previously measured by fluorescence resonance energy transfer with a donor linked to SH1 and an acceptor to SH2. In the present study the distribution of the distances between the two thiols was recovered from frequency-domain fluorometry. In the native state and in the presence of ligands such as MgADP, pyrophosphate, orthovanadate (Vi) and actin, we found wide distributions of the separations between SH1 and SH2 (11-16 A) comparable to that found in the random-coil state (20 A). These results suggest that the SH1-SH2 segment has a high degree of conformational flexibility even in native S1. The flexibility is not much affected by the physiological state of S1. However, the ligands MgADP, Vi and MgADP + Vi decrease significantly the mean SH1-SH2 distance from 27 to 17 A with the effect of MgADP+ Vi being the most pronounced. The anisotropy decay of donor-labeled S1 is biphasic with two rotational correlation times. The long component is decreased by these ligands from 289 to 93 ns, suggesting a more compact symmetric structure of S1 in the presence of the ligands. The complex S1(MgADP)Vi has been shown to be a stable analogue of S1(MgADP)Pi, an unstable intermediate that is generated in the actomyosin ATPase cycle during muscle contraction. Since the power stroke of muscle is accompanied by release of Pi from S1(MgADP)Pi, the present results are consistent with a model in which force generation can be accompanied by transition of S1 from a highly symmetric or compact structure to a more extended structure.     

Transient kinetic analysis of N-phenylmaleimide-reacted myosin subfragment-1

Alkylation of myosin's Cys-707 (SH1) and Cys-697 (SH2) has profound consequences for myosin's ability to interact with actin and hydrolyze MgATP. Pre-steady-state measurements of myosin-S1 alkylated at SH1 and SH2 by N-phenylmaleimide (NPM) in the presence of ATP were taken to identify the steps of the reaction that are altered. It was found that the rate constant most affected by this modification is the apparent rate of the ATP hydrolysis step. This rate constant is reduced 20000-fold, an effect comparable in magnitude to the effect of the same modification on the binding of MgATP to S1 or acto-S1 [Xie, L., and Schoenberg, M. (1998) Biochemistry 37, 8048]. In contrast, the rate constants of phosphate release and dissociation of acto-S1 by ATP were reduced <20-fold. For unmodified S1, the enhancement of fluorescence seen after addition of ATP had the same rate constant as the ATP hydrolysis step (S1.ATP if S1.ADP.Pi) measured by single-turnover experiments in a quench-flow experiment. This is consistent with results previously observed [Johnson, K. A., and Taylor, E. W. (1978) Biochemistry 17, 3432]. However, NPM-modified S1 exhibited virtually no fluorescence enhancement upon ATP binding. This provides further evidence that M.ATP is the predominant intermediate of NPM-S1-catalyzed ATP hydrolysis.     

Adenine nucleotide-binding sites on mitochondrial F1-ATPase: studies of the inactive complex formed upon binding ADP at a catalytic site.

ADP-induced inhibition of mitochondrial F1-ATPase has been studied. It is shown that in the presence of magnesium and the absence of light, the photoaffinity ADP analog, 2-azido-ADP, induces a reversible inhibition of native F1 that is indistinguishable from that obtained with ADP. Photolysis of the inactive complex results in the predominant labeling of a catalytic-site peptide identified previously (Cross et al., 1987, Proc. Natl. Acad. Sci. USA 84, 5715-5719). Dissociation of the inactive complex formed between F1 and ADP is biphasic with a rapid azide-insensitive phase followed by a slow azide-sensitive phase (k approximately 3 x 10(-3) s-1). It is also shown that incubation of the ADP-inhibited enzyme with EDTA or phosphate does not result in release or migration of ADP from the catalytic site. However, it does convert the complex to a form that reactivates in the presence of 100 microM ATP at a rate too rapid to observe using manual mixing.     

Transient kinetics of the interaction of 1,N6-ethenoadenosine 5'-triphosphate with myosin subfragment 1 under normal and cryoenzymic conditions: a comparison with adenosine 5'-triphosphate

The kinetics of the interaction of the fluorescent analogue 1,N6-ethenoadenosine 5'-triphosphate (epsilon-ATP) with myosin subfragment 1 (S1) were studied at 15 and -7.5 degrees C with 40% ethylene glycol as cryosolvent. Two techniques were used: fluorescence stopped flow and rapid flow-quench. When S1 is mixed with epsilon-ATP in a stopped-flow apparatus, biphasic fluorescence transients are obtained which are difficult to assign. Chemical sampling by the rapid-flow-quench method led to the chemical identity and the kinetics of interconversion of key intermediates, and by this method the optical signals were assigned and information about the cleavage and release of products was obtained. The data were interpreted by a shortened form of the Bagshaw-Trentham scheme for myosin adenosinetriphosphatase: M + ATP K1 in equilibrium M.ATP k2----M*.ATP k3 in equilibrium k3 M**.ADP.Pi k4----M + ADP + Pi The constants obtained were compared with those for ATP under identical conditions. In agreement with Rosenfeld and Taylor [Rosenfeld, S. S., & Taylor, E. W. (1984) J. Biol. Chem. 259, 11920-11929] we find that epsilon-ATP is bound tightly to S1 and that the chemical step is slower than with ATP. We show that the fast fluorescence transient is due to the tight binding of epsilon-ATP with K1 = 32 microM and k2 = 58 s-1 at 15 degrees C. With ATP these values are 8 microM and 16 s-1, respectively. There is a large difference in the delta H for k2: 50 kJ.mol-1 for epsilon-ATP and 119 kJ.mol-1 for ATP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antibody and peptide probes of interactions between the SH1-SH2 region of myosin subfragment 1 and actin's N-terminus.

The negatively charged residues in the N-terminus of actin and the 697-707 region on myosin subfragment 1 (S-1), containing the reactive cysteines SH1 and SH2, are known to be important for actin-activated myosin ATPase activity. The relationship between these two sites was first examined by monitoring the rates of SH1 and SH2 modification with N-ethylmaleimide in the presence of actin and, secondly, by testing for direct binding of SH1 peptides to the N-terminal segment on actin. While actin alone protected SH1 from N-ethylmaleimide modification, this effect was abolished by an antibody against the seven N-terminal amino acids on actin, F(ab)(1-7), and was greatly reduced when the charge of acidic residues at actin's N-terminus was altered by carbodiimide coupling of ethylenediamine. Neither F(ab)(1-7) nor ethylenediamine treatment reversed the effect of F-actin on SH2 reactivity in SH1-modified S-1. These results show a communication between the SH1 region on S-1 and actin's N-terminus in the acto-S-1 complex. To test whether such a communication involves the binding of the SH1 site on S-1 to the N-terminal segment of actin, the SH1 peptide IRICRKG-NH2(4+) was used. Cosedimentation experiments revealed the binding of three to six peptides per actin monomer. Peptide binding to actin was affected slightly, if at all, by F(ab)(1-7). The antibody also did not change the polymerization of G-actin by the peptides. The peptides caused a small reduction in the binding of S-1 to actin and did not change the binding of F(ab)(1-7).(ABSTRACT TRUNCATED AT 250 WORDS)