Ralph G. Yount

[11] Chemical modification of myosin by active-site trapping of metal-nucleotides with thiol crosslinking reagents

Publisher Summary It has been discovered that crosslinking of two thiols in myosin in the presence of MgADP leads to stable and stoichiometric trapping of MgADP at the active site. This has led to the proposal that myosin contains a jaw-like active site structure that closes upon binding of MgADP. Closure of this jaw is proposed to juxtapose two sulfhydryl groups, the so-called SH-1 and SH-2, so that they may be crosslinked and lock shut the active-site cleft. Active-site trapping can be accomplished with at least eight different thiol crosslinking reagents with rigid crosslink spans that vary from 14 A to 2 A. Myosins broad substrate specificity allows this technique to be used to trap a wide range of metal nucleotides. Metal nucleotide complexes can be stably trapped and stored under conditions where the rate of dissociation has half-times on the order of several days. This technique can thus be used to probe the microenvironment and structure of the force-generating ATPase site without the need to synthesize ATP analogs that must be covalently attached to the active site. This chapter focuses first on the general requirements for active-site trapping. Second, the methodology that has been employed to establish thiol crosslinking and to demonstrate concomitant stable trapping of metal nucleotides is discussed. In addition, the specific crosslinking reagents and reaction conditions that have been found to be successful or unsuccessful in active-site trapping are described in the chapter. Finally, some of the applications of this technique are discussed, these include trapping of spectroscopically interesting metals and nucleotides as well as the trapping of a new photoaffinity ATP analog that ensures specific photoincorporation into the active site of myosin.

X-ray structures of the Dictyostelium discoideum myosin motor domain with six non-nucleotide analogs.

The three-dimensional structures of the truncated myosin head from Dictyostelium discoideum myosin II complexed with dinitrophenylaminoethyl-, dinitrophenylaminopropyl-, o-nitrophenylaminoethyl-,m-nitrophenylaminoethyl-,p-nitrophenylaminoethyl-, ando-nitrophenyl-N-methyl-aminoethyl-diphosphate·beryllium fluoride have been determined to better than 2.3-Å resolution. The structure of the protein and nucleotide binding pocket in these complexes is very similar to that of S1dC·ADP·BeF x (Fisher, A. J., Smith, C. A., Thoden, J., Smith, R., Sutoh, K., Holden, H. M., and Rayment, I. (1995) Biochemistry 34, 8960–8972). The position of the triphosphate-like moiety is essentially identical in all complexes. Furthermore, the alkyl-amino group plays the same role as the ribose by linking the triphosphate to the adenine binding pocket; however, none of the phenyl groups lie in the same position as adenine in S1dC·MgADP·BeF x , even though several of these nucleotide analogs are functionally equivalent to ATP. Rather the former location of adenine is occupied by water in the nanolog complexes, and the phenyl groups are organized in a manner that attempts to optimize their hydrogen bonding interactions with this constellation of solvent molecules. A comparison of the kinetic and structural properties of the nanologs relative to ATP suggests that the ability of a substrate to sustain tension and to generate movement correlates with a well defined interaction with the active site water structure observed in S1dC·MgADP·BeF x .

Mechanics of glycerinated muscle fibers using nonnucleoside triphosphate substrates

We have investigated the ability of the photoaffinity, nonnucleotide ATP analogues, 2-[(4-azido-2-nitrophenyl) amino] ethyl triphosphate (NANTP) and 2-[(4-azido-2-nitrophenyl) amino] propyl triphosphate (PrNANTP), to support active contraction in glycerinated rabbit psoas fibers. At millimolar concentrations, in the absence of calcium, both analogues relaxed fibers. In the presence of calcium, MgNANTP produced isometric tension and stiffness that were one-half to two-thirds the values obtained in MgATP. Maximum shortening velocity and the calcium-activated, myofibrillar catalyzed rate of hydrolysis were approximately the same for MgNANTP as for MgATP. With MgNANTP as the substrate, increasing concentrations of the diphosphate analogue, MgNANDP, inhibited shortening velocity but did not change isometric tension. The addition of increased concentrations of orthophosphate (P) decreased tension while shortening velocity increased. Thus, the effects of the hydrolysis products of NANTP were quite similar to those observed previously for ADP and P in the presence of MgATP. Taken together, these observations show that MgNANTP binds to, and functions in the active site of myosin in a manner quite analogous to MgATP. Thus, the aryl azido group should serve as a valid photoaffinity label for the purine portion of the active site. In contrast, MgPrNANTP, which differs from MgNANTP only in an extra CH2 spacer between the nitrophenyl ring and the triphosphate moiety did not support isometric tension or active shortening in the presence of calcium. Fiber stiffness increased in the presence of calcium and MgPrNANTP, with a calcium-activated, myofibrillar MgPrNANTPase which was about half that obtained with MgATP. Thus, in the presence of MgPrNANTP, cross-bridges appeared to be cycling through states that were attached to actin, but not producing force.

The essential light chains constitute part of the active site of smooth muscle myosin

Myosin, a major contractile protein, characteristically possesses a long coiled-coil α-helical tail and two heads. Each head contains both an actin binding site and an ATPase site and is formed from the NH2-terminal half of one of the two heavy chains (relative molecular mass, Mr, 200,000) and a pair of light chains; the so-called regulatory and essential light chains of approximately Mr 20,000 each. Recently we have identified1 Trp 130 of the myosin heavy chain from rabbit skeletal muscle as an active-site amino-acid residue after labelling with a new photoaffinity analogue of ADP, N-(4-azido-2-nitrophenyl)-2-aminoethyl diphosphate (NANDP)2. Nonspecific labelling was eliminated by first trapping NANDP at the active site with thiol crosslinking agents3. Exclusive labelling of the heavy chains with no labelling of the light chains agreed with previous findings4,5 that the heavy chains alone contain the actin-activated Mg-ATPase activity of rabbit skeletal myosin. Here we report similar photolabelling experiments with smooth muscle myosin (chicken gizzard) in which 3H-NANDP is trapped at the active site with vanadate6 and which show that both the heavy chains and the essential light chains are labelled. The results indicate that both chains contribute to the ATP binding site and represent the first direct evidence for participation of the essential light chains in the active site of any type of myosin.

Vanadate-mediated photocleavage of myosin

Publisher Summary Skeletal myosin is known to form a very stable transition state-like complex with MgADP and vanadate ions (V i ). During the irradiation ADP and V i are released simultaneously with a concomitant four-fold increase in the Ca 2+ -ATPase activity and an increase in the UV absorbance of myosin subfragment 1 (S1) over unirradiated controls. This modification is the result of the vanadate-promoted photooxidation of the fl-hydroxymethyl group of a serine to an aldehyde. This aldehyde can tautomerize to an enol. Photocleavage of myosin at active site yields a stable photomodified myosin, which, in the presence of excess V i and MgADP, will reform a new stable MgADP-V i complex at the active site. After purification of the new complex by removal of excess V i and MgADP by centrifugal gel filtration, irradiation leads to specific cleavage of the heavy chain. The heavy chain of S1 is cleaved at two sites when irradiated in the presence of miUimolar vanadate (in the absence of Mg 2+ or ADP. Mocz 6 has also reported a third site of vanadate-mediated photocleavage near the COOH terminus of the myosin heavy chain. An important consideration in photocleavage studies is that various polyvanadates, such as di-, tetra-, and pentavanadate, are in rapid equilibria with monovanadate at total vanadate concentrations in the millimolar range.

Synthesis of non-nucleotide ATP analogues and characterization of their chemomechanical interaction with muscle fibres

SummaryTo probe the substrate requirements for the actomyosin chemomechanical interaction, the effects of a series of eight new non-nucleotide ATP analogues on actomyosin-catalysed hydrolysis rates and on fibre mechanics have been investigated. These analogues have substitutions of new functional groups at the 2- and 4- positions of the ATP analogues, 2-[(4-azido-2-nitrophenyl)amino]ethyl triphosphate (NANTP), and 3-[(4-nitrophenyl)amino]propyl triphosphate (PrNANTP). Previous work has shown NANTP but not PrNANTP will support active tension and shortening in skinned muscle fibres in a manner almost identical to ATP. Here all 2- and 4- phenyl substituted analogues had myosin subfragment 1 (S1) NTPase hydrolysis rates higher than ATP and the rates were stimulated by addition of actin. In general, the replacement of the 4-azido group of NANTP with-H,-NO2 or-NH2 had small effects on fibre mechanics while replacement of 2-NO2 group with-H or-NH2 dramatically lowered the ability of the new analogues to support active tension and shortening. All PrNANTP-based analogues were ineffective in supporting active tension or shortening. We found no correlation between S1 or actoS1 NTPase rates and any mechanical parameters. However, for all analogues there was a strong correlation between the maximal velocity of shortening (Vmax) and isometric tension (Po). A three-state, chemomechanical model is proposed in which the analogues effect the transition rate into a strongly-bound, force-producing crossbridge state to account for this correlation. These studies identify 2-[(2-nitrophenyl)amino]ethyl triposphate as the chemically simplest ATP analogue which closely mimics the effect of ATP in skinned muscle fibres.

Adenosine-5′-sulfatopyrophosphate, an analogue of adenosine triphosphate: I. Preparation, properties, and mode of cleavage by snake venoms☆

Abstract An improved and simplified procedure has been worked out for the synthesis and isolation of adenosine-5′-sulfatopyrophosphate, a sulfate analogue of adenosine triphosphate. The sulfate analogue has two isomeric forms in which the sulfate grouping is on either the α- or β-phosphates of adenosine diphosphate. The apparent formation constants of adenosine-5′-sulfatopyrophosphate (mixed isomers) and adenosine-5′-sulfatophosphate with Mg ++ and Ca ++ have been measured. These values are low but comparable to similar constants measured by other workers for the trianionic form of adenosine triphosphate and the dianionic form of adenosine diphosphate, respectively. Both isomers of the sulfate analogue are cleaved by enzymes from the venom of Crotalus atrox and Vipera russellii . Phosphosulfate has been identified as one of the products indicating cleavage between the two phosphate groupings. A similar type of cleavage appears to be catalyzed by an acetone powder of Escherichia coli ; E. coli alkaline phosphatase, however, has no effect on the analogue. Crude potato apyrase slowly hydrolyzes the sulfate analogue but apparently by an enzyme other than the apyrase. Enzyme-free controls in almost every case were stable, some for periods as long as 54 hours at 25 °C and pH 8. This stability is in marked contrast to previously published results, and work reported here indicates the analogue is suitable for enzymic studies.

Resolution of multiple fluorescence lifetimes in heterogeneous systems by phase-modulation fluorometry☆

Procedures are described for the treatment of phase and modulation lifetime data in fluorescent systems having multiexponential decay. All computer procedures (called FIT programs) arise from the lifetime resolution theory for phase-modulation measurements (Weber, G. (1981) J. Phys. Chem. 85, 949-953). The programs most successful in resolving heterogeneous lifetimes use a Monte Carlo approach in which phase and modulation lifetime data at three modulation frequencies are simultaneously utilized. These programs are shown to have more utility than the final closed form procedure presented by Weber (1981). The FIT routines are simple and require little computer time while yielding excellent results. To illustrate the applicability of these programs, defined binary (carbazole and pyrene) and ternary systems (carbazole, pyrene and POPOP) were examined. In most cases, the resolved lifetimes were within 5% of the independently measured value and the fractional fluorescence contributions were within 10% of that expected. These results demonstrate that phase-modulation measurements analyzed by appropriate computer programs are capable of solving for lifetimes in both binary and, in selected cases, ternary systems. An example is given from the recent literature (Dalbey, R., Weiel, J. and Yount, R.G. (1983) Biochemistry 22, 4696-4706) in which the above programs allowed the resolution of both binary and ternary lifetimes of a dansyl label on myosin, where Förster energy transfer was occurring. These lifetimes were used to quantify changes in distances between two activity-related thiols on myosin upon the addition of Mg-ATP or its analogs.

Adenosine-5′-sulfatopyrophosphate, an analogue of adenosine triphosphate: II. Interaction with myosin, actomyosin, and muscle fibers☆

Abstract The ATP analogue, adenosine-5′-sulfatopyrophosphate (ADP-sulfate), is not a substrate for either myosin or actomyosin and will not replace ATP as an energy source for contraction of glycerol-extracted muscle fibers or superprecipitation of actomyosin gels. Preincubation of actomyosin with ADP-sulfate inhibits superprecipitation at all ATP concentrations. Similar treatment activates actomyosin ATPase activity at high ATP concentrations but inhibits at low ATP concentrations. Adding ATP and ADP-sulfate together eliminates these effects. Myosin Ca ++ - and Mg ++ -moderated ATPase activity is uncompetitively inhibited by ADP-sulfate indicating that it does not bind to the normal ATP binding site. The inhibition is reversible as judged by the absence of permanent effects on myosin ATPase activity. A rapid mixing device has been used to mix actomyosin and ATP reproducibly for assaying superprecipitation by the increase in turbidity at 660 mμ. This method has the advantage that the resulting “contracted” gel particles are so small that no mixing is necessary for consistent absorbancy readings.

Subfragment 1: The first crystalline motor

The solution of the crystal structure of subfragment I (Sl) from chicken skeletal myosin as recently reported by Ivan Rayment and co-workers in Science (Rayment et al., 1993a) represents the start of a new era in muscle research. This landmark structure combined with the earlier solution of the crystal structure of G-actin by Wolfgang Kabsch and the Holmes group in Heidelberg (Kabsch et al., 1990) and the resultant atomic model of F-actin by Ken Holmes and co-workers (Holmes et al., 1990) means that the molecular structures of the two major players in muscle contraction are now known. This information has already lead to dramatic new insights (Rayment et al., 1993b) by fitting the Sl and actin structures into the envelope of the Sl decorated F-actin structure calculated by 3D reconstruction of cryoelectron micrographs (Milligan et al., 1990). But first, why was Rayment successful in solving the Sl structure when many other laboratories were not? Several reasons stand out. First was the decision to use Sl with two intact light chains prepared with papain, a preparation developed in the Lowey laboratory, rather than the more universally studied smaller chymotryptic SI in which the regulatory light chain is missing. In the papain Sl crystals the regulatory light chains form important contacts between adjacent molecules and likely play a critical role in the crystallization. A second factor was Rayment’s hunch that if he modified the c-amino groups of all surface lysines, the resulting Sl would still have the correct molecular structure but would crystallize more readily. He was right. While initial attempts at acetylating the lysines led to too much heterogeneity, reductive methylation (suggested by D. Winkelmann) proved to be the key. This mild modification preserves the net positive charge of lysine and simply increases the bulk and hydrophobicity of the e-amino group. About 97% of the lysines in Sl were converted into e-dimethyl lysines without detectable alteration of other residues. Without this alteration, no diffraction size crystals have been grown. Some six years were spent optimizing this modification (e.g., it requires the use of dimethylaminob-