John C. Seidel

Rotational dynamics of spin-labeled F-actin in the sub-millisecond time range.

The rotational motions of F-ai HMM and S-l are equally effective, on a molar basis, in slowing this rotation and both produce their maximal effect at a ratio of about one molecule of HMM or S- 1 per ten actin monomers. With chymotryptic S-1, the effect is partially reversed at higher concentrations. With S- 1 prepared with papain in the presence of Mg2 + : the reversal is smaller, while with HMM or myosin there is no reversal at higher concentrations. Tropomyosin slightly decreases the aetin rotational mobility, and the addition of HMM to the actin-tropomyosin complex produces a further slowing. The rotational correlation time for acto-HMM is the same whether the spin-label is on actin or HMM, indicating that the rotation of the head region of HMM when bound to F-a&in is controlled by a mode of rotation within the F-actin filaments.

Submillisecond rotational dynamics of spin-labeled myosin heads in myofibrils.

The rotational motion of crossbridges, formed when myosin heads bind to actin, is an essential element of most molecular models of muscle contraction. To obtain direct information about this molecular motion, we have performed saturation transfer EPR experiments in which spin labels were selectively and rigidly attached to myosin heads in purified myosin and in glycerinated myofibrils. In synthetic myosin filaments, in the absence of actin, the spectra indicated rapid rotational motion of heads characterized by an effective correlation time of 10 microseconds. By contrast, little or no submillisecond rotational motion was observed when isolated myosin heads (subfragment-1) were attached to glass beads or to F-actin, indicating that the bond between the myosin head and actin is quite rigid on this time scale. A similar immobilization of heads was observed in spin-labeled myofibrils in rigor. Therefore, we conclude that virtually all of the myosin heads in a rigor myofibril are immobilized, apparently owing to attachment of heads to actin. Addition of ATP to myofibrils, either in the presence or absence of 0.1 mM Ca2+, produced spectra similar to those observed for myosin filaments in the absence of actin, indicating rapid submillisecond rotational motion. These results indicate that either (a) most of the myosin heads are detached at any instant in relaxed or activated myofibrils or (b) attached heads bearing the products of ATP hydrolysis rotate as rapidly as detached heads.

The conformation of myosin during the steady state of ATP hydrolysis: Studies with myosin spin labeled at the S1 thiol groups☆

Abstract MgATP produces a large change in the ESR spectrum of myosin that has been spin labeled at the S1 thiol groups. This change, indicative of increased mobility of the label, persists during the steady state of the hydrolysis of ATP, and when ATP has been depleted the spectrum changes to one identical with that observed on adding MgADP. It appears that the binding or hydrolysis of ATP by myosin in the presence of Mg induces a conformational change in myosin that persists during the steady state. If, as proposed by Taylor and coworkers (1,2) the predominant species present during the steady state is the myosin-ADP complex, then the conformation of myosin in this complex depends on whether the complex was formed with added ADP or in the course of the hydrolysis of ATP.

Electron spin resonance of myosin spin labeled at the St thiol groups during hydrolysis of adenosine triphosphate

Abstract On the addition of Mg2+ and ATP the electron spin-resonance spectrum of the spin label, N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)-iodoacetamide, selectively bound to the S1 thiol groups of myosin changes from the one characteristic of strong immobilization to one indicating weaker immobilization. The latter spectrum persists during the steady state of hydrolysis of ATP; when hydrolysis is complete it changes to a spectrum identical with that produced by ADP. This third spectrum indicates a mobility between that of the label on myosin in the absence of ATP and that found during the steady state. The same results are obtained with heavy meromyosin or subfragment-1. The appearance of the spectrum typical of the steady state requires the presence of a divalent cation; either Ca2+ or Mg2+ is effective. It also seems to require hydrolysis of ATP since it is not observed in the absence of activating cations, when hydrolysis has been inhibited with N-ethylmaleimide, or when nonhydrolyzable analogs of ATP are used. One of these, β,γ-imino-adenosinetriphosphate, produces the same spectral change as ADP. These different spectra have been interpreted in terms of the kinetic scheme developed by Lymn and Taylor for native myosin in which the rate-limiting step follows the rapid hydrolysis of the terminal phosphate of ATP. Ourpresent observation of an “initial burst” of Piliberation with S1-labeled myosin justifies the application of this scheme. According to this scheme the intermediate responsible for the steady-state spectrum contains the products of ATP hydrolysis but its spectrum is distinct from the complex formed by adding products. This suggests the presence of two spectrally distinct myosin-product complexes. The changes in esr spectra probably reflect localized conformational changes in the head of the myosin molecule. Reducing the pH, temperature, or salt concentration substantially reduces the mobility of the spin labels during the hydrolysis of ATP, suggesting that the conformation of myosin during the steady state may depend on the temperature, pH, and concentration of salt. Alternatively, the spectral changes may be brought about by a change in the relative concentrations of two or more spectrally distinct steady-state intermediates. Changes in these parameters have little or no effect on spectra recorded in the absence of substrate.

The effects of actin on the electron spin resonance of spin-labeled myosin☆

Abstract Myosin and heavy meromyosin have been spin labeled at either the S 1 or S 2 thiol groups, and their interaction with F-actin has been studied by electron spin resonance, both in the absence of substrate and during the hydrolysis of ATP. The spectrum of myosin labeled at either group indicates strong immobilization of the label. In the absence of substrate, actin added to S 1 -labeled myosin slightly increases the separation of the outer spectral peaks, indicating a decrease in the mobility of the spin label. Actin also reduces the microwave power required to saturate the esr signal of S 1 -labeled myosin or heavy meromyosin. The latter phenomenon is a more sensitive measure of the actin-myosin interaction than the spectral change seen in the absence of saturation. This suggests that saturation measurements may provide a more sensitive method of detecting changes in the environment of slowly tumbling nitroxide radicals than spectral measurements carried out in the absence of saturation. The decrease in the amplitude of the spectrum on adding actin at saturating microwave power was used to determine the stoichiometry of the interaction between actin and heavy meromyosin. This decrease is maximal when 2 moles of actin monomer are added per mole of heavy meromyosin and is reversed when actin and myosin are dissociated by ATP. During the steady state hydrolysis of ATP, actin had no detectable effect on the spectrum of S 1 -labeled myosin. It can be concluded that spin labels bound to the S 1 groups are in a region of the myosin molecule that is affected by the interaction with actin. Actin does not affect the rate at which the bound spin label is reduced by dithiothreitol nor does the spin labeling of S 1 groups affect the activation by actin of the ATPase activity of myosin. These findings suggest that the most likely mechanism by which actin alters the mobility of labels on S 1 groups involves a change in the conformation of myosin. If a spin label is bound to the S 2 thiol groups rather than the S 1 groups, then actin has no detectable effect on the spectrum either in the presence or absence of ATP.

Activation by actin of ATPase activity of chemically modified gizzard myosin without phosphorylation

The ATPase activity of myosin from chicken gizzard measured in the presence of either Mg2+ or Ca2+ is increased in the absence of dithiothreitol or upon reaction with Cu2+, o-iodosobenzoate, or N-ethylmaleimide. Iodosobenzoate or Cu2+ produce no change in K+(EDTA)-ATPase while N-ethylmaleimide produces a decrease. These treatments also make the actin-activated ATPase insensitive to Ca2+ when assayed in the presence of tropomyosin and a partially purified myosin light chain kinase. Phosphorylation of N-ethylmaleimide modified myosin remains dependent on Ca2+ and therefore appears not to be required for activation by actin of the ATPase activity of modified myosin.

Chymotryptic heavy meromyosin from gizzard myosin: A proteolytic fragment with the regulatory properties of the intact myosin

Summary Proteolytic cleavage of gizzard myosin using α-chymotrypsin under conditions which produce HMM when applied to skeletal muscle myosin yields an enzymatically active fragment which behaves like the original myosin in the its ATPase activity is activated by actin but only with the participation of Ca 2+ and additional regulatory proteins. In contrast, papain-subfragment-1 from gizzard myosin has an actin-activated ATPase that is dependent neither on Ca 2+ nor on regulatory proteins. The chymotryptic fragment can be phosphorylated by a partially purified preparation of light chain kinase, and contains intact 20,000 and 17,000-dalton light chains and a heavy chain with an apparent molecular weight of approximately 120,000, based on gel electrophoresis in sodium dodecyl sulfate.

The effects of nucleotides and Mg2+ on the electron spin resonance spectra of myosin spin labeled at the S2 thiol groups

Abstract Myosin has been spin labeled at the S2 thiol groups, whose modification leads to loss of Ca2+-ATPase activity, with N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl) maleimide after blocking of more rapidly reacting groups with NEM or DTNB. A loss of K+-ATPase activity and an increase in Ca2+-ATPase accompany the reaction with NEM or DTNB; subsequent spin labeling produces a loss of Ca2+-ATPase activity which is linearly related to the amount of spin label bound and is complete with 2.1 moles of label bound per 500,000 g of myosin. After sequential reaction with DTNB and the spin label, Ca2+-ATPase activity can be restored by treatment with DTT with no change in the ESR spectrum. The S2 groups differ from S1 groups in their reactivity; an iodoacetamide spin label which reacts rapidly and preferentially with S1 groups of native myosin does not react readily with S2 groups of NEM- or DTNB-blocked myosins. In addition, the rate of reaction of S1 groups with the iodoacetamide label is unaffected by ADP while the reaction of S2 groups with the maleimide label is greatly accelerated by ADP. Spin labels bound to S2 groups are strongly immobilized. Their mobility is increased by ATP, ITP, ADP, or PPi, but this effect is somewhat smaller than that previously observed with myosin labeled at the S1 groups. The change in the ESR spectrum of S2 labeled myosin induced by ATP reflects binding of the nucleotide since it does not require hydrolysis and the same effect is produced by ADP. The additional spectral change observed during ATP hydrolysis with S1-labeled myosin is not observed with S2-labeled myosin. If S2 groups are labeled with a series of maleimide labels in which the number of atoms between the maleimide and nitroxide rings differ, increasing the distance between the two rings of the spin label results in a transition from strong immobilization to weak immobilization. Adenosine triphosphate appears to affect the mobility of only those labels which are strongly immobilized.

The effect of divalent metal binding on the electron spin resonance spectra of spin-labeled actin. Evidence for spin-spin interactions involving manganese. II.

Abstract The electron spin resonance spectra of actin spin labeled with the sulfhydryl reagent, N -(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl) maleimide, in which the divalent metal was Mg 2+ , Ca 2+ , or Mn 2+ were compared. The shapes of the spectra of spin-labeled G-actin were the same for each metal, but with Mn 2+ the size of the signal was smaller. Maximum reduction in the signal, about 50%, was obtained on replacing 1 mole of Mg 2+ or Ca 2+ per mole of actin by Mn 2+ . This effect was reversed if the Mn 2+ was again replaced by Ca 2+ or Mg 2+ . These results appear to reflect a spin-spin interaction between the unpaired electron of the nitroxide radical and those of Mn 2+ . Approximate calculations gave a value of 16–23 A for the distance between magnetic centers in G- or F-actin. Polymerization of actin did not alter the effect on the size of the electron spin resonance spectrum of replacing Mg 2+ by Mn 2+ . Thus, on polymerization the distance between the spin label and Mn 2+ within an actin monomer does not change, nor do detectable magnetic interactions occur in F-actin between the spin label on one actin monomer and Mn 2+ bound to an adjacent monomer.

Saturation transfer electron paramagnetic resonance study of the mobility of myosin heads in myofibrils under conditions of partial dissociation

The rotational motion of rigidly spin-labeled myosin heads of glycerinated myofibrils as reflected in saturation-transfer EPR spectra behaves to a first approximation as though the heads consist of two populations with different rotational motions. An immobilized fraction has a correlation time (tau 2) of approximately 0.5 ms, comparable to that of spin-labeled subfragment-1 (S1) bound to thin filaments, while a mobile fraction has a tau 2 of 10 microseconds, comparable to that of the heads of purified myosin filaments. The effects of nonhydrolyzable ATP analogues, potassium pyrophosphate (PPi), or adenylyl imidodiphosphate, Ca2+, temperature, or ionic strength on the spectra can be analyzed in terms of the fraction of myosin heads immobilized by attachment to thin filaments, without requiring changes in the motion of either attached or detached heads.