Nucleotide-induced changes in the heat capacity of beef cardiac myosin

The heat of reaction of ATP with beef cardiac myosin has been determined in a flow microcalorimeter at two different temperatures. The reaction heat obtained by extrapolation to infinite flow rate is interpreted to be the heat of formation of the steady-state set of myosin-nucleotide complexes. The large temperature dependence of this heat, an apparent change in heat capacity, could be caused by an isomerization between two myosin conformations. The enthalpies and heat capacities of binding of ADP, AMP, and pyrophosphate have also been measured and are discussed in terms of this model.     

Nucleotide-induced changes in the proteolytically sensitive regions of myosin subfragment 1

Limited proteolytic digestions of myosin subfragment 1 (S-1) with elastase, subtilisin, papain, and thermolysin yield fragments that correspond within 1-2K daltons to the 25K, 50K, and 20K fragments produced by trypsin. While papain and thermolysin cut preferentially at the 26K/70K junction, elastase and subtilisin cleave both the 26K/70K and the 75K/22K junctions in S-1. Using the above proteases as conformational probes, we have previously demonstrated that the binding of actin is sensed at both the 26K/50K and the 50K/22K junctions [Applegate, D., & Reisler, E. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 7109-7112]. We report here that the binding of nucleotides at the active site is also sensed at both junctions. Both 2 mM MgADP and 5 mM MgATP slow the rate of elastase and subtilisin cleavage of the 95K heavy chain. With elastase, the 3-fold decrease in the rate of cleavage induced by nucleotides is evidenced at both the 26K/50K and the 50K/22K junctions. The analysis of subtilisin digestions is complicated by Mg nucleotide induced cleavage at a new site to produce a 91K fragment. Using N-methyl-6-anilinonaphthalene-2-sulfonyl chloride (MnsCl) to fluorescently label the 26K peptide, we demonstrate that the additional cleavage site is approximately 4K daltons from the N-terminal portion of the 95K heavy chain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nucleotide-induced conformations in the neck region of dimeric kinesin

The neck region of kinesin constitutes a key component in the enzyme's walking mechanism. Here we applied cryoelectron microscopy and image reconstruction to investigate the location of the kinesin neck in dimeric and monomeric constructs complexed to microtubules. To this end we enhanced the visibility of this region by engineering an SH3 domain into the transition between neck linker and neck coiled coil. The resulting chimeric kinesin constructs remained functional as verified by physiology assays. In the presence of AMP-PNP the SH3 domains allowed us to identify the position of the neck in a well defined conformation and revealed its high flexibility in the absence of nucleotide. We show here the double-headed binding of dimeric kinesin along the same protofilament, which is characterized by the opposite directionality of neck linkers. In this configuration the neck coiled coil appears fully zipped. The position of the neck region in dimeric constructs is not affected by the presence of the tubulin C-termini as confirmed by subtilisin treatment of microtubules prior to motor decoration.     

Nucleotide-induced states of myosin subfragment 1 cross-linked to actin

Actomyosin interactions and the properties of weakly bound states in carbodiimide-cross-linked complexes of actin and myosin subfragment 1 (S-1) were probed in tryptic digestion, fluorescence, and thiol modification experiments. Limited proteolysis showed that the 50/20K junction on S-1 was protected in cross-linked acto-S-1 from trypsin even under high-salt conditions in the presence of MgADP, MgAMPPNP, and MgPPi (mu = 0.5 M). The same junction was exposed to trypsin by MgATP and MgATP gamma S but mainly on S-1 cross-linked via its 50K fragment to actin. p-Phenylenedimaleimide-bridged S-1, when cross-linked to actin, yielded similar tryptic cleavage patterns to those of cross-linked S-1 in the presence of MgATP. By using p-nitrophenylenemaleimide, it was found that the essential thiols of cross-linked S-1 were exposed to labeling in the presence of MgATP and MgATP gamma S in a state-specific manner. In contrast to this, the reactive thiols were protected from modification in the presence of MgADP, MgAMPPNP, and MgPPi at mu = 0.5 M. These modifications were compared with similar reactions on isolated S-1. Experiments with pyrene-actin cross-linked to S-1 showed enhancement of fluorescence intensity upon additions of MgATP and MgATP gamma S, indicating the release of the pyrene probe on actin from the sphere of S-1 influence. The results of this study contrast the "open" structure of weakly bound actomyosin states to the "tight" conformation of rigor complexes.     

Simultaneous observation of tail and head movements of myosin V during processive motion

Processive stepping of myosin Va (myoV) has been tracked by monitoring either the tail position (center of mass) or the position of one or both heads. Here, we combine these two approaches by attaching a quantum dot to one of the motor domains and a bead to the tail. Using laser trapping and total internal reflection microscopy, the position of one head and the tail are observed simultaneously as myoV moves processively on an actin filament bundle against the resistive load of the laser trap. The head moves one step (73 ± 10 nm) for every two steps of the tail (35 ± 9 nm). One tail step occurs concurrently with quantum dot-labeled head movement, whereas the other occurs with movement of the unlabeled head, consistent with a hand-over-hand model. Load increases the probability of the motor taking a back step. The back step is triggered by the motor taking a shorter forward step (head step, 68 ± 11 nm; tail step, 32 ± 10 nm), likely one actin monomer short of its preferred binding site. During a back step, the motor reverses its hand-over-hand motion, with the leading head detaching and reattaching to one of multiple actin sites behind the trailing head. After a back step, the motor can correct its mistake and step processively forward at resistive loads <0.7 piconewton or stall or detach at higher loads. Back stepping may provide a mechanism to ensure efficient cargo delivery even when myoV encounters obstacles within the actin cytoskeletal meshwork or when other motors are attached to the same cargo.     

The myosin converter domain modulates muscle performance

Myosin is the molecular motor that powers muscle contraction as a result of conformational changes during its mechanochemical cycle. We demonstrate that the converter, a compact structural domain that differs in sequence between Drosophila melanogaster myosin isoforms, dramatically influences the kinetic properties of myosin and muscle fibres. Transgenic replacement of the converter in the fast indirect flight muscle with the converter from an embryonic muscle slowed muscle kinetics, forcing a compensatory reduction in wing beat frequency to sustain flight. Conversely, replacing the embryonic converter with the flight muscle converter sped up muscle kinetics and increased maximum power twofold, compared to flight muscles expressing the embryonic myosin isoform. The substitutions also dramatically influenced in vitro actin sliding velocity, suggesting that the converter modulates a rate-limiting step preceding cross-bridge detachment. Our integrative analysis demonstrates that isoform-specific differences in the myosin converter allow different muscle types to meet their specific locomotion demands.     

Effect of adenosine 5'-[beta,gamma-imido]triphosphate on myosin head domain movements.

Conventional and saturation transfer electron paramagnetic resonance spectroscopy (EPR and ST EPR) was used to study the orientation of probe molecules in muscle fibers in different intermediate states of the ATP hydrolysis cycle. A separate procedure was used to obtain ST EPR spectra with precise phase settings even in the case of samples with low spectral intensity. Fibers prepared from rabbit psoas muscle were labeled with isothiocyanate spin labels at the reactive thiol sites of the catalytic domain of myosin. In comparison with rigor, a significant difference was detected in the orientation-dependence of spin labels in the ADP and adenosine 5'-[beta,gamma-imido]triphosphate (AdoPP[CH2]P) states, indicating changes in the internal dynamics and domain orientation of myosin. In the AdoPP[CH2]P state, approximately half of the myosin heads reflected the motional state of ADP-myosin, and the other half showed a different dynamic state with greater mobility.     

Postural adjustments to head movements in the cat

Head movements induced by motor cortex stimulation in the cat are accompanied by variations in the vertical force exerted by each limb. These postural responses were found to show stereotyped patterns: with head dorsiflexions an increase was observed in the force exerted by the anterior limbs and a decrease at the posterior limb level. From comparison between the latencies of the force variations, the beginning of head acceleration, and EMG activity in the limb extensor muscles, it was concluded that triggering of these postural responses is not reflex, but depends on the same command as the movement itself. This early response might be a means of avoiding the downward movement of the trunk which would otherwise result from the reaction force corresponding to the upward head movement.     

Evidence for inserted sequences in the head region of nonmuscle myosin specific to the nervous system. Cloning of the cDNA encoding the myosin heavy chain-B isoform of vertebrate nonmuscle myosin.

The complete amino acid sequence of a vertebrate nonmuscle myosin heavy chain-B isoform (MHC-B, 1976 amino acids, 229 kDa) has been deduced by using cDNA clones from chicken brain libraries. The chicken nonmuscle MHC-B shows overall similarity in primary structure to other MHCs in the areas contributing to the ATP-binding site and actin-binding site. Similar to other nonsarcomeric MHC IIs, there is a short uncoiled tail sequence at the carboxyl terminus of the molecule. It is in the uncoiled tail sequence that the greatest number of differences in amino acids sequence between MHC-A and B were found, which allowed generation of isoform-specific antibodies. These antibodies were used to determine the relative content of MHC-A and MHC-B in various tissues. During the cloning of the cDNA encoding chicken brain MHC-B, we found a 63-nucleotide insertion encoding 21 amino acids located in the head region of the MHC near to the actin-binding site and a 30 nucleotide insertion encoding 10 amino acids near to the ATP-binding site. Analysis using S-1 nuclease showed that both inserts are expressed in a tissue-dependent manner; mRNA containing the inserts is present in tissues of the nervous system, but is absent from other non-muscle cells, which contain the noninserted isoform of MHC-B. Similar inserts were found in corresponding positions in human cerebellar mRNA. Antibodies raised against a peptide synthesized based on the 21 amino acid insert found in chickens recognize a MHC isoform in the same tissues that are enriched for the mRNA. These insertions appear to be a mechanism for generating additional MHC-B isoforms specific to the nervous system.     

Nucleotide-induced changes in the interaction of myosin subfragment 1 with actin: detection by antibodies against the N-terminal segment of actin

The binding of myosin subfragment 1 (S-1) to actin in the presence and absence of nucleotides was determined under conditions of partial saturation of actin, up to 80%, by Fab(1-7), the antibodies against the first seven N-terminal residues on actin. In the absence of nucleotides, the binding constant of S-1 to actin (2 x 10(7) M-1) was decreased by 1 order of magnitude by Fab(1-7). The binding of S-1 to actin caused only limited displacement of Fab, and between 30 and 50% of actin appeared to bind both proteins. In the presence of MgAMP.PNP, MgADP, and MgPPi and at low S-1 concentrations, the same antibodies caused a large decrease in the binding of S-1 to actin. However, the binding of S-1.nucleotide to actin in the presence of Fab(1-7) increased cooperatively with the increase in S-1 concentration. Also, in contrast to rigor conditions, there was no indication for the binding of Fab(1-7) and S-1.nucleotide to the same actin molecules. These results show a nucleotide-induced transition in the actomyosin interface, most likely related to the different roles of the N-terminal segment of actin in the binding of S-1 and S-1.nucleotide. The possible implications of these findings to the regulation of actomyosin interactions are discussed.     

Thiol reactivity as a sensor of rotation of the converter in myosin

Smooth muscle myosin has two reactive thiols located near the C-terminal region of its motor domain, the “converter”, which rotates by ∼70° upon the transition from the “nucleotide-free” state to the “pre-power stroke” state. The incorporation rates of a thiol reagent, 5-(((2-iodoacetyl)amino)ethyl)aminonaphthalene-1-sulfonic acid (IAEDANS), into these thiols were greatly altered by adding ATP or changing the myosin conformation. Comparisons of the myosin structures in the pre-power stroke state and the nucleotide-free state explained why the reactivity of both thiols is especially sensitive to a conformational change around the converter, and thus can be used as a sensor of the rotation of the converter. Modeling of the myosin structure in the pre-power stroke state, in which the most reactive thiol, “SH1”, was selectively modified with IAEDANS, revealed that this label becomes an obstacle when the converter completely rotates toward its position in the pre-power stroke state, thus resulting in incomplete rotation of the converter. Therefore, we suggest that the limitation of the converter rotation by modification causes the as-yet unexplained phenomena of SH1-modified myosin, including the inhibition of 10S myosin formation and the losses in phosphorylation-dependent regulation of the basic and actin-activated Mg-ATPase activities of myosin.     

Nucleotide-induced specific fluorescent labeling of the 23-kDa NH2-terminal tryptic peptide of myosin ATPase by the serine-reactive reagent 9-anthroylnitrile

The fluorescent reagent 9-anthroylnitrile (ANN) reacted preferentially with serine among various amino acids tested. When the myosin subfragment-1 (S-1) was incubated with ANN, the 9-anthroyl (AN) group was covalently incorporated into the S-1 heavy chain. The incorporation of the AN group was enhanced by the presence of ATP and ADP. In the presence of ATP, 0.98 mol of the AN group was maximally incorporated into S-1. The resulting S-1 derivative exhibited four absorption maxima in the range of 300-400 nm and fluoresced strongly with an emission maximum at 462 nm upon excitation at 390 nm. The spectral properties were similar to those of the AN-derivatives of serine and polyserine. When 0.98 mol of the AN group was incorporated into S-1, the K+- and Ca2+-ATPase activities decreased to 30%, while the Mg2+-ATPase activity increased to 220% of the original value. Tryptic digestion of the labeled S-1 revealed that the AN group was attached only to the NH2-terminal 23-kDa tryptic peptide of the S-1 heavy chain. Neither the 20-nor the 50-kDa peptide was labeled with ANN. The results suggest that a serine residue, which becomes more reactive in the presence of the nucleotide, is located in the 23-kDa tryptic peptide of S-1.     

A conformational transition in the myosin VI converter contributes to the variable step size

Myosin VI (MVI) is a dimeric molecular motor that translocates backwards on actin filaments with a surprisingly large and variable step size, given its short lever arm. A recent x-ray structure of MVI indicates that the large step size can be explained in part by a novel conformation of the converter subdomain in the prepowerstroke state, in which a 53-residue insert, unique to MVI, reorients the lever arm nearly parallel to the actin filament. To determine whether the existence of the novel converter conformation could contribute to the step-size variability, we used a path-based free-energy simulation tool, the string method, to show that there is a small free-energy difference between the novel converter conformation and the conventional conformation found in other myosins. This result suggests that MVI can bind to actin with the converter in either conformation. Models of MVI/MV chimeric dimers show that the variability in the tilting angle of the lever arm that results from the two converter conformations can lead to step-size variations of ∼12 nm. These variations, in combination with other proposed mechanisms, could explain the experimentally determined step-size variability of ∼25 nm for wild-type MVI. Mutations to test the findings by experiment are suggested.     

Coordination of eye and head movements in cats

Horizontal displacements of gaze in cats with unrestrained head were studied using the magnetic search coil method. Three types of eye-head coordination were found when cats oriented gaze towards visual targets. Maximal velocities of gaze, head and eye movements in orbits depend linearily on amplitudes of their displacements in the range of up to 20 degrees. Gaze velocity reached its top level in about 0.3 of complete time of movement execution. Data support the idea of saccadic-vestibular summation during coordinated eye-head movements in cats.     

Sensory processing in the vestibular nuclei during active head movements

Many secondary vestibular neurons are sensitive to head on trunk rotation during reflex-induced and voluntary head movements. During passive whole body rotation the interaction of head on trunk signals related to the vestibulo-collic reflex with vestibular signals increases the rotational gain of many secondary vestibular neurons, including many that project to the spinal cord. In some units, the sensitivity to head on trunk and vestibular input is matched and the resulting interaction produces an output that is related to the trunk velocity in space. In other units the head on trunk inputs are stronger and the resulting interaction produces an output that is larger during the reflex. During voluntary head movements, inputs related to head on trunk movement combine destructively with vestibular signals, and often cancel the sensory reafferent consequences of self-generated movements. Cancellation of sensory vestibular signals was observed in all of the antidromically identified secondary vestibulospinal units, even though many of these units were not significantly affected by reflexive head on trunk movements. The results imply that the inputs to vestibular neurons related to head on trunk rotation during reflexive and voluntary movements arise from different sources. We suggest that the relative strength of reflexive head on trunk input to different vestibular neurons might reflect the different functional roles they have in controlling the posture of the neck and body.     

A unified theory of the control of actin and myosin in nonmuscle movements

A theory is advanced that all nonmuscle movements involving actin and myosin are controlled by Ca⁺⁺ flows across membranes as determined by chemical and electrical processes at those membranes. The mechanisms of amoeboid motion, cytoplasmic streaming, and other movements are discussed, and their underlying chemical control reactions are analyzed. Several predictions and experimental tests are suggested. This theory links motility with recent findings on excitable membranes and cyclic AMP.