Myosin step size

Uyeda, Taro Q.P.; Kron, Stephen J.; Spudich, James A.

doi:10.1016/0022-2836(90)90287-v

Cited by 439 publications

(211 citation statements)

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“…Concentrations up to 1 mM ATP effected no more than 25% release of G680V-S1 from actin in the co-sedimentation assay. This was surprising, since the K m of Dictyostelium S1 for ATP is on the order of 100 M as measured by actin-activated ATPase (17) and the strongly bound state of the cycle has been calculated to be on the order of 5% of the total, at least for rabbit skeletal muscle myosin II (21). If this holds for Dictyostelium, in the presence of millimolar ATP we would expect the majority of S1 to be free.…”

Section: Resultsmentioning

confidence: 99%

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Cold-sensitive Mutants G680V and G691C ofDictyostelium Myosin II Confer Dramatically Different Biochemical Defects

Patterson

Ruppel

et al. 1997

Journal of Biological Chemistry

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Cold-sensitive myosin mutants represent powerful tools for dissecting discrete deficiencies in myosin function. Biochemical characterization of two such mutants, G680V and G691C, has allowed us to identify separate facets of myosin motor function perturbed by each alteration. Compared with wild type, the G680V myosin exhibits a substantially enhanced affinity for several nucleotides, decreased ATPase activity, and overoccupancy or creation of a novel strongly actin-binding state. The properties of the novel strong binding state are consistent with a partial arrest or pausing at the onset of the mechanical stroke. The G691C mutant, on the other hand, exhibits an elevated basal ATPase indicative of premature phosphate release. By releasing phosphate without a requirement for actin binding, the G691C can bypass the part of the cycle involving the mechanical stroke. The two mutants, despite having alterations in glycine residues separated by only 11 residues, have dramatically different consequences on the mechanochemical cycle.The study of the family of molecular motors recently has been invigorated by the availability of crystal structures of both the myosin (1-5) and kinesin family members (6, 7). This information, combined with a wealth of biochemical characterization of these motors, provides a platform on which to build a complete understanding of the mechanics of these pivotal cellular components. In a complementary approach, we have been generating conditionally functional (cold-sensitive) mutants of myosin II in the Dictyostelium system (8, 9). In our isolation of cold-sensitive myosin mutants, we uncovered two mutations in a biochemically renowned region of the protein, a helical element that houses two highly reactive cysteines in rabbit myosin.This region of myosin was first characterized because of the chemical accessibility of two cysteines contained within it (positions 697 (SH2) and 707 (SH1) in rabbit myosin). The crystal structure of chicken pectoralis muscle S1 displays these cysteines in a bent ␣-helix, separated from one another by 18 Å and facing opposite sides of the molecule (2). However, crosslinking experiments using rabbit skeletal muscle myosin demonstrate that during the stroking cycle, the two approach more closely than in the absence of nucleotide (10) and can be directly joined by a disulfide linkage, indicative of a separation of only about 3 Å and a substantially altered geometry (11). The conformational changes are not purely local, in that either cysteine can cross-link with residues in other parts of the structure (for example, SH1-Cys 522 , and SH2-Cys 540 in rabbit (12, 13)) during the cycle. Chemical modification studies have demonstrated that addition of bulky hydrophobic groups to the cysteines dramatically changes the properties of the myosin molecule, particularly with regard to the promiscuity and activity of its ATPase. The physiological substrate of myosin is Mg 2ϩ -ATP which supports only low-level ATPase activity in the absence of actin. However, if provided with Ca 2...

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Section: Resultsmentioning

confidence: 99%

“…Essentially, ATPase activity was measured by liberation of radioactive P i from [␥-32 P]ATP by a modification of the method of Clarke and Spudich (20). In vitro movement assays were performed by observation of fluorescently labeled actin filaments over nitrocellulose surfaces coated with myosin as described by Uyeda et al (21).…”

Section: Methodsmentioning

confidence: 99%

Cold-sensitive Mutants G680V and G691C ofDictyostelium Myosin II Confer Dramatically Different Biochemical Defects

Patterson

Ruppel

et al. 1997

Journal of Biological Chemistry

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“…Finally, we would like to point out that, although single molecule detection has been reported and opens up new possibilities with regard to mechanochemical coupling studies of motor proteins [4], the major discrepancies in the literature concern sliding over assemblies of myosin heads at low loads [6,7]. Thus, it may be desirable to follow the kinetics of nucleotide turnover by a few hundred adjacent myosin heads during unloaded sliding.…”

Section: Discussionmentioning

confidence: 99%

“…However, there is no direct evidence of how many such strokes may be derived from the hydrolysis of a single ATP molecule. Under conditions of low load where actin filaments are sliding near their maximum velocity, there remains a discrepancy in the estimation of the distance travelled per ATP molecule hydrolyzed as calculated from bulk assays [6,7].…”

Section: Introductionmentioning

confidence: 99%

Measurement of nucleotide exchange kinetics with isolated synthetic myosin filaments using flash photolysis

Conibear

Bagshaw

1996

FEBS Letters

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The kinetics of nucleotide release have been measured at the level of isolated synthetic myosin filaments. This was achieved by displacing a rhodamine nucleotide analog from a filament by flash photolysis of caged-ATP with selective observation using total internal reflectance fluorescence microscopy. The procedure gave improved time resolution over previously used flow methods. Kinetics were measured both in the presence of the ADP analog and during steady-state hydrolysis of the ATP analog. These studies open the way for kinetic measurements during actin filament sliding on myosin tracks.

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“…The movement was tracked by fitting the fluorescent spots with a two-dimensional Gaussian distribution function. On the other hand, in myosin II experiments, we performed the in vitro actin gliding assay [12] (Fig. 1) and measured the sliding velocity between the actin filament and myosin II molecules.…”

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Fluctuation Analysis of Mechanochemical Coupling Depending on the Type of Biomolecular Motors

et al. 2008

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Mechanochemical coupling was studied for two different types of myosin motors in cells: myosin V, which carries cargo over long distances by as a single molecule; and myosin II, which generates a contracting force in cooperation with other myosin II molecules. Both mean and variance of myosin V velocity at various [ATP] obeyed Michaelis-Menten mechanics, consistent with tight mechanochemical coupling. Myosin II, working in an ensemble, however, was explained by a loose coupling mechanism, generating variable step sizes depending on the ATP concentration and realizing a much larger step (200 nm) per ATP hydrolysis than myosin V through its cooperative nature at zero load. These different mechanics are ideal for the respective myosin's physiological functions.PACS numbers: 87.16. Nn, 05.10.Gg, Molecular motors play essential roles in various physiological functions, such as muscle contraction, molecular transport, cell motility and cell division through displacements powered by the chemical energy of ATP hydrolysis. The primary goal of molecular motor biophysical studies is to understand how chemical energy is converted into mechanical movement, i.e. mechanochemical coupling. Single molecule techniques are a powerful tool to investigate chemical reactions and mechanical movements by molecular motors because they can directly observe the elemental reaction process that is undetectable in ensemble average measurements [1,2]. This is especially true for motors like myosin V, which function as a single molecule or in cooperation with a small number of other myosin V molecules [3]. By combining single molecule studies with in vitro kinetics studies, myosin V is thought to produce regular 36 nm steps coupled to one ATP hydrolysis [4]. This means that the mechanochemical coupling of myosin V is a one-to-one relationship, i.e. "tight coupling", although no direct measurement has shown this relationship. In contrast, little is known about the mechanochemical coupling of ensemble functioning motors, such as myosin II in muscle. Because of its poor stepping ability, mechanical steps are easily buried in thermal noise. This makes direct detection of individual mechanical events difficult [5], leaving most to discuss only the average displacement [6]. Furthermore, because in vivo myosin II work in ensemble, single molecule characteristics may not accurately reflect the mechanochemical coupling of myosin II. In fact, multi-molecule systems show various characteristics unpredictable from single molecule studies [7,8]. Therefore, the mechanochemical coupling of myosin II is still poorly understood.In order to examine mechanochemical coupling of both kinds of molecular motors, in this report, we investigated the characteristics of fluctuation in myosin V and myosin II motility and discuss their mechanochemical coupling consistently. Since these fluctuations contain the underlying molecular process, we can extract pertinent information of mechanochemical coupling from fluctuation analysis, as Schnitzer et al. did to show one kines...

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Myosin step size

Cited by 439 publications

References 26 publications

Cold-sensitive Mutants G680V and G691C ofDictyostelium Myosin II Confer Dramatically Different Biochemical Defects

Cold-sensitive Mutants G680V and G691C ofDictyostelium Myosin II Confer Dramatically Different Biochemical Defects

Measurement of nucleotide exchange kinetics with isolated synthetic myosin filaments using flash photolysis

Fluctuation Analysis of Mechanochemical Coupling Depending on the Type of Biomolecular Motors

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