Measurement of ATPase Activities of Myosin at the Level of Tracks and Single Molecules

Conibear, Paul B.; Kuhlman, Philip A.; Bagshaw, Clive R.

doi:10.1007/978-1-4684-6039-1_3

Cited by 18 publications

(30 citation statements)

References 26 publications

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“…The biosensor (Fig. 1) contains a fluorescently labeled (Alexa Fluor 488, the FRET donor; Invitrogen) chicken-gizzard smooth muscle regulatory light chain (cgRLC) exchanged onto skeletal muscle HMM (SI Appendix) and a fluorescent nucleotide (Cy3-ATP, the FRET acceptor) bound to the myosin nucleotide binding pocket (17)(18)(19). We used the cgRLC because it contains a single reactive Cys and can be exchanged efficiently with the native nonlabeled RLC under conditions that do not affect ATPase activity (SI Appendix, Fig.…”

Section: Significancementioning

confidence: 99%

Direct real-time detection of the structural and biochemical events in the myosin power stroke

Muretta

Rohde

Johnsrud

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

156

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A principal goal of molecular biophysics is to show how protein structural transitions explain physiology. We have developed a strategic tool, transient time-resolved FRET [(TR) 2 FRET], for this purpose and use it here to measure directly, with millisecond resolution, the structural and biochemical kinetics of muscle myosin and to determine directly how myosin's power stroke is coupled to the thermodynamic drive for force generation, actin-activated phosphate release, and the weak-to-strong actin-binding transition. We find that actin initiates the power stroke before phosphate dissociation and not after, as many models propose. This result supports a model for muscle contraction in which power output and efficiency are tuned by the distribution of myosin structural states. This technology should have wide application to other systems in which questions about the temporal coupling of allosteric structural and biochemical transitions remain unanswered.FRET | myosin | power stroke | phosphate release | structural kinetics M yosin family proteins use ATP hydrolysis to generate force and movement required for normal physiology. They drive muscle contraction, help control cell division and cellular motility, move organelles through the cytoplasm, and are important elements of the cellular mechanical-sensing machinery (1, 2). The key to understanding how myosin and related enzymes function in cells, and how to modulate their activity to treat disease, is to determine how the protein's structural dynamics and biochemical kinetics are coupled. Although high-resolution crystal structures provide best-guess snapshots of protein structure over a range of biochemical states, determining the physiological relevance of these snapshots remains one of the central challenges of structural biophysics.How myosin generates force remains debated despite more than 50 y of intense research (1-3). The most popular current model (2, 4) proposes that after ATP hydrolysis, myosin interacts weakly with actin and this interaction initiates an ordered series of structural and biochemical transitions that culminate in the dissociation of hydrolyzed phosphate, followed by the isomerization of the actin-binding interface to a state that binds actin with nanomolar affinity and then the rotation of the myosin lightchain domain (LCD) toward the plus end of the actin filament. This rotation converts the thermodynamic energy of phosphate release and actin binding into mechanical energy that performs work. A number of results question this model, however, including spectroscopic data showing that a structural transition in the myosin relay helix, hypothesized to be coupled to LCD rotation, precedes P i release (5) and force development precedes P i release in muscle fibers (6).Determining how these events take place in solution and in cells is an important question, because (i) differences in the mechanics of different myosins likely reflect differences in how the biochemical and structural transitions described above are coordinated (4); (ii) disea...

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Section: Significancementioning

confidence: 99%

Direct real-time detection of the structural and biochemical events in the myosin power stroke

Muretta

Rohde

Johnsrud

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

156

View full text Add to dashboard Cite

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“…The multipoint attachments of myosin ¢laments to the surface also ensures they are well immobilized and less likely to detach and reattach during the assay than monomeric myosin (cf. Conibear et al 1998). Although myosin monomers may dissociate and re-bind to immobilized ¢laments, given that exchange clearly occurs between ¢laments in suspension, we calculated that the expected rate is in the order of one molecule per minute per micrometre of ¢lament and, therefore, at low labelling ratios (e.g.…”

Section: Discussionmentioning

confidence: 98%

“…Our optical set-up has been described in detail (Conibear et al 1998;Bagshaw & Conibear 1999). Cy3 £uorophores were excited with a frequency-doubled Nd-YAG laser (mGreen 4301^050, Uniphase) and Cy5 was excited with an HeNe laser (05LHR927, Melles Griot).…”

Section: (B) Total Internal Re£ection £Uorescence Microscopymentioning

confidence: 99%

“…The development of £uorescence microscopy techniques with single-molecule sensitivity o¡ers an opportunity of re-examining this question. Furthermore, it is now possible to resolve single turnovers of myosin ATPase at the level of single molecules by examining the lifetime of £uorescent ATP analogues bound to the myosin active site (Funatsu et al 1995;Conibear et al 1998;Ishijima et al 1998;Oiwa et al 1999). However, £uctuations in £uores-cence can arise via photochemical or photophysical mechansims (Moerner & Orrit 1999).…”

Section: Introductionmentioning

confidence: 99%

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Myosin monomer density and exchange in synthetic thick filaments investigated using fluorescence microscopy with single molecule sensitivity

Conibear

Bagshaw

2000

Proc. R. Soc. Lond. B

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The number of myosin molecules in synthetic thick ¢laments, prepared by dialysis at 0.12 M NaCl and pH 7.0, was estimated to be between 400 and 800 molecules per micrometre under conditions appropriate for in vitro motility assays. This estimate was based on a number count of Cy3-labelled myosin molecules incorporated into ¢laments at a nominal ratio of 1:1000. At this dilution, single £uorescent spots were resolved corresponding to individual labelled myosins. The spots usually bleached with a oneor two-step pro¢le but, in around 30% of the cases, £uctuations were observed indicating that additional photophysical or photochemical events had occurred. Myosin molecules were shown to exchange between ¢laments in suspension on a time-scale of several hours at 4 8C, but the reaction was only 75% complete after 48 h, suggesting a non-exchangeable core. However, myosin exchange does not appear to be the predominant source of the £uctuations in £uorescence intensity in the single molecule assays.

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“…Ideally many more events should be counted, however to record even 30 events from a single myosin active site is quite a challenge (see below for limitations). To date, sufficient data have generally been acquired by pooling events from several tens of molecules [1,11]. The problem becomes more severe if there are indications of two or more populations of enzyme molecules with different kinetic properties, or if the enzyme switches between states of differing activity on a time scale which is longer than the turnover time.…”

Section: Single Molecule Kinetics Versus Macroscopic Kineticsmentioning

confidence: 99%

Single-Molecule Enzymology: Critical Aspects Exemplified by Myosin ATPase Activity

Bagshaw

Conibear

2000

Single Mol.

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The myosin ATPase provides a model system for developing single-molecule enzyme assays because of its slow turnover rate and low K m for fluorescent ATP analogs. While the average lifetime of fluorescence spots arising from Cy3-nucleotide bound to single immobilised myosin sites is close to that expected from the catalytic activity measured in bulk preparations, quantitative comparisons suggest other processes may contribute to fluctuations. Here we review the possible factors involved. We also discuss the benefits and difficulties of measuring the same molecule over repeated cycles. The potential complexity of these assays is illustrated by an example in which Cy3-labelled substrates are apparently turned over by two different myosin molecules in the same bulk environment, but they show inherently different kinetics.

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Measurement of ATPase Activities of Myosin at the Level of Tracks and Single Molecules

Cited by 18 publications

References 26 publications

Direct real-time detection of the structural and biochemical events in the myosin power stroke

Direct real-time detection of the structural and biochemical events in the myosin power stroke

Myosin monomer density and exchange in synthetic thick filaments investigated using fluorescence microscopy with single molecule sensitivity

Single-Molecule Enzymology: Critical Aspects Exemplified by Myosin ATPase Activity

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