The self-assembly, elasticity, and dynamics of cardiac thin filaments.

Solutions of intact cardiac thin filaments were examined with transmission electron microscopy, dynamic light scattering (DLS), and particle-tracking microrheology. The filaments self-assembled in solution with a bell-shaped distribution of contour lengths that contained a population of filaments of much greater length than the in vivo sarcomere size ( approximately 1 mum) due to a one-dimensional annealing process. Dynamic semiflexible modes were found in DLS measurements at fast timescales (12.5 ns-0.0001 s). The bending modulus of the fibers is found to be in the range 4.5-16 x 10(-27) Jm and is weakly dependent on calcium concentration (with Ca2+ > or = without Ca2+). Good quantitative agreement was found for the values of the fiber diameter calculated from transmission electron microscopy and from the initial decay of DLS correlation functions: 9.9 nm and 9.7 nm with and without Ca2+, respectively. In contrast, at slower timescales and high polymer concentrations, microrheology indicates that the cardiac filaments act as short rods in solution according to the predictions of the Doi-Edwards chopsticks model (viscosity, eta approximately c(3), where c is the polymer concentration). This differs from the semiflexible behavior of long synthetic actin filaments at comparable polymer concentrations and timescales (elastic shear modulus, G' approximately c(1.4), tightly entangled) and is due to the relative ratio of the contour lengths ( approximately 30). The scaling dependence of the elastic shear modulus on the frequency (omega) for cardiac thin filaments is G' approximately omega(3/4 +/- 0.03), which is thought to arise from flexural modes of the filaments.

Myosin VI: a multifunctional motor.

Myosin VI moves towards the minus end of actin filaments unlike all the other myosins so far studied, suggesting that it has unique properties and functions. Myosin VI is present in clathrin-coated pits and vesicles, in membrane ruffles and in the Golgi complex, indicating that it has a wide variety of functions in the cell. To investigate the cellular roles of myosin VI, we have identified a variety of myosin VI-binding partners and characterized their interactions. As an alternative approach, we have studied the in vitro properties of intact myosin VI. Previous studies assumed that myosin VI existed as a dimer but our biochemical characterization and electron microscopy studies reveal that myosin VI is a monomer. Using an optical tweezers force transducer, we showed that monomeric myosin VI is a non-processive motor with a large working stroke of 18 nm. Potential roles for myosin VI in cells are discussed.

A second generation apparatus for time-resolved electron cryo-microscopy using stepper motors and electrospray.

We describe here a second generation apparatus for studying transient reaction conformations in macromolecules and their complexes by electron cryo-microscopy. Reactions are trapped by rapid freezing in times ranging from a few milliseconds to tens of seconds after initiation. Blotting of the electron microscope grid and freezing it in liquid ethane uses computer controlled microstepping motors. For the fastest time resolution, a blotted grid containing a thin film of one reactant is sprayed with small droplets containing a second reactant just before freezing. The spray is produced electrically (electrospray), which gives a dense cloud of droplets <1 microm in diameter from the 1-2 microl of solution required per grid. A second method in which two solutions are first mixed by turbulent flow and then blotted prior to freezing is used for reactions with time courses >1s.

Role of titin in vertebrate striated muscle.

Titin is a giant muscle protein with a molecular weight in the megaDalton range and a contour length of more than 1 microm. Its size and location within the sarcomere structure determine its important role in the mechanism of muscle elasticity. According to the current consensus, elasticity stems directly from more than one type of spring-like behaviour of the I-band portion of the molecule. Starting from slack length, extension of the sarcomere first causes straightening of the molecule. Further extension then induces local unfolding of a unique sequence, the PEVK region, which is named due to the preponderance of these amino-acid residues. High speeds of extension and/or high forces are likely to lead to unfolding of the beta-sandwich domains from which the molecule is mainly constructed. A release of tension leads to refolding and recoiling of the polypeptide. Here, we review the literature and present new experimental material related to the role of titin in muscle elasticity. In particular, we analyse the possible influence of the arrangement and environment of titin within the sarcomere structure on its extensible behaviour. We suggest that, due to the limited conformational space, elongation and compression of the molecule within the sarcomere occur in a more ordered way or with higher viscosity and higher forces than are observed in solution studies of the isolated protein.

Stability and folding rates of domains spanning the large A-band super-repeat of titin.

Titin is a very large (>3 MDa) protein found in striated muscle where it is believed to participate in myogenesis and passive tension. A prominent feature in the A-band portion of titin is the presence of an 11-domain super-repeat of immunoglobulin superfamily and fibronectin-type-III-like domains. Seven overlapping constructs from human cardiac titin, each consisting of two or three domains and together spanning the entire 11-domain super-repeat, have been expressed in Escherichia coli. Fluorescence unfolding experiments and circular dichroism spectroscopy have been used to measure folding stabilities for each of the constructs and to assign unfolding rates for each super-repeat domain. Immunoglobulin superfamily domains were found to fold correctly only in the presence of their C-terminal fibronectin type II domain, suggesting close and possibly rigid association between these units. The domain stabilities, which range from 8.6 to 42 kJ mol(-1) under physiological conditions, correlate with previously reported mechanical forces required to unfold titin domains. Individual domains vary greatly in their rates of unfolding, with a range of unfolding rate constants between 2.6 x 10(-6) and 1.2 s(-1). This variation in folding behavior is likely to be an important determinant in ensuring independent folding of domains in multi-domain proteins such as titin.

Flexibility and extensibility in the titin molecule: analysis of electron microscope data.

Muscle elasticity derives directly from titin extensibility, which stems from three distinct types of spring-like behaviour of the I-band portion of the molecule. With progressively greater forces and sarcomere lengths, the molecule straightens and then unfolds, first in the PEVK-region and then in individual immunoglobulin domains. Here, we report quantitative analysis of flexibility and extensibility in isolated titin molecules visualized by electron microscopy. Conformations displayed by molecules dried on a substrate vary from a random coil to rod-like, demonstrating highly flexible and easily deformable tertiary structure. The particular conformation observed depends on the "wettability" of the substrate during specimen preparation: higher wettability favours coiled conformations, whereas lower wettability results in more extended molecules. Extension is shown to occur during liquid dewetting. Statistical methods of conformational analysis applied to a population of coiled molecules gave an average persistence length 13.5(+/-4.5) nm. The close correspondence of this value to an earlier one from light-scattering studies confirms that conformations observed by microscopy closely reflected the equilibrium conformation in solution. Analysis of hydrodynamic forces exerted during dewetting also indicates that the force causing straightening of the molecules and extension of the PEVK-region is in the picoNewton range, whereas unfolding of the immunoglobulin and fibronectin domains may require forces about tenfold higher. The microscope data directly illustrate the relationship between titin conformation and the magnitude of applied force. They also suggest the presence of torsional stiffness in the molecule, which may affect considerations of elasticity.

Titin and the sarcomere symmetry paradox.

Titin is thought to play a major role in myofibril assembly, elasticity and stability. A single molecule spans half the sarcomere and makes interactions with both a thick filament and the Z-line. In the unit cell structure of each half sarcomere there is one thick filament with 3-fold symmetry and two thin filaments with approximately 2-fold symmetry. The minimum number of titin molecules that could satisfy both these symmetries is 12. We determined the actual number of titin molecules in a unit cell from scanning transmission electron microscopy mass measurements of end-filaments. One of these emerges from each tip of the thick filament and is thought to be the in-register aggregate of the titin molecules associated with the filament. The mass per unit length of the end-filament (17.1 kDa/nm) is consistent with six titin molecules not 12. Thus the number of titin molecules present is insufficient to satisfy both symmetries. We suggest a novel solution to this paradox in which four of the six titin molecules interact with the two thin filaments in the unit cell, while the remaining two interact with the two thin filaments that enter the unit cell from the adjacent sarcomere. This arrangement would augment mechanical stability in the sarcomere.

A role for C-protein in the regulation of contraction and intracellular Ca2+ in intact rat ventricular myocytes.

1. C-protein is a major component of muscle thick filaments whose function is unknown. We have examined for the first time the role of the regulatory binding domain of C-protein in modulating contraction and intracellular Ca2+ concentration ([Ca2+]i) in intact cardiac myocytes. 2. Rat ventricular myocytes were reversibly permeabilised with the pore-forming toxin streptolysin O. Myosin S2 (which binds to the regulatory domain of C-protein) was introduced into cells during permeabilisation to compete with the endogenous C-protein-thick filament interaction. 3. Introduction of S2 into myocytes increased contractility by approximately 30%, significantly lengthened the time to peak of the contraction and the time to half-relaxation, but had no effect on [Ca2+]i transient amplitude. 4. Our data are consistent with increased myofilament Ca2+ sensitivity when there is reduced binding of C-protein to myosin near the head-tail junction. 5. We propose that the effects of introducing S2 into intact cardiac cells can be equated with the consequences of selectively phosphorylating C-protein in vivo, and that the regulation of contraction by C-protein is mediated by the effects of crossbridge cycling on the Ca2+ affinity of troponin C.

Extensibility in the titin molecule and its relation to muscle elasticity.

Studies of the origins of muscle passive tension have revealed a direct relationship between elasticity and the mechanical properties of the titin molecule. 'Molecular combing' has made it possible to visualize with high resolution changes in the configuration and structure of isolated titin caused by mechanical forces. The differential extensibility seen in individual molecules is consistent with the important role suggested for the PEVK-region in muscle elasticity. An additional factor emphasizing compliance of this part of the molecule in muscle may relate to the arrangement of the titin filament system in the sarcomere, in particular to titin interactions with thick and thin filaments. The branching of titin network near the PEVK-region suggests that, in addition to conferring extensibility, it may also be important in facilitating the transition of titin intermolecular interactions between the arrays of thick and thin filaments.

Two-headed binding of a processive myosin to F-actin.

Myosins are motor proteins in cells. They move along actin by changing shape after making stereospecific interactions with the actin subunits. As these are arranged helically, a succession of steps will follow a helical path. However, if the myosin heads are long enough to span the actin helical repeat (approximately 36 nm), linear motion is possible. Muscle myosin (myosin II) heads are about 16 nm long, which is insufficient to span the repeat. Myosin V, however, has heads of about 31 nm that could span 36 nm and thus allow single two-headed molecules to transport cargo by walking straight. Here we use electron microscopy to show that while working, myosin V spans the helical repeat. The heads are mostly 13 actin subunits apart, with values of 11 or 15 also found. Typically the structure is polar and one head is curved, the other straighter. Single particle processing reveals the polarity of the underlying actin filament, showing that the curved head is the leading one. The shape of the leading head may correspond to the beginning of the working stroke of the motor. We also observe molecules attached by one head in this conformation.

Titin: a molecular control freak.

Recent studies of the giant protein titin have shed light on its roles in muscle assembly and elasticity and include the surprising findings described here. We now know that the titin kinase domain, which has long been a puzzle, has a novel regulation mechanism. A substrate, telethonin, has been identified that is located over one micron away from the kinase domain in mature muscle. Single-molecule studies have demonstrated the fascinating process of reversible mechanical unfolding of titin. Lastly, and most surprisingly, it has been claimed that titin controls assembly and elasticity in chromosomes.

Observation of transient disorder during myosin subfragment-1 binding to actin by stopped-flow fluorescence and millisecond time resolution electron cryomicroscopy: evidence that the start of the crossbridge power stroke in muscle has variable geometry.

The mechanism of binding of myosin subfragment-1 (S1) to actin in the absence of nucleotides was studied by a combination of stopped-flow fluorescence and ms time resolution electron microscopy. The fluorescence data were obtained by using pyrene-labeled actin and exhibit a lag phase. This demonstrates the presence of a transient intermediate after the collision complex and before the formation of the stable "rigor" complex. The transient intermediate predominates 2-15 ms after mixing, whereas the rigor complex predominates at time >50 ms. Electron microscopy of acto-S1 frozen 10 ms after mixing revealed disordered binding. Acto-S1 frozen at 50 ms or longer showed the "arrowhead" appearance characteristic of rigor. The most likely explanation of the disorder of the transient intermediate is that the binding is through one or more flexible loops on the surfaces of the proteins. The transition from disordered to ordered binding is likely to be part of the force-generating step in muscle.

A computer-controlled spraying-freezing apparatus for millisecond time-resolution electron cryomicroscopy.

Apparatus is described for the kinetic investigation of biological reactions by electron cryomicroscopy with time resolution on the order of milliseconds. This involves layering a grid with one reactant and then spraying on a second reactant immediately before freezing. Two-stage mixing can be achieved by mixing two solutions, holding them in a delay line for a preset interval, and then spraying the aged solution onto a grid carrying a third reactant. The individual steps of these procedures are under software control and can be adjusted independently. Spray-freezing is widely applicable since solutions of small molecules, proteins, and protein assemblies can be delivered as aerosols. Thus the method can be used to study both the effects of small molecules on macromolecules and for monitoring protein-protein interactions. It may also be useful in other situations, for instance in light microscopy.

Flexibility within myosin heads revealed by negative stain and single-particle analysis.

Electron microscopy of negatively stained myosin has previously revealed three discrete regions within the heads of the molecule. However, despite a probable resolution of approximately 2 nm, it is difficult to discern directly consistent details within these regions. This is due to variability in both head conformation and in staining. In this study, we applied single-particle image processing and classified heads into homogeneous groups. The improved signal-to-noise ratio after averaging these groups reveals substantially improved detail. The image averages were compared to a model simulating negative staining of the atomic structure of subfragment-1 (S1). This shows that the three head regions correspond to the motor domain and the essential and regulatory light chains. The image averages were very similar to particular views of the S1 model. They also revealed considerable flexibility between the motor and regulatory domains, despite the molecules having been prepared in the absence of nucleotide. This flexibility probably results from rotation of the regulatory domain about the motor domain, where the relative movement of the regulatory light chain is up to 12 nm, and is most clearly illustrated in animated sequences (available at http://www.leeds.ac.uk/chb/muscle/myosinhead.htm l). The sharply curved conformation of the atomic model of S1 is seen only rarely in our data, with straighter heads being more typical.

Elasticity and unfolding of single molecules of the giant muscle protein titin.

The giant muscle protein titin, also called connectin, is responsible for the elasticity of relaxed striated muscle, as well as acting as the molecular scaffold for thick-filament formation. The titin molecule consists largely of tandem domains of the immunoglobulin and fibronectin-III types, together with specialized binding regions and a putative elastic region, the PEVK domain. We have done mechanical experiments on single molecules of titin to determine their visco-elastic properties, using an optical-tweezers technique. On a fast (0.1s) timescale titin is elastic and force-extension data can be fitted with standard random-coil polymer models, showing that there are two main sources of elasticity: one deriving from the entropy of straightening the molecule; the other consistent with extension of the polypeptide chain in the PEVK region. On a slower timescale and above a certain force threshold, the molecule displays stress-relaxation, which occurs in rapid steps of a few piconewtons, corresponding to yielding of internal structures by about 20 nm. This stress-relaxation probably derives from unfolding of immunoglobulin and fibronectin domains.

Direct visualization of extensibility in isolated titin molecules.

"Molecular combing" induced by a receding meniscus has been shown to extend individual titin molecules. Electron microscopy reveals that both ends of the molecule tend to attach to a mica substrate, probably due to their local positive charges. This leaves the remainder of the molecule free to be straightened and extended by forces of up to approximately 800 pN. A small region in the I-band part of the molecule, which probably corresponds to sequence high in P, E, V and K residues, is the most compliant and appears to extend by an unfolding of the polypeptide chain. Other parts of the molecule are also capable of extension. These mechanical extensions in titin are probably reversible.

Titin as a scaffold and spring. Cytoskeleton.

The complete sequence of the giant muscle protein titin has been determined. It provides further insight into how titin may act both as a scaffold and a spring to specify and maintain muscle structure.

Studies of the interaction between titin and myosin.

The interaction of titin with myosin has been studied by binding assays and electron microscopy. Electron micrographs of the titin-myosin complex suggest a binding site near the tip of the tail of the myosin molecule. The distance from the myosin head-tail junction to titin indicates binding 20-30 nm from the myosin COOH terminus. Consistent with this, micrographs of titin-light meromyosin (LMM) show binding near the end of the LMM molecule. Plots of myosin- and LMM-attachment positions along the titin molecule show binding predominantly in the region located in the A band in situ, which is consistent with the proposal that titin regulates thick filament assembly. Estimates of the apparent dissociation constant of the titin-LMM complex were approximately 20 nM. Assays of LMM cyanogen bromide fragments also suggested a strong binding site near the COOH terminus. Proteolysis of a COOH-terminal 17.6-kD CNBr fragment isolated from whole myosin resulted in eight peptides of which only one, comprising 17 residues, bound strongly to titin. Two isoforms of this peptide were detected by protein sequencing. Similar binding data were obtained using synthetic versions of both isoforms. The peptide is located immediately COOH-terminal of the fourth "skip" residue in the myosin tail, which is consistent with the electron microscopy. Skip-4 may have a role in determining thick filament structure, by allowing abrupt bending of the myosin tail close to the titin-binding site.

Millisecond time resolution electron cryo-microscopy of the M-ATP transient kinetic state of the acto-myosin ATPase.

The structure of the AM-ATP transient kinetic state of the acto-myosin ATPase cycle has been examined by electron microscopy using frozen-hydrated specimens prepared in low ionic strength. By spraying grids layered with the acto-S1 complex with ATP immediately before freezing, it was possible to examine the structure of the ternary complex with a time resolution of 10 ms. Disordered binding of the S1 was observed, suggesting more than one attachment geometry. This could be due to the presence of more than one biochemical intermediate, or to a single intermediate binding in more than one conformation.