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This meeting is being held to celebrate a great advance that was made forty years ago by Professor Setsuro Ebashi. This was the discovery of the substance “troponin”, a component of the thin filaments of striated muscle of vertebrates. A few years earlier, Ebashi had given direct evidence that calcium ions, at a concentration of a few micromolar, cause contraction of actomyosin preparations. This was a remarkable achievement, since at that time calcium chelators were not available, and the level of calcium impurities had to be kept down by extreme care in preparation of his solutions. This observation was made while Ebashi was spending a year in the laboratory of Fritz Lipmann in New York. On his way back to Japan in 1960, he passed through Britain and took the opportunity of coming to Cambridge to visit Alan Hodgkin and myself. He told us of his observations, and also said that Lipmann had been unwilling to accept that anything as simple as a calcium ion could perform such an important and specific function, and that this had delayed Ebashi’s publication of his results. Later, Ebashi told me that he had been much encouraged by my enthusiastic response to what he told us.

I first met Dr. Setsuro Ebashi in 1954, when he was an instructor (or assistant professor) in the Department of Pharmacology, the University of Tokyo, and I was an undergraduate student. Our group of students was doing some experiments on dogs in the corner of the department, which Professor Ebashi had kindly allowed us to use. He was quite kindhearted, but when talking with him one immediately recognized his brilliance, and his penetrating eyes aroused a feeling of awe in us. I did not realize it then, but this was the time when he was establishing the fact that the essential principle of Marsh’s relaxing factor is not a soluble enzyme such as creatine kinase or myokinase, but a microsomal ATPase described by Kielley and Myerhof in 1948.

First of all, I should like to express my gratitude for the invitation, making it possible for me to participate in the celebration of the fortieth anniversary of the discovery of troponin by Professor Ebashi and his colleagues. This discovery opened up new vistas of the regulation of striated muscle contraction and the role of ionized calcium in it.

A Ca-sensitizing protein factor first isolated from minced muscle showed some similarity to the previously found tropomyosin in amino acid composition and was thus considered to be a native form of tropomyosin (; ; ; ). In 1965, however, a new protein was found in this protein factor in addition to tropomyosin and named troponin (). The discovery of troponin triggered a new era of the molecular biology of the regulation of muscle contraction. Troponin was shown to be the Ca-receptive protein for the Ca-sensitive contraction in striated muscle. In the absence of Ca, troponin in association with tropomyosin suppresses the contractile interaction between myosin and actin, and this suppression is removed by an action of Ca on troponin to activate the contraction (). An electron microscopic study revealed that troponin is distributed along the thin filament at regular intervals of about 40 nm, and this finding led to the construction of a model of thin filament as an ordered assembly of troponin, tropomyosin and actin (; ). By these studies, the molecular basis of the Ca -regulation of muscle contraction was established.

In 2003, we published the crystal structures of the core domains of human cardiac muscle troponin (). Thus, for the first time we were able to visualize the architecture of the molecule; and to see how the three components (TnC, TnI and TnT) fold together to form the troponin molecule. Moreover, our molecular switch mechanism was confirmed, which was previously proposed () based on the crystal structure of a much smaller complex (the full length TnC in complex with TnI (1–47), a short N-terminal fragment of TnI, both from rabbit skeletal muscle troponin). Namely, a C-terminal TnI segment (117–126 in the rabbit skeletal troponin sequence), directly downstream from the “inhibitory region” (104–115), forms an amphipathic α-helix and interacts with the hydrophobic pocket of the TnC N-lobe in a [Ca]-dependent manner. This binding removes the inhi-bitory region from actin-tropomyosin, and thereby relieves the inhibitory action of TnI.

Muscle contraction as an event manifest by the sliding of myosin filaments along actin filaments was first proposed about fifty years ago by H. Huxley and J. Hanson (). This theory built a foundation for muscle research at the molecular level. A decade later the discovery of troponin by Professor S. Ebashi (; ) highlighted the importance of regulation of muscle contraction and sparked numerous experimental studies of the mysterious protein troponin whose properties are now becoming understood at satisfying resolution.

When Galvani discovered the electrical regulation of muscle contraction science began an inexorable transformation. Observation of an inorganic trigger for a physiological event presaged the end of vitalism, the beginning of electrochemistry, and over 400 years of research into the first demonstrable biochemical machine: striated muscle. This molecular machine has been studied in various contexts, ranging from holistic (live muscle) to reductionist (purified molecules). Generations of scientists have, collectively, disassembled and reassembled the contractile apparatus of striated muscle, demonstrating an increasingly complete understanding of its function. In the process, high resolution structures have been determined for most components of this machine. Given the relative orientation of these proteins in a muscle fiber, visualization of muscle contraction at the atomic level seems attainable. This effort is complicated by the inherent properties of proteins, specifically, proteins with conformationally heterogeneous native ensembles.

Muscle contraction, in general, is regulated by the intracellular calcium-ion concentration. Ca-regulation in skeletal or cardiac muscle of vertebrate is mediated at the level of thin filaments consisting of actin, tropomyosin, and troponin. However, pure actin filaments themselves activate contraction irrespective of calcium concentration. Troponin, together with tropomyosin, is required to regulate contraction. Troponin consists of three subunits: inhibitory TnI, Ca-binding TnC, and tropomyosin-binding TnT. Troponin has an elongated shape and forms two structural regions, which are a long tail region containing the N-terminal region of TnT (TnT (chicken skeletal residues 1–164 of TnT)) and a globular head region containing TnI, TnC, and the C-terminal region of TnT [TnT (chicken skeletal residues 165–263 of TnT)]. The globular head region plays a central role in regulating muscle contraction.

Cellular movement and function have long been known to depend on the actin cytoskeleton and its regulation. The actin cytoskeleton is the ultimate target of numerous cellular signaling pathways. The first signaling system understood in any detail was that of vertebrate skeletal muscle. Setsuro Ebashi, celebrated by this volume, was a pioneer through his role in showing that the calcium ion is the physiological regulator of muscle contraction followed by his landmark discovery and naming of troponin as the calcium ion receptor that regulates contraction through its interaction with tropomyosin and actin. Early work in the field is summarized in his remarkable 1968 review with M. Endo (). There they put forth the evidence for a pathway by which activation of the muscle by an action potential would ultimately result in a contractile response consequent to the binding of calcium ion released from the sarcoplasmic reticulum to troponin bound to tropomyosin on the actin filament. The concept of a signaling cascade is now central to any thinking about signaling pathways as we attempt to understand such mechanisms at the molecular level. Whereas troponin is found only in striated muscles, tropomyosin is expressed in virtually all eucaryotic cells and is recognized to be a universal actin filament regulator, versatile in its function despite its deceptively simple coiled coil structure. In this chapter we give an overview of tropomyosin’s multiple regulatory roles and insights into aspects of the structural basis for its functions, focusing on vertebrate forms. As such, this is a personal view rather than a comprehensive review that can be found elsewhere (; ).

The regulation of muscle contraction by the thin filament proteins tropomyosin (Tm) and troponin (Tn) has remained an area of interest since the proteins were first discovered 40 years ago., Although we have learnt a great deal about the proteins themselves and the mechanism by which they regulate muscle contraction some aspects of the mechanism remain to be adequately explained. Our interest is in the cooperativity of the calcium regulatory process and this remains poorly understood and several different models have been proposed. At it simplest the essence of the problem can be simply outlined. In skeletal muscle the binding of calcium to the two regulatory sites of TnC is required for activation of muscle contraction. Isolated TnC binds the calcium cooperatively, as might be expected for a two-calcium-ion switch, with a hill coefficient () of between 1 & 2., In contrast the calcium activation of isometric force in a skinned muscle fibre occurs with a much larger hill coefficient, leading to the idea that cooperativity extends beyond the single actinTmTn structural unit of the thin filament. Some models of muscle activation suggest the whole filament switches as a single unit while studies of the purified proteins in solution tend to indicate more limited cooperativity extending to only the nearest neighbour actinTmTn units. In this paper the reasons why the nature of the cooperativity remains a problem will be explored together with an overview of what our recent studies of the proteins in solution have revealed about thin filament cooperativity.