- Muscle converts chemical energy into work
- ATP fuels muscle contraction
This section gives a brief glimpse at how our knowledge of the molecular mechanics of muscle came about. Although this means that it is not strictly syllabus material, it does help put the material in this section in perspective. It also shows how much work has gone into this subject, because after all, we wouldn't want you thinking it has all been plain sailing!
OK, so this is no fairy tale, but muscle research has been around for over 2000 years, so it is difficult to know where to begin. If we take the involvement of chemistry as a starting point, then it has been noted as early as 1842 by Schwann that muscles are chemo-mechanical devices, which means that they convert chemical energy into mechanical work. At that point however, no one understood where the chemical energy came from and it took until 1931, before Lohmann established that the fuel that muscles use was actually ATP. Up until this point a number of different possibilities had been investigated, for example de-amination had been thought to be one possibility because dissected muscle smells of ammonia when left for a few days.
Also in the early 1930. s Straub made the discovery that contraction involved both Actin and Myosin, but it was 1939 before Engelhardt and Lyubimova established that it was in fact the myosin which actually uses the ATP.
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The structure of striated muscle was also very misunderstood for a long time. It was thought that this was unlikely to be important because smooth muscle could contract despite having no such structure. This assumption proved to be very wrong though as the way in which these proteins are organized in skeletal muscle is a powerful key to understanding their operation. In all types of muscle both actin and myosin form polymers, long chains that are organized in skeletal muscle in a very specific matrix. In the early 1940's the exact arrangement of these was unknown, but it was known that you could recreate these filaments in a test-tube. It was at this stage that Andrew Szent-Györgyi then carried out the experiment that really brought the knowledge of the time together. He showed that he could recreate contraction in a test tube by placing synthetic actin and myosin filaments together in the presence of ATP. Myosin on its own however, could not contract, therefore contraction is brought about by the interaction of actin, myosin and ATP.
It wasn. t until the late 1950. s though that the first clues as to how this might be happening appeared. Up until this point it had been believed for a long time that the myosin filaments caused muscle shortening by actually shrinking themselves. The real solution to a large part of this problem however, came through structural studies by both Ralph Neidergerke and Huxley Andrew in Hieldelberg and by Hugh Huxley and Jean Hanson in Cambridge. These studies came in the forms of x-ray diffraction, light microscopy and electron microscopy and they allowed detailed pictures of the sarcomere to be seen for the first time. What they showed was that it consisted of an ordered matrix of two sets of inter-digitating fibres. These two sets of fibres are called the thick and thin filaments and are made predominantly of myosin and actin respectively. The model that both of these groups proposed was that, during contraction the two sets filaments slide past each other so that they overlap more, making the overall length of the sarcomere shorter.
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| A short animation describing the muscle fibre to the sarcomere. | An electron micrograph of skeletal muscle showing the repeating pattern of the sarcomere. |
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So at this stage it was known that myosin utilized the ATP to actively push the Actin past it, and in 1957 Hugh Huxley developed images of even closer detail showing the contacts between the Actin and Myosin namely the cross-bridges. He then went on the next year to describe the crossbridge theory in which these contacts repeatedly knocked into the actin pushing it past it.
Demonstrating and proving this theory proved to be very difficult, mainly because only 15-25% of these crossbridges are in contact with the actin at any one point during contraction. What is possible though is to remove the ATP causing the heads to remain attached, this is known as placing the muscle in rigor. In 1965 Ken Holmes and his associates, did just this in the x-ray diffractor and showed the reflections indicating the crossbridges at right angles increased in rigor, and dropped off on the addition of ATP. This was the first indication of these crossbridges actually moving.
The next set of advances took place aside from the structure, in the field of enzyme kinetics, researchers such as Lymn, Taylor and Trentham demonstrated some of the enzymatic properties of myosin and its protolytic fragments HMM and S1. These included the fact that when myosin is not in the presence of actin then both ATP and the hydrolyzed products of ATP (ADP and Pi) will remain bound to the myosin in almost equal measure. From this you can deduce a number of things.
From this sort of information an entire kinetic scheme was developed, such as the one shown below. However, what this actually does is to lay out all of the various possible reaction pathways. The aim is then to find all of the relevant rate constants, to establish which is the most likely path. To this end a whole plethora of techniques were used, from fluorescent analogues of ATP to ADP and Pi detection systems. However the ideal situation is to match this kinetic data to the structural information from the proteins themselves.
Up until the early 1990. s the only structural viewpoint of muscle contracting had come from x-ray diffraction. This technique had by this point been greatly enhanced to the point that movements could be detected on the time scale of just 200µs. The detection of these movements using this method are dependent on the amount of mass moving at any one time, and it was using this approach that an important discrepancy was uncovered. The disagreement concerned the amount of mass moving in a muscle at any one point, because it was known that only 15-25% of myosin heads were interacting with actin at any one point, but the amount of mass seen to be actually moving was substantially less than this.
This meant that the swinging cross-bridge model had to be refined slightly, and one of the first to suggest such an alteration was Roger Cooke. He suggested in 1986 that rather than the whole cross-bridge moving that most of the movement actually came by the tail of the molecule acting as a lever-arm. In order to test this theory though the structure of the components involved would have to known in more detail.
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| A cartoon diagram of myosin and its proteolytic fragments | A simple animation of the lever arm in action. |
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The first breakthrough in this area came in 1990 when Wolfgang Kabsch and his associates solved the crystal structure of the actin monomer and then combined this information with cryo-electron microscope images, to create a model of filamentous actin. Then in 1994 Ivan Rayment's group solved the structure of chicken skeletal S1. This gave researchers their first look at the molecule at the centre of muscle contraction. However, the only problem with crystal structures as a whole is that they are static pictures, and the whole point of myosin is that it moves. The first S1 structure was in rigor, i.e. there was no nucleotide present, and over the past 6 years a range of structures have been solved to try and show how the molecule carries out its task. The first couple of structures to be released after Rayment's both showed different isoforms of myosin (those of chicken smooth muscle and brush border myosin), both in the presence of ADP. These structures gave some of the first real evidence towards the lever-arm hypothesis as they showed the molecule with the tail region rotated about the motor domain by about 35°.
This did not show the full extent of the power-stroke but it did show where the pivot point or the start of the lever arm might be. Another set of experiments that gave positive evidence towards this idea was the in-vitro motility assay. This gave an important means of testing variant myosin. s that had been created by a new wave of molecular biology techniques. One such experiment devised by Mike Anson and colleagues, showed that by artificially increasing the length of the lever arm, the speed with which actin was transported across the myosin heads was proportionally increased.
Rayment then published 3 new myosin structures, from the slime mould Dictyostelium Discoideum this time using the nucleotide analogues ADP· BeF, ADP· AlF, and ADP· Vanadate. All of these structures had the disadvantage that the lever arm itself was missing, however, the Vanadate structure showed that the converter domain had swung around by a huge 70°. This could be interpreted as showing the full power stroke, but the lack of the lever arm and the lack of resolution in some key areas of the structures still left many unanswered questions. Other groups were trying to answer some of these questions by other methods than crystallography. These included single molecule studies where, using laser trapping technology workers such as Justin Malloy and his colleagues hoped not only to measure the distance of a power stroke created by a myosin head but also the force that this produced. Other techniques tried to measure this in the muscle fibre by techniques such as fluorescence polarization or electron spin labelling of the myosin lever arm region. The benefit of techniques such as these is that they try to measure the changes that take place in the muscle fibre itself, because it must be remembered that this is a mechanical system and mechanical factors have a large influence over how the system operates. For example, it has recently been shown that the release of ADP from the myosin head is dependent on the strain placed on the fibre. All of these factors can then be combined in the final model of how muscle actually works. The exact structural details will always be important in creating such a model however, and Carolyn Cohen and her colleagues have recently released a series of structures of scallop myosin, which finally give this kind of detail, with the lever arm included.
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So is this the story of muscle contraction finally concluded? Well, the simple answer is no, because not only are we only beginning to place all of this structural information into the context of the muscle fibre, but we also have to understand more fully what is happening in the way that muscle is actually controlled. This may sound like just some of the fine print, but the different isoforms of myosin helped to create a firmer picture of myosin moving. Therefore it should also mean that a fuller understanding why these isoforms are different and how they are controlled should also lead to a more accurate model of how these molecules work.