Molecular mechanism of muscle regulation by troponin

Muscle contraction is triggered by a brief pulse of calcium inside each muscle cell. The calcium binds to a protein called troponin in the muscle thin filaments, producing a change in the filament structure. This allows the motor protein myosin in the nearby thick filaments to bind to and pull on the thin filaments, causing the muscle to shorten. The same process occurs in all the muscles that move the skeleton and in the heart, where a pulse of calcium triggers each heart beat.

Our aim is to determine the molecular structural changes caused by binding of calcium to troponin, and to discover how they control muscle contraction. There have been many previous studies of calcium binding to isolated fragments of troponin, but we will take a radically different approach, in which the structural changes in troponin will be measured inside a working muscle cell. This is necessary to preserve the normal transient interactions between the muscle proteins that take place during a single contraction. We will label isolated troponin with fluorescent probes, and replace the native troponin in muscle cells by the labelled troponin. We can then use the polarisation of the fluorescent light to follow structural changes in troponin inside the cell. We have developed and refined this approach over the last ten years, and tested it on other muscle proteins, so we are confident that the method can reveal how troponin controls muscle contraction.

It is important to understand the normal mechanism of muscle control to understand how it fails in muscle disease, which can help in designing drugs and therapies. The mechanism is essentially the same in skeletal muscles and in the heart. Muscle weakness impairs the quality of life of many elderly people, and heart disease is a major threat to human health. Some inherited heart diseases are caused by specific errors in the structure of troponin, but we don?t know how these cause the disease because we don?t know how troponin works normally. This project aims to provide this knowledge.

Normal function of all cells and tissues depends on many proteins interacting in a complicated but coordinated way, and it is often difficult to understand how a defect in one protein leads to a complex set of symptoms. The approach we are developing to measure changes in protein structures inside cells should help to overcome this general limitation of current biomedical research.

The specific aim of this project is to determine the molecular mechanism of muscle regulation as it occurs in the cellular environment. Contraction of skeletal muscle in response to a motor nerve impulse, and of the myocardium during a single heartbeat, is triggered by a brief pulse of Ca2+ ions, which bind to troponin on the actin-containing thin filaments of muscle. The resulting changes in the structure of troponin and its partner regulatory protein tropomyosin control the interaction between myosin and actin that drives muscle contraction. We will use a novel technique, Fluorescence for In Situ Structure (FISS), to elucidate the structural changes in troponin and the mechanism of regulation of muscle contraction in situ. Double-cysteine mutants of troponin components will be labelled with a bifunctional rhodamine, and exchanged into demembranated skeletal muscle cells. The intracellular orientation of the rhodamine dipoles will be measured by polarised fluorescence. Thus we will determine the in situ orientation and conformation of the key functional domains of troponin, and their changes in response to binding Ca2+. Myosin itself plays an active role in the regulatory mechanism, and the FISS technique provides a unique opportunity to unravel the roles of Ca2+ and myosin binding. It will also allow us to determine the time courses of the structural changes in response to a brief pulse of Ca2+ and relate them to the physiological rates of activation and relaxation. These studies will reveal the dynamic molecular structural changes underlying the physiological regulation of muscle contraction. The main features of this mechanism are common to skeletal and cardiac muscle, so the results will underpin a wide range of physiological, pharmacological and pathological studies on both muscle types, with relevance to heart disease and to skeletal muscle weakness in the elderly. A more general aim of this project is to develop the FISS technique for wider application to elucidate conformational changes in intact macromolecular complexes in other cell types. FISS can reveal molecular structural changes in intact cells on the physiological timescale, and these capabilities will be required to understand the molecular bases of many cell functions and the pathologies related to them. This approach can thus help to bridge the generic gap between molecular- and cell-level approaches in biomedicine that currently hinders structure-based development of new drugs and therapies.