Bio-physical studies of motor proteins

Molecular motors are widespread in biology. Muscle contractions are the result of billions of motors called myosins travelling along tracks called microtubules. In analogy to a petrol engine which burns petrol to fuel its motion producing carbon dioxide and other pollutants, molecular motors fuel their motion by altering the structure of a molecule which is then regenerated by a separate process elsewhere in the cell. In this sense molecular motors are a truly renewable energy source, and it is an ultimate aim to harness the unique power of these motors to replace conventional motors. The effects of the molecular motors this fellowship aims to study are less tangible, but their biological function is arguably even more valuable. These motors travel, or translocate, along DNA or proteins. DNA is an incredibly long molecule (the human genome is over two metres long, but only a millionth of a millimetre wide) and holds the unique genetic information for a person. Molecular motors must continually translocate along this molecule, sometimes simply get to another region, or more commonly whilst performing a function, such as reading the genetic code encrypted within DNA or checking for damage. DNA is a very well defined structure; the double helix means DNA motors travel along a very smooth road and can thus take regular steps. Another set of motors, that travel along proteins, do not have this luxury, proteins are far less structured and present a rocky track that the motor must travel along, exactly how these motors cope with this challenging terrain is unknown. Whilst the 'DNA road' is smooth, it is also busy. DNA is a double helix, each strand oriented in an opposite direction, meaning that a motor can travel in either direction. This is further complicated since the DNA must be compacted to fit within a cell that measures less than a hundredth of a millimetre, achieved by wrapping the DNA up onto spools, which will impede the passage of DNA motor proteins. So not only do we wish to understand the molecular mechanisms of the motors (i.e. their Haynes manual) but also how their interactions with other motor proteins (i.e. their Highway Code). Conventional biochemical approaches use solutions containing many thousands of these proteins which are mixed with the required fuel and 'track' i.e. DNA or protein and the accumulation of a specific reaction product is monitored. Many thousands of molecules are required as detection of these tiny molecules is very hard. However in this approach problems rapidly arise due to desynchronisation. At the beginning of the experiment all molecules should be in the same state, waiting for their fuel. But once their fuel is added different molecules will take longer to get going, stall, or take different routes and thus an average over all molecules (which is what the experiment will report) may not detect subtle sub-populationsThis fellowship will adopt a different approach by using an Atomic Force Microscope (AFM) to address individual molecules rather than whole poplulations. The AFM works by using by using a sharp tip mounted on a soft cantilever to manipulate or image a molecular motor that has been secured to a surface. By securing the motors track (i.e. DNA or protein) to the AFM tip and dangling it above the motor we can measure how the enzyme translocates the track and how this translocation reacts to an externally applied force, a kind of molecular fishing. Additionally the AFM tip can be scanned over the motor to build up a 3 dimensional image, normally these images take a long time to collect, typically much slower than the times over which these motors work. I aim to develop technologies that will speed up the imaging rates such that processes occurring within fractions of a second can be observed, and movies of these processes can be built up. In our 'movies' all the action is played out on a set which is a fraction of the width of a human hair.