Reflection anisotropy spectroscopy as a new tool for linking macromolecular conformation to biological function: applications in biological redox chem

Living things are made up of a subtle combination of molecules that store the instructions for carrying out biological functions and molecules that carry out these instructions. The first type of molecules are called nucleic acids (DNA) and the second type are called proteins. The nucleic acids code the instructions for assembling proteins from smaller molecules called amino acids. Once assembled the proteins fold up into three dimensional shapes and the shapes of proteins play a crucial role in their functional behaviour. Sometimes a determination of the shape of a protein provides considerable insight into its function and this observation gives rise to the suggestion that 'if you want to understand function study form.' Considerable advances have been made over the last fifty years in determining the shapes of proteins using the technique of protein crystallography. In this technique a single crystal of the protein is studied using x-ray diffraction and an analysis of the diffraction pattern yields the shape of the protein. However some of the important functions carried out by proteins can only occur if the protein changes shape as a result of some stimulus. A very common stimulus for such shape changes is the transfer of electrons from one part of a biological system to another and these processes are called electron transfer reactions. They play a very important part in the regulation of the behaviour of proteins in living things. It is almost impossible to study these shape changes using x-ray diffraction since to do so it is necessary to grow a crystal and then arrange for all the molecules in the crystal to change shape in the same way at the same time while taking a series of x-ray diffraction patterns. As a result of this difficulty there is very little information on the changes induced in the shapes of proteins by electron transfer reactions or indeed by any other stimuli. In this research programme we are proposing a novel way of studying changes in the shapes of proteins induced by electron transfer reactions. We will exploit a novel optical technique called reflection anisotropy spectroscopy (RAS). RAS was developed in the late 1980's as a method of monitoring the growth of semiconductors. In the 1990's it was applied to the study of metal surfaces and of molecules adsorbed on metal surfaces in ultra high vacuum. Recently it has been applied to the study of biological molecules adsorbed at metal-liquid interfaces. We have shown that RAS is able to determine the orientation of small molecules adsorbed at metal liquid interfaces and to monitor, in real time, the interaction between adsorbed molecules as the potential is changed on the electrode of an electrochemical cell. Most importantly in preliminary experiments we have shown that the technique can follow the changes in shape of large proteins adsorbed at metal-liquid interfaces as electron transfer reactions occur as a result of variations in the potential applied to the metal electrode. This opens up a new way of measuring the timescales of the changes in the shape of proteins that result from electron transfer interactions. That is the aim of this grant application. We know of no other way of obtaining this important information.

We will develop instrumentation based on reflection anisotropy spectroscopy to measure the kinetics and multi-component nature of conformation changes linked to function of protein molecules in real time. We have shown that RAS has the potential to provide this important information. In RAS the intensity of light reflected from a surface is measured. The light is incident normal to the surface and the reflected light is measured as close to normal reflection as possible, usually just a few degrees off from the incident beam. Generally, due to the large penetration depth of light into solids, this arrangement would result in 99% of the reflected light arising from the bulk of the material and only 1% from the surface or from molecules adsorbed on the surface. In RAS a geometrical arrangement is chosen which makes it possible to eliminate the contribution to the signal from the bulk and ensure that 100% of the light intensity that is measured arises from the surface and from molecules adsorbed on the surface. This is achieved by measuring the difference in reflection from two directions at right angles in the surface of linearly polarized light at normal incidence to, and near normal reflection from, the surface. The measurement is equivalent to determining the rotation of the plane of polarization on reflection of normal incident light. We will develop an improved RAS instrument and combine RAS measurements with directed engineering of conformationally active redox proteins to investigate the potential of RAS to follow complex conformational events in real time. The major advance in this programme is the first application of RAS to such studies. We will integrate fully the RAS method with the more traditional techniques of electrochemistry, atomic force microscopy (AFM) and adsorption studies using a quartz crystal microbalance. We will thus provide a new and valuable research tool for probing conformational motion coupled to biological function..