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

点击翻译按钮获取中文摘要

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.