BS3335 UAEB - PROTEINS IN HEALTH & DISEASE - BIOPHYSICAL TOOLS

In this course, we will learn to use biophysical techniques to find out information about different types of proteins, in terms of concentration, secondary structure, oligomeric state, reconstitution systems using both detergent and lipid, purity, function and inhibition. The students will be provided with a set of initial problems to solve. To solve these problems, a hands-on approach will adopted that will make use of a range of different biophysical techniques. Each of these techniques will provide different types of information about the samples. An important aspect that will be emphasized is to learn the advantages and disadvantages of each technique, and to be able to design experiments knowing which techniques should be used first, and which last. Each daily session will consist of two parts (morning and afternoon). Each part will start will a 30 min briefing and will end up with a collation of results session and discussion. All days will have associated a pre-recorded video lecture for support, but a live 30 minute briefing will precede all sessions. In the first part we will look at ways to determine protein concentration using visible and UV light, and to measure the size of larger particles such as liposomes or detergent micelles using dynamic light scattering. The second part will look at how to obtain secondary structure information to assess proper folding, from infrared spectroscopy and circular dichroism. The third part of the course will focus on size and oligomerization of molecules, and how this can be determined. To do this, we will use standard centrifugation techniques and also analytical ultracentrifugation (velocity and sedimentation). The fourth part will look at function, using kast kinetics (stopped flow), fluorescence and channel activity in model lipid bilayers. Inhibition by small molecules will be tested and dose response plots will be used to determine the affinity of the drugs.

Prerequisites Tree