Laser Aligned Protein Beams

The goal of this project is to detennine the atomic structnre of proteins which are difficult or impossible to crystallize, such as membrane proteins, using new diffraction techniques. In collaboration with ProfP. Fromme we plan to solve the following proteins, for which she has expression and (in some cases) crystal growth facilities. Photosystem I (PSI); the PSI-Light-harvesting complex; Outer envelope protein (OEPI6); ATP-synthase; gp41 of the HIV virus. We request continued support for graduate student M Hunter for two years. The two new diffraction techniques we are developing are all based on our aerojet hydrated protein-beam injector (figure 2 below), which provides a singlefile stream of micron-sized droplets containing crystallites or molecules. The solution can be delivered by syringe pump to the jet, which runs across a synchrotron beam. The jet is driven by a piezo oscillator, which will allow us to . sychronize it with the LCLS free-electron X-ray laser at SLAC. Under recent CBST support our graduate student M. Hunter has become expert in preparing protein molecule or crystallite solutions for the aerojet. For crystallites of Photosystem 1 membrane protein (29nm cell), this involves cyanobacterium cell growth, protein extraction and crystal growth (if needed). This is a long and complex procedure. The development of the aerojet (which Hunter assisted) is described in De Ponte et al (2008). It uses high-pressure gas focussing of the buffer/protein solution to allow a small droplet beam to be formed from a bigger nozzle, sufficiently large to avoid clogging. This aerojet is the only design (unlike electrospray, Rayleigh or neubulizers) which produces uncharged species in very high concentration ( a single-file stream, not a mist) which can be contolled electronically (for sychrbnization), which runs indefinitely without clogging.