The aim of this proposal is to use the interaction between the large membrane protein Photosystem I and ferredoxin (Fd) as a model system to study and further develop the new method of laser-induced femtosecond nano-crystallography. The goal of the proposal is to determine if the dynamics of the excited state of protein molecules can be resolved by studying the inter-protein electron transfer and the conformational changes that finally lead to the undocking of ferredoxin from the Photosystem I/Fd complex. This method development has the potential to revolutionize the determination of the structure and dynamics of protein complexes. The proposal is based on a breakthrough in the field that has been achieved in December 2009, when an important step toward the Grand Challenge goal of making molecular movies and perhaps "damage-free" atomic-resolution protein crystallography was achieved at the DOE's Linac Coherent Light Source (LCLS) at Stanford (to be submitted to Nature 2010). The LCLS is the world's first (pulsed) hard X-ray laser, where we obtained several million single-shot X-ray diffraction patterns, each from a single nanocrystal of the large membrane protein complex Photosystem I (PSI). We showed for the first time that the "diffract-before-destroy" principle, shown previously for inorganic materials, is also valid for fragile protein crystals. Using 3 and 70 fs X-ray laser pulses at 2 kV (8) we have obtained strong diffraction, extending beyond the detector limit of 9 Angstroms. The femtosecond pulse duration is far shorter than the time taken for atoms (or most electrons) to move, so that radiation damage is outrun, and the pulse terminates before damage begins. The patterns were read out from a CCD detector 30 times per second, the repetition rate of the LCLS. The mean size of the crystallites was about 17 molecules on a side. The fully hydrated crystallites are fired in vacuum across the LCLS beam in a continuous, ten microndiameter water jet. We will use the ASU-designed liquid jet protein-beam injector which was used for our successful experiments in Dec 2009. This injector (IDBR 0555845) produces a continuous stream of fully hydrated crystals that allows single-shot diffraction patterns to be read out at about the same rate (30 Hz) as the repetition rate of the LCLS. This 100% hit rate is critical in order to obtain the millions of patterns needed from a stream of protein crystals to allow reconstruction of a three-dimensional charge-density map at each time step. The first of the set of proposed experiments will take place at the June (2kV) beamtime (approved against intense international competition) and in Dec. 2010 when harder X-rays (at 1.5 ) will be available. We expect that, using damage-free 3fs pulses, it will be possible to achieve near-atomic resolution. The cocrystallization of PSI with ferredoxin (Fd) has already been achieved and the method to grow nanocrystals will be extended form our work on Photosystem I to the growth of optimized nano- PSI/ferredoxin crystals. A laser pulse will be used to excite the nanocrystals of PSI/Fd in the jet, followed by snap-shot diffraction after a delay of 1-10 microseconds. The intellectual merit of the proposal lies in the fact that photosynthesis at PSI (and PSII), by converting solar energy to chemical energy, sustains our biosphere. In particular, the reduction of ferredoxin by PSI provides the electrons for the reduction of NADP, which in turn is used as NADPH with ATP to reduce CO2 to carbohydrates, a controlling factor in global warming. This work will provide a detailed atomic mechanism of the conformational change which occurs when, due to the arrival of an electron, ferredoxin undocks from PSI. Ferredoxin is responsible for the entire redox regulation of plant chloroplasts. The work is transformative and involves a radically new approach. The broader impacts resulting from this work include: i) Training of the postdo
|Effective start/end date||9/1/10 → 8/31/14|
- NSF: Directorate for Biological Sciences (BIO): $590,221.00
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