This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The specificity of interactions between pathogen and host allows both invasion (when the pathogen recognizes the host) and immunological recognition (when the host recognizes the pathogen. These projects investigate both types of interactions, and include both the membrane-spanning region as well as the separated soluble region of the proteins. The initial structures have not yet been published. The first project encompasses the interaction between human toll like receptors (TLRs) and bacterial outer membrane porins. We have grown initial crystals of the bacterial porins that are TLR ligands that diffract to 2.3 ? resolution. These crystals are small in two of the three dimensions such that the large beam size at most synchrotrons with tunable wavelengths (>0.1 mm) limits the quality of our data. We are unable to see any diffraction using our home generator. Since these porins do not have significant sequence similarity to porins of known structure, molecular replacement has failed. The protein does not have sufficient methionines to perform Se-Met MAD and we are collecting derivatives. From 50 data sets collected so far, we have identified two derivatives. The first is a Lu derivative with a single site, and during this beamtime, we propose to do a full 4-wavelength MAD on Lu-soaked crystals in order to improve the phases. The second derivative is an iodide derivative with 4 sites. These crystals diffracted poorly (4 A resolution) and we propose bringing crystals that vary the soaking concentration and times in order to improve the diffraction limit to better than 3.5 A resolution. In addition, we will bring several other heavy atom soaked crystals (see above). The second project investigates the opposite pathogenic recognition phenomenon - i.e. the recognition of host siaylic acids by the bacterial receptor. Crystals have been grown of the recognition region of the bacterial receptor (GspB). Like the porin crystals, these grow as thin rods and lack sufficient methionines to determine the structure by Se-Met. In addition, we believe that the small size of these crystals is precluding observation of any diffraction on our home generator. We are at the beginning of searching for heavy atom derivatives;although data have been collected from crystals soaked with 5 distinct compounds, no derivatives have yet been identified. We will bring native protein to determine if a smaller beam size will improve the quality of the data set, and in addition, we will bring the above mentioned derivative soaks for MAD data collection. The final project is of the integral-membrane bacterial complex II homolog quinol:fumarate reductase. These crystals grow as long rods. The best data set collected on the home generator is at 6A resolution, while the best data set collected at the synchrotron is at 2.7 A resolution. Our current project is to determine a co-structure with a partner protein. Only one co-structure of membrane proteins in complex with a partner protein has been reported in the literature to date. Thus the significance of this structure from a technique development standpoint would be great. In addition, the biological significance of this complex is immense.
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