STRUCTURE DETERMINATION ON THE LYMPHOKINE GM-CSF

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DESCRIPTION: (Adapted from Applicant's Abstract). The objective of this research is to gain knowledge of the three-dimensional structures of human, simian and murine granulocyte macrophage-colony stimulating factors (GM-CSF). The investigators claim to have obtained human GM-CSF crystals which diffract to at least 2.5 A and are well suited to structure studies at high resolution. The simian and murine forms have not yet produced X-ray quality crystals. The technique of X-ray crystallography will be applied to crystals of each of these three proteins. Using heavy atom isomorphous replacement methods and/or the multiple wavelength anomalous dispersion technique, one of the structures will be solved at 3 A resolution or better, to allow tracing of the path of the peptide chain and model building. At this point initial phases will be improved by use of the probable three-fold informational redundancy caused by the non-crystallographic symmetry. The molecular replacement method will then use this model to solve the other two structures and those of specifically-designed mutants. All structures will be refined at the highest resolution possible to ensure maximum accuracy of the models. The structural knowledge will be used in combination with results from mutagenesis experiments and other structure/function studies to map the structural features required for GM-CSF activity. The investigators believe that such information will lead to the structure-based design of agonist and antagonist molecules and that it will also give insight into the practical implications of heterogeneity known to be present in recombinant GM-CSF molecules and guide site-directed mutagenesis experiments aimed at removing structural heterogeneities and improving pharmacological properties.