SELENIUM &BROMINE DOUBLE EDGE MAD EXPERIMENT FOR AN HSF DNA COMPLEX

Stanford University, Stanford CA

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Abstract

We have used multiple anomalous disperson (MAD) to determine the structure of a complex between the DNA binding domain of the heat shock transcription factor (HSF) along with a 12-mer of DNA containing its cognate binding site. Data was collected on crystals containing selenomethionine-substituted protein and bromouracil-substituted DNA. The present model contains complete DNA, most of the protein, and 100 water molecules. The protein monomers are missing residues from the N- and C-terminii, as well as from the flexible loop. The current R value is 21.0% (free R is 29.3%) for all data between 6.0 and 2.5 E. There are no Ramachandran outliers, the r.m.s deviations are 0.009 E for bond lengths and 1.5540 for bond angles. The structure shows that the DNA is contacted solely along with phosphate backbone, with the majority of contacts coming from the putative recognition helix of the DNA binding domain. We are in the process of interpreting the biological implications of the structure.