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Probing the Structure and Function of the Helix‐5 Water‐Mediated Network in H‐Ras
Author(s) -
Sanchez Jaquelyn,
Marcus Kendra,
Mattos Carla
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.893.12
Subject(s) - mutant , gtpase , chemistry , mutagenesis , helix (gastropod) , polyethylene glycol , crystallization , crystallography , membrane , wild type , fast protein liquid chromatography , monomer , biophysics , biochemistry , biology , gene , enzyme , organic chemistry , ecology , snail , polymer
H‐Ras is a monomeric G‐protein that is bound to the inner plasma membrane of the cell. The intimate interaction formed between the protein and the membrane plays an important role in its structural and functional properties such as its activation/inactivation through GTPase activity. This study tests the hypothesis that the conserved helix 5 water‐mediated network acts as a link between the two distant regions of the protein1. Two residues within the helix 5 network that mediate interactions with conserved water molecules were mutated using site‐directed mutagenesis: D154A and T158A. The mutants were purified using Fast Protein Liquid Chromatography (FPLC) and used in crystallization trials. The hanging drop method was used to set up trays with various reservoir mixtures of polyethylene glycol and calcium acetate in a screen around the conditions where wild type Ras crystals are known to grow. Crystals were obtained for both mutants and X‐ray data collected for RasD154A. The structure shows the mutated side chain and impairment of the water network. Data collection is ongoing for the RasT158A crystals. In the future, hydrolysis rate studies will be conducted to test whether the observed change in structure has an effect on the reaction rate of GTP to GDP catalyzed by Ras. (1) B Kearney et al. J. Mol. Biol. 426, 611‐629 (2014)