Crystallization of phosphono-CheY from Thermotoga maritima

摘要: An analog to the active form of signal transduction protein CheY is produced, purified, and crystallized herein. The analog, which was named phosphono-CheY, is synthesized mimic in vivo known as P-CheY; P-CheY only has a half-life ~30 seconds, making it extremely difficult study. P- is produced by phosphorylation an aspartate residue on histidine kinase CheA. Subsequently, binds flagellar motor FliM switches the flagella’s rotational direction. becomes dephosphorylated phosphatase CheZ (in E. coli) or phosphatases CheC/D, CheX, FliY/N T. maritima) change the flagella rotation back its previous state. binding release from FliM causes bacteria exhibit periods smooth swimming tumbling motions which drive bacterium out harsh environments into nutrient rich ones; these events define bacterial chemotaxis. phosphono-CheY synthesized order replace the labile P-O bond with stable P-C bond; this will allow for crystallographic data assays be performed without degradation the protein. produced reacting phosphonomethyltriflate (PMT) presence 3.0 equivalents triethylamine 125 mM Ca2+. Typically, this reaction result 45-70% conversion meaning a purification step must subsequently. Purification successful using cation exchange HPLC 50 sodium acetate buffer at pH = 5.3. gradient was run from 0 11.7% mobile phase B over 47 minutes pure phosphono-CheY eluted 31 23 minutes. Crystallization trials modeled after those for unmodified maritima were applied diffraction quality crystals were grown wells that contained following: PEG 3400 (26%)/100 HEPES (pH = 7.0)/.2 M (NH4)SO4/15 MgCl2, (28%)/100 4.5)/.2 M (NH4)SO4/15 4000 7.0)/.2 MgCl2.