Crystallization of phosphono-CheY from Thermotoga maritima

UNCW Author/Contributor (non-UNCW co-authors, if there are any, appear on document)
Cory Bottone (Creator)
The University of North Carolina Wilmington (UNCW )
Web Site:
Christopher Halkides

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

Additional Information

A Thesis Submitted to the University of North Carolina Wilmington in Partial Fulfillment Of the Requirements for the Degree of Master of Science
Language: English
Date: 2009
Crystallization, Proteins--Synthesis, Thermophilic microorganisms--Research
Proteins -- Synthesis
Thermophilic microorganisms -- Research

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