Crystallization of phosphono-CheY from Thermotoga maritima
- UNCW Author/Contributor (non-UNCW co-authors, if there are any, appear on document)
- Cory Bottone (Creator)
- Institution
- The University of North Carolina Wilmington (UNCW )
- Web Site: http://library.uncw.edu/
- Advisor
- 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.
Crystallization of phosphono-CheY from Thermotoga maritima
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Created on 1/1/2009
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Additional Information
- Publication
- Thesis
- 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
- Keywords
- Crystallization, Proteins--Synthesis, Thermophilic microorganisms--Research
- Subjects
- Proteins -- Synthesis
- Thermophilic microorganisms -- Research
- Crystallization