Over-expression, purification and characterization of the oligomerization dynamics of an invertebrate mitochondrial creatine kinase
| Autor: | W. Ross Ellington, Gregg G. Hoffman |
|---|---|
| Rok vydání: | 2005 |
| Předmět: |
Gene isoform
Sarcomeres Light Size-exclusion chromatography Biophysics Creatine Kinase Mitochondrial Form Magnesium Chloride lac operon Gene Expression Biology Chaetopterus variopedatus Biochemistry Analytical Chemistry Dynamic light scattering Transition state analog Enzyme Stability Escherichia coli Animals Creatine Kinase MB Form Protein Isoforms Scattering Radiation Histone octamer Particle Size Protein Structure Quaternary Molecular Biology Creatine Kinase Nitrates Temperature Polychaeta biology.organism_classification Recombinant Proteins Adenosine Diphosphate Isoenzymes Molecular Weight Kinetics Protein Subunits biology.protein Chromatography Gel Creatine kinase Electrophoresis Polyacrylamide Gel Chickens |
| Zdroj: | Biochimica et biophysica acta. 1751(2) |
| ISSN: | 0006-3002 |
| Popis: | Mitochondrial creatine kinase (MtCK) plays a central role in energy homeostasis within cells that display high and variable rates of ATP turnover. Vertebrate MtCKs exist primarily as octamers but readily dissociate into constituent dimers under a variety of circumstances. MtCK is an ancient protein that is also found in invertebrates including sponges, the most primitive of all multi-cellular animals. We have cloned, expressed, and purified one of these invertebrate MtCKs from a marine polychaete worm, Chaetopterus variopedatus (CVMtCK). Size exclusion chromatography and dynamic light scattering (DLS) were used to characterize oligomeric state in comparison with that of octameric chicken sarcomeric isoform (SarMtCK). At protein concentrations1 mg/ml, CVMtCK was predominantly octameric (90%). When diluted to 0.1 mg/ml, CVMtCK dissociated into dimers much more rapidly than SarMtCK when observed under identical conditions. The rate of dissociation for both MtCKs increased as temperature rose from 2 to 28 degrees C, and in CVMtCK, fell at higher incubation temperatures. The fraction of octameric CVMtCK at equilibrium increased with temperature and then fell. Temperature transition studies showed that octamers and dimers were rapidly interconvertible on a similar time scale. Importantly, when CVMtCK was converted to the transition state analog complex (TSAC), both size exclusion chromatography and DLS showed that there was minimal dissociation of octamers into dimers while SarMtCK octamers were highly unstable as the TSAC. These results clearly show distinct differences in octamer stability between CVMtCK and SarMtCK, which could impact function under physiological circumstances. Furthermore, the large yield of recombinant protein and high stability of CVMtCK in the TSAC suggest that this protein might be a good target for crystallization efforts. |
| Databáze: | OpenAIRE |
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