Over-expression, purification and characterization of the oligomerization dynamics of an invertebrate mitochondrial creatine kinase.
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 concentrations >1 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.
Department of Biological Science, Florida State University Tallahassee, FL 32306-4370, USA.
Gregg G
GG
Hoffman
W Ross
WR
Ellington
D000244:Adenosine Diphosphate / Q000737:chemistry; D000818:Animals; D002645:Chickens; D002850:Chromatography, Gel; D003402:Creatine Kinase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D052279:Creatine Kinase, MB Form; D052281:Creatine Kinase, Mitochondrial Form; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D007527:Isoenzymes / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D007700:Kinetics; D008027:Light; D015636:Magnesium Chloride / Q000737:chemistry; D008970:Molecular Weight; D009566:Nitrates / Q000737:chemistry; D010316:Particle Size; D011077:Polychaeta / Q000201:enzymology* / Q000235:genetics; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics; D020836:Protein Structure, Quaternary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D012518:Sarcomeres / Q000201:enzymology; D012542:Scattering, Radiation; D013696:Temperature
D007527:Isoenzymes; D009566:Nitrates; D020033:Protein Isoforms; D021122:Protein Subunits; D011994:Recombinant Proteins; D015636:Magnesium Chloride; D000244:Adenosine Diphosphate; C031618:sodium nitrate; D003402:Creatine Kinase; D052279:Creatine Kinase, MB Form; D052281:Creatine Kinase, Mitochondrial Form