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Calcium-induced conformational changes in the regulatory domain of the human mitochondrial ATP-Mg/Pi carrier.

Harborne SP, Ruprecht JJ, Kunji ER - Biochim. Biophys. Acta (2015)

Bottom Line: Careful analysis by SEC confirmed that although the regulatory domain crystallised as dimers, full-length ATP-Mg/Pi carrier is monomeric.Detailed bioinformatics analyses of different EF-hand states indicate that upon release of calcium, EF-hands close, meaning that the regulatory domain would release the amphipathic α-helix.We propose a mechanism for ATP-Mg/Pi carriers in which the amphipathic α-helix becomes mobile upon release of calcium and could block the transport of substrates across the mitochondrial inner membrane.

View Article: PubMed Central - PubMed

Affiliation: The Medical Research Council, Mitochondrial Biology Unit, Cambridge Biomedical Campus, Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 0XY, UK.

No MeSH data available.


Related in: MedlinePlus

Full-length HsAPC-1 is monomeric.A) SEC trace for full-length HsAPC-1 and protein quantification by SDS-PAGE gel (inset). B) Contributions of protein, detergent and lipid to the total mass. C) SEC trace for crudely isolated HsAPC-1 RD. Peak 1 was found to contain a contaminant, and identified as the 56.1 kDa E2 component of the L. lactis pyruvate dehydrogenase complex (Supplementary Fig. 1). In panels A and C the elution volume of molecular weight standards are indicated and annotated with the mass of the standard. D) Protein from each of the peaks 2 and 3 run on an SDS-PAGE gel in the presence or absence of DTT.
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f0015: Full-length HsAPC-1 is monomeric.A) SEC trace for full-length HsAPC-1 and protein quantification by SDS-PAGE gel (inset). B) Contributions of protein, detergent and lipid to the total mass. C) SEC trace for crudely isolated HsAPC-1 RD. Peak 1 was found to contain a contaminant, and identified as the 56.1 kDa E2 component of the L. lactis pyruvate dehydrogenase complex (Supplementary Fig. 1). In panels A and C the elution volume of molecular weight standards are indicated and annotated with the mass of the standard. D) Protein from each of the peaks 2 and 3 run on an SDS-PAGE gel in the presence or absence of DTT.

Mentions: To clarify this issue further, an independent experimental analysis of the oligomeric state of the HsAPC-1 was conducted, first on the full-length HsAPC-1 (Fig. 3). The protein was expressed in yeast, solubilised in LMNG, purified and analysed by size exclusion chromatography (SEC) (Fig. 3A). Compared to molecular weight standards, the total mass of the HsAPC-1 protein:detergent:lipid micelle was 223.5 kDa. An experimental assessment of the individual contributions to the total mass revealed that 153.2 kDa could be attributed to detergent, 18.7 kDa could be attributed to lipid, with the remaining mass, 51.2 kDa, being attributed to protein. Full-length HsAPC-1 has a theoretical molecular mass of 53.4 kDa, demonstrating that the protein is monomeric in detergent (Fig. 3B).


Calcium-induced conformational changes in the regulatory domain of the human mitochondrial ATP-Mg/Pi carrier.

Harborne SP, Ruprecht JJ, Kunji ER - Biochim. Biophys. Acta (2015)

Full-length HsAPC-1 is monomeric.A) SEC trace for full-length HsAPC-1 and protein quantification by SDS-PAGE gel (inset). B) Contributions of protein, detergent and lipid to the total mass. C) SEC trace for crudely isolated HsAPC-1 RD. Peak 1 was found to contain a contaminant, and identified as the 56.1 kDa E2 component of the L. lactis pyruvate dehydrogenase complex (Supplementary Fig. 1). In panels A and C the elution volume of molecular weight standards are indicated and annotated with the mass of the standard. D) Protein from each of the peaks 2 and 3 run on an SDS-PAGE gel in the presence or absence of DTT.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4562336&req=5

f0015: Full-length HsAPC-1 is monomeric.A) SEC trace for full-length HsAPC-1 and protein quantification by SDS-PAGE gel (inset). B) Contributions of protein, detergent and lipid to the total mass. C) SEC trace for crudely isolated HsAPC-1 RD. Peak 1 was found to contain a contaminant, and identified as the 56.1 kDa E2 component of the L. lactis pyruvate dehydrogenase complex (Supplementary Fig. 1). In panels A and C the elution volume of molecular weight standards are indicated and annotated with the mass of the standard. D) Protein from each of the peaks 2 and 3 run on an SDS-PAGE gel in the presence or absence of DTT.
Mentions: To clarify this issue further, an independent experimental analysis of the oligomeric state of the HsAPC-1 was conducted, first on the full-length HsAPC-1 (Fig. 3). The protein was expressed in yeast, solubilised in LMNG, purified and analysed by size exclusion chromatography (SEC) (Fig. 3A). Compared to molecular weight standards, the total mass of the HsAPC-1 protein:detergent:lipid micelle was 223.5 kDa. An experimental assessment of the individual contributions to the total mass revealed that 153.2 kDa could be attributed to detergent, 18.7 kDa could be attributed to lipid, with the remaining mass, 51.2 kDa, being attributed to protein. Full-length HsAPC-1 has a theoretical molecular mass of 53.4 kDa, demonstrating that the protein is monomeric in detergent (Fig. 3B).

Bottom Line: Careful analysis by SEC confirmed that although the regulatory domain crystallised as dimers, full-length ATP-Mg/Pi carrier is monomeric.Detailed bioinformatics analyses of different EF-hand states indicate that upon release of calcium, EF-hands close, meaning that the regulatory domain would release the amphipathic α-helix.We propose a mechanism for ATP-Mg/Pi carriers in which the amphipathic α-helix becomes mobile upon release of calcium and could block the transport of substrates across the mitochondrial inner membrane.

View Article: PubMed Central - PubMed

Affiliation: The Medical Research Council, Mitochondrial Biology Unit, Cambridge Biomedical Campus, Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 0XY, UK.

No MeSH data available.


Related in: MedlinePlus