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Recombinant production and purification of the subunit c of chloroplast ATP synthase.

ABSTRACT: In chloroplasts, the multimeric ATP synthase produces the adenosine triphosphate (ATP) that is required for photosynthetic metabolism. The synthesis of ATP is mechanically coupled to the rotation of a ring of c-subunits, which is imbedded in the thylakoid membrane. The rotation of this c-subunit ring is driven by the translocation of protons across this membrane, along an electrochemical gradient. The ratio of protons translocated to ATP synthesized varies according to the number of c-subunits (n) per oligomeric ring (c(n)) in the enzyme, which is organism dependent. Although this ratio is inherently related to the metabolism of the organism, the exact cause of the c(n) variability is not well understood. In order to investigate the factors that may contribute to this stoichiometric variation, we have developed a recombinant bacterial expression and column purification system for the c? monomeric subunit. Using a plasmid with a codon optimized gene insert, the hydrophobic c? subunit is first expressed as a soluble MBP-c? fusion protein, then cleaved from the maltose binding protein (MBP) and purified on a reversed phase column. This novel approach enables the soluble expression of an eukaryotic membrane protein in BL21 derivative Escherichia coli cells. We have obtained significant quantities of highly purified c? subunit using these methods, and we have confirmed that the purified c? has the correct alpha-helical secondary structure. This work will enable further investigation into the undefined factors that affect the c-ring stoichiometry and structure. The c-subunit chosen for this work is that of spinach (Spinacia oleracea) chloroplast ATP synthase.

SUBMITTER: Lawrence RM

PROVIDER: S-EPMC4839536 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature

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Recombinant production and purification of the subunit c of chloroplast ATP synthase.

Lawrence Robert M RM Varco-Merth Benjamin B Bley Christopher J CJ Chen Julian J-L JJ Fromme Petra P

Protein expression and purification 20101030 1

In chloroplasts, the multimeric ATP synthase produces the adenosine triphosphate (ATP) that is required for photosynthetic metabolism. The synthesis of ATP is mechanically coupled to the rotation of a ring of c-subunits, which is imbedded in the thylakoid membrane. The rotation of this c-subunit ring is driven by the translocation of protons across this membrane, along an electrochemical gradient. The ratio of protons translocated to ATP synthesized varies according to the number of c-subunits ( ...[more]

PMID: 21040791

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Project description:New findingsWhat is the central question of this study? Humans with obesity have lower ATP synthesis in muscle along with lower content of the β-subunit of the ATP synthase (β-F1-ATPase), the catalytic component of the ATP synthase. Does lower synthesis rate of β-F1-ATPase in muscle contribute to these responses in humans with obesity? What is the main finding and its importance? Humans with obesity have a lower synthesis rate of β-F1 -ATPase and ATP synthase specific activity in muscle. These findings indicate that reduced production of subunits forming the ATP synthase in muscle may contribute to impaired generation of ATP in obesity.AbstractThe content of the β-subunit of the ATP synthase (β-F1 -ATPase), which forms the catalytic site of the enzyme ATP synthase, is reduced in muscle of obese humans, along with a reduced capacity for ATP synthesis. We studied 18 young (37 ± 8 years) subjects of which nine were lean (BMI = 23 ± 2 kg m-2 ) and nine were obese (BMI = 34 ± 3 kg m-2 ) to determine the fractional synthesis rate (FSR) and gene expression of β-F1 -ATPase, as well as the specific activity of the ATP synthase. FSR of β-F1 -ATPase was determined using a combination of isotope tracer infusion and muscle biopsies. Gene expression of β-F1 -ATPase and specific activity of the ATP synthase were determined in the muscle biopsies. When compared to lean, obese subjects had lower muscle β-F1 -ATPase FSR (0.10 ± 0.05 vs. 0.06 ± 0.03% h-1 ; P < 0.05) and protein expression (P < 0.05), but not mRNA expression (P > 0.05). Across subjects, abundance of β-F1 -ATPase correlated with the FSR of β-F1 -ATPase (P < 0.05). The specific activity of muscle ATP synthase was lower in obese compared to lean subjects (0.035 ± 0.004 vs. 0.042 ± 0.007 arbitrary units; P < 0.05), but this difference was not significant after the activity of the ATP synthase was adjusted to the β-F1 -ATPase content (P > 0.05). Obesity impairs the synthesis of β-F1 -ATPase in muscle at the translational level, reducing the content of β-F1 -ATPase in parallel with reduced capacity for ATP generation via the ATP synthase complex.

Dataset Information

Recombinant production and purification of the subunit c of chloroplast ATP synthase.

Publications

Recombinant production and purification of the subunit c of chloroplast ATP synthase.

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