In:
Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 109, No. 25 ( 2012-06-19)
Abstract:
The current study provides insight into the construction and the operational principles of a molecular nanomotor. Because the c-ring stoichiometries appear constant in an organism, changing the gear of the ATP synthase by engineering the c-ring stoichiometry potentially harbors intriguing biotechnological possibilities for the future. The proposed strategy for altering the ion-to-ATP ratio could be applicable to other organisms. For example, it eventually could be used to optimize the cell's energy balance on demand in biomass producers such as plants or photosynthetic bacteria. We were able to redesign the stoichiometry of the ATP synthase c 11 ring from the bacterium Ilyobacter tartaricus by a simple mutagenesis approach. The c 11 ring was selected for this work because of its exceptional biochemical robustness and its known 3D structure. We hypothesized that the rotor ring stoichiometry would be altered by selected mutations within a tight interaction motif established between the N-terminal helices of two adjacent c-subunits. This motif consists of a repetitive pattern of conserved glycine residues (GxGxGxGxG), which also appears in other membrane proteins with interacting α-helices. To test this hypothesis, we carried out site-directed mutagenesis of I. tartaricus c-rings, in which specific glycine residues were replaced by alanines, and expressed these c-ring mutants in Escherichia coli . The purified mutants first were assessed by a gel-based electrophoretic separation method. Mutants that diverged noticeably from the wild-type migration pattern were selected, reconstituted into proteoliposomes, and further investigated by electron microscopy and atomic force microscopy. As visually confirmed by these methods, the mutations altered wild-type c 11 stoichiometry ( Fig. P1 B ) to c 〈 11 , c 12 ( Fig. P1 C ), c 13 , c 14 , and c 〉 14 . Molecular dynamics simulations of wild-type and mutant c-rings suggest that energetic and geometric perturbations in the interface between c-subunits underlie these alternative stoichiometries. Thus, we conclude that the glycine-rich motif in the c-subunit is a critical determinant of the rotor ring stoichiometry in I. tartaricus and that c-rings of diverse stoichiometries can, in principle, derive from the same c-subunit mutant. Moreover, protein interactions revealed by surface plasmon resonance spectroscopy demonstrated that the assembly of the F 1 F o complex is independent of the size of the c-ring. Furthermore, we show that the mutant rotors can be incorporated into functional ATP synthases and preserve the catalytic properties of the wild-type enzyme. Last, we also show that with the increased c-ring stoichiometry, the mutant ATP synthases are able to operate at a lower ion motive force threshold than the wild-type. This remarkable result demonstrates that the enzymes with larger c-rings indeed operate at a higher ion-to-ATP ratio—that is, in a different gear. We focus on the F o rotor, which consists of a ring-shaped assembly of 8–15 identical copies of a small protein known as the “c-subunit.” Each c-subunit contributes to creating a binding site for an ion, namely, H + or Na + ( 3 , 4 ). The c-ring can rotate back and forth stochastically, but the difference in the concentration of the coupling ion across the membrane causes it to rotate preferentially in one direction. As this rotation happens, ions are released sequentially on one side of the membrane and loaded onto the ring from the other via two independent ion pathways. Although the number of translocated ions varies across different organisms, three ATP molecules are produced invariably in F 1 after a full rotation of the c-ring. Therefore, the stoichiometry of the c-ring determines the theoretical ion-to-ATP ratio. ATP is the universal energy currency in all organisms and must be produced continuously by the enzyme F 1 F o -ATP synthase ( Fig. P1 A ). This membrane protein complex uses the energy stored in a transmembrane ion gradient to power a unique rotational mechanism, and then converts this mechanical work into the chemical energy stored in the form of ATP. Two structurally separate but tightly coupled molecular motor complexes, F 1 and F o , operate in concert to translocate ions across the membrane in F o to synthesize ATP from ADP and inorganic phosphate in F 1 . The ion-to-ATP ratio ( Fig. P1 B and C ) describes the number of ions required to synthesize each ATP molecule ( 1 , 2 ) and is analogous to the gearing ratio of a mechanically coupled rotary motor. Changing the ion-to-ATP ratio in ATP synthase therefore would be comparable to a gear-shift in an engine. Here, we report the successful alteration of the gear ratio of an ATP synthase by introducing mutations in its amino acid sequence.
Type of Medium:
Online Resource
ISSN:
0027-8424
,
1091-6490
DOI:
10.1073/pnas.1120027109
Language:
English
Publisher:
Proceedings of the National Academy of Sciences
Publication Date:
2012
detail.hit.zdb_id:
209104-5
detail.hit.zdb_id:
1461794-8
SSG:
11
SSG:
12
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