Structural investigations on the ATP-synthase from chloroplasts
Final Report Abstract
The ATP-synthase is one of the most remarkable molecular machines, which drives ATPsynthesis by translocating protons along a potential difference. This is achieved by a rotational mechanism, which requires a rotating part and a stator. Many of the structural details of the coupling mechanism are only poorly understood, because no crystal structures of the whole complex or similarly important of the membrane part that translates a potential difference of protons into a rotational movement exists. We have taken on the challenge to crystallize the ATPsynthase from chloroplasts and obtained crystals diffracting to low resolution in the last reporting period. Building on this success we systematically optimized a number of different factors. We paid special attention to the use of different detergents, which are added to stabilize the complex outside of the membrane. It turned out that some of the sampled detergents promoted controlled disruption of the complex, whereas others were useful for purifying a structurally intact complex as verified biochemically and by electron microscopy. Furthermore, we have shown that our purification protocol removed all lipids from the ATPsynthase, even those lipids which are thought to be tightly bound and essential for function. With the optimal detergents and re-added lipids systematic crystallization trials were performed. However, crystals remained small and of limited quality. Alternatively, we tried purifying the membrane-part for further crystallization trials. However, we only achieved incomplete separation of membrane part and extrinsic part, which totally compromised the ability of the sub-complex to crystallize. Unfortunately, despite numerous efforts we were not able to cystallize the ATPsynthase for further structural studies. Alternatively, we used electron cryo microcopy and image processing to determine a low resolution structure of the ATPsynthase from chloroplasts. This structure revealed how membrane part and soluble part are structurally coupled. The structure shows a surprisingly big gap between the rotor and the membrane anchor of the stator. By generating homology models for most of the subunits and fitting them into the EM-density, we could generate a pseudo-atomic model of the ATPsynthase from chloroplasts. This model still leaves a number of open questions. However, the model is a reliable basis for designing future experiments (e.g. design of FRET labels) to further explore function and dynamics of the ATPsynthase.
Publications
- Mellwig, C. and Böttcher, B. (2003) A unique resting position of the ATPsynthase from chloroplasts. J Biol Chem, 278, 18544-18549.
