Abstract
Correct protein folding is the foundation for all cellular processes. Often this is not a spontaneous process; instead various molecular chaperones are required for the correct folding of a range of different proteins. Chaperones prevent aggregation and promote the correct fate of the protein in vivo. The best-studied chaperone system
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is the GroEL-GroES machine from Escherichia coli. GroEL is composed of two heptameric rings stacked back to back. GroES, the co-chaperonin, consists of one heptameric ring that serves as a lid on the double ring GroEL structure, thereby providing a safe folding environment for the substrate protein. Viral intruders, such as bacteriophage T4 hijack this chaperonin complex. The T4 phage requires the chaperonin to fold its major capsid protein, gp23, however in a very special way. Instead of using the host co-chaperonin GroES it uses gp31, a co-chaperonin that is encoded by bacteriophage T4 itself. So the question arises: What are the unique properties that gp31 possesses, which makes it absolutely essential for the folding of the T4 major capsid protein? It is difficult to study the different steps in the chaperonin-assisted folding pathway, as this is a dynamic process of a large non-covalent protein complex. In recent years, mass spectrometry has allowed the analysis of larger protein complexes. We set out to use native mass spectrometry to analyze the GroEL-gp31 chaperonin complex with the aim of obtaining information that would provide further insight into the molecular mechanism by which this folding machine facilitates the folding of the T4 viral capsid protein. The initial step of the folding cycle is the binding of gp23 to the GroEL chaperonin. The generally accepted view was that the chaperonin complex could only bind one substrate molecule at the time. However we revealed that the GroEL complex could bind up to two non-native gp23 substrates simultaneously. We also showed it was possible to obtain properly refolded gp23 by using the GroEL-gp31 complex. This mass spectrometric approach thus provided us with unique information about the chaperonin complex that so far had not been revealed by any other method. We next investigated the substrate binding properties of the chaperonin complex by using a combination of native mass spectrometry and tandem mass spectrometry. The latter is a powerful technique that can be used to dissect protein complexes in the gas-phase in a sequential fashion and allows the identification of the building blocks, and potentially also the topology of the complex, its quaternary structure and stability. We showed that the stability and the conformational changes of the chaperonin complex induced by substrate binding are dependent on the type of substrate that binds. Interestingly, the chaperonin complex is only able to distinguish between different substrates, when both rings are present and inter-ring communication is possible.
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