Seminar

Structural and mechanistic studies of large macromolecules by NMR

Yiannis Gelis, PhD

Solution state NMR methodologies are routinely used to study proteins and protein complexes with molecular weights below 50kDa. Still, a combination of novel isotope labeling schemes and Methyl-TROSY has allowed obtaining high resolution structural, functional and dynamic insights to biologically important macromolecular assemblies in range of MDa. SecA is a dimeric 204 kDa protein that plays a central role in bacterial protein secretion, the fundamental cellular process by which proteins are transported across or integrated into a membrane with the expenditure of metabolic energy extracted from ATP hydrolysis. In the context of binary or ternary complexes it interacts with all the translocation components, including both the signal sequence and the mature domain of preproteins, the chaperone SecB, the protein conducting channel SecYEG and translating ribosomes. Building on methyl labeling strategies and paramagnetic spin labeling it was possible to determine the high-resolution structure of a SecA-signal sequence complex, by NMR. The recognition of signal sequences by their cognate receptors is the first and most decisive step during protein translocation. The structure, together with an extensive thermodynamic analysis and in vivo experiments revealed the details of a promiscuous recognition and the first steps of protein translocation. The same approach can be used for the study of other large macromolecular complexes such as of SecA with translating ribosomes and the Hsp90 machinery.