Outer membrane proteins: structure, folding and dynamics as seen by liquid and solid state NMR

Title Outer membrane proteins: structure, folding and dynamics as seen by liquid and solid state NMR
Lecturer Prof. Alain Milon(CNRS and Université de Toulouse-UPS)
Language English
Date&Time 11/10/2014 (Mon) 11:00~12:00
Venue Large seminar room
 Outer membrane proteins play a diversity of fundamental roles in gram negative bacteria, as well as in corynebacteriales. These can be structural components, transporters, epitopes for specific cell – cell
recognition mechanisms, enzymes, and as such they are interesting targets for antibacterial drug development. During this talk I will address two topics recently developed in our laboratory, the structure –
dynamics of the outer membrane proteins A from Klebsiella pneumoniae (kpOmpA) and protein channels from the outer membrane of Corynebacterium glutamicum (PorA-PorH).
 The transmembrane domain of kpOmpA possesses four long extracellular loops which exhibit substantial sequence variability throughout OmpA homologues in Enterobacteria. These loops are responsible for the
immunological properties of the protein, such as cellular and humoral recognitions. Using liquid state NMR we have determined the 3D structure of kpOmpA in DHPC micelles (J. Mol. Biol. 2009). In a micellar
environment, a complex dynamical behavior has been observed: a rigid barrel core, ms motion at the micellar-water interface, and sub-ns motions within the loops. Using solid state NMR relaxation measurements and proteolysis experiments, we have demonstrated the persistence of this complex motional behavior in E. coli polar lipid bilayers (Biochim. Biophys. Acta, 2012). Using single molecule force spectroscopy (with D. Muller and A. Engel) we have shown that kpOmpA is able to unfold and refold reversibly its b-barrel core (Structure 2012). Recent advances on characterizing this protein structure and dynamics involve: a) characterizing the structure of its C-terminal domain and its interaction with the peptido-glycane; b) analyzing NMR spectra of N-terminal membrane domain in liposomes using MAS at 1 GHz and 60 kHz spinning frequency (with G. Pintacuda); c) comparing the NMR spectra of the various domains and the full length protein in solution, in liposomes and in intact cell envelopes using cellular solid state NMR as established in (Proc. Natl. Acad. Sci. USA 2012).
 The outer membrane of corynebacteriales (C. glutamicum, M. tuberculosis) is made of very specific lipids named mycolic acids. We have purified and characterized the phase behavior of TDM and TMM too major lipids from C. glutamicum outer membrane. We have further expressed and characterized functionally PorA and PorH two major proteins from this outer membrane which are post translationally modified by a mycolic acid and form ion channels in this membrane (J. Biol. Chem. 2011, Biochim. Biophys. Acta 2013, FEBS Lett. 2013).

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