PhD projects


Structural Biology of Membrane Proteins

PhD project:

Structure – dynamics - function of OmpA protein from klebsiella pneumoniae;

A joint ssNMR – AFM – EM – SMFS approach

Principal Investigator:

Prof A. Milon, IPBS, 205 rte de Narbonne, 31077 Toulouse, France; alain.milon|

In collaboration with:

Prof. A. Engel, M.E. Müller Institute for Microscopy, Biozentrum, University of Basel, 4056 Basel, Switzerland; andreas.engel|

Dr. D. Muller, Professor of Cellular Machines, Center of Biotechnology, University of Technology Tatzberg 47-51, D-01307 Dresden, Germany; mueller|

kpOmpA is a membrane protein belonging to the outer membrane protein A family. Its transmembrane domain (216 aa) presents a significant homology with E. coli OmpA (80 %), whose three dimensional structure has been determined by X-ray crystallography and by NMR(1). The E. Coli homologue can function as an adhesin and invasin, participate in biofilm formation, act as both an immune target and evasin, and serves as a receptor for several bacteriophages. It is assumed that most of these functions involve the four protein loops that emanate from the protein to the outside of the cell(2). The difference between kpOmpA and E. coli OmpA is mostly concentrated in the extra cellular loops which are larger in the case of kpOmpA. kpOmpA was shown to activate macrophages and dendritic cells through the TLR2 dependent pathway, and these larger loops are supposed to play a specific role in the interactions with the immune system(3, 4). Thus the structure and dynamics of these loops is of prime functional significance. However, the structures determined so far both by NMR and by X-ray diffraction have been obtained on recombinant membrane domains, purified and refolded in detergent solutions, and they display a significant degree of mobility in these conditions. The question thus arises of their structural and functional behaviour in more physiological conditions.

The group of A. Milon has recently determined the 3D structure of kpOmpA membrane domain by solution state NMR and have established efficient protocols for the expression, purification, stable isotope labelling of kpOmpA membrane domain(5).  The protein has been refolded in a variety of detergent solutions and lipid bilayers(6).  The group possess all the methodologies required to characterise protein structure and dynamics by liquid and solid state approaches(7-11). Preliminary data have been obtained by solid state NMR methods on kpOmpA membrane domain reconstituted at a high lipid/protein ration in lipid bilayers.

Figure Figure: 2D 13C-13 C spin diffusion MAS spectrum
of kpOMPA in lipid bilayers
(u-15N, 13C)-KpOMPA, L/P = 1/30;
700 MHz CPMAS- 3.2mm;
293K, nr = 13 kHz

The group of A. Engel is a leading expert in electron microscopy (EM) and atomic force microscopy (AFM) analyses of the structure and dynamics of membrane proteins(12, 13).  The group of D. Muller has recently developed efficient approaches to characterise the unfolding landscape of membrane proteins by single-molecule force spectroscopy (SMFS)(14). SMFS has been applied so far on transmembrane alpha-helical proteins, and deserve to be applied on beta barrel membrane proteins such as kpOmpA membrane domains.

This PhD project is focused on understanding the structure, dynamics, function and energetics of kpOmpA by combining ssNMR, EM, AFM and SMFS. A central question for the protein function concerns the specific role of the extra cellular loops. This implies various activities:

- Molecular biology – protein biochemistry (A. Milon’s group, 3 months)

The N-terminal membrane domain and the entire protein will be compared. Indeed, although the N-terminal domain has been shown to be folded as a typical 8 stranded β-barrel, it has been assumed that the C-terminal domain may insert into the membrane to form a 16-stranded β-barrel to account for the channel activity of this protein(2). Various construction will be expressed, in order to perform single point mutations, or to add tags required for SMFS studies.  All the technology for producing these proteins with high yield is already available in the group.

- Incorporation of kpOmpA into model and natural membranes; preparation of 2D crystals; EM and AFM characterisations (A. Engel’s group, 6 months)

The recombinant proteins will be reconstituted at high protein/lipid ratio in lipid bilayers (N-terminal domain and entire protein). They will also be expressed in E. coli strains devoid of endogenous porins(15) in order to prepare natural outer membranes containing kp-OmpA. These membranes will be analysed by electron diffraction to check for the presence of 2D crystals. The resolution attainable from this approaches depends on the sample, however, even relatively low resolution structures should be sufficient to distinguish between the 16 stranded and the 8 stranded barrels.

- Solid state NMR of kpOmpA (A. Milon’s group, 15 months)

After having completed the NMR structure of kpOmpA membrane domain in detergent by solution state NMR(5), we will use MAS solid state NMR techniques to solve the 3D structure of the proteins reconstituted in lipid bilayers (including natural E. coli, LPS containing, outer membranes). This will greatly benefit from the availability of 2D crystals developed in A. Engel’s group, known to provide an optimum sensitivity and resolution. A particular focus will be placed on characterizing the loops dynamics, from15N relaxation studies(9,16). One major issue is to determine whether the slow (ms) and fast (ns) dynamics which have been observed in detergent solutions is still present in bilayers. These experiments will be performed on our 500 MHz and 700 MHz solid state NMR Bruker avance spectrometers, using triple resonance MAS probes.

- SMFS characterisation of kpOmpA in membranes (D. Muller’s group, 12 months)

SMFS provides a quantitative view of molecular interactions establishing the energetic landscape of membrane proteins. Here, we will analyse the molecular interactions of kpOmpA as a function of the environment (lipid bilayers of different composition, various detergents), and as a function of the sequence (comparisons of the entire proteins with the N-terminal domain, comparison between various mutants). Having mapped the kpOmpA structure with these interactions we will then investigate how they change upon binding to the myloid A protein and to sugars from different cells to which kpOmpA serves as a receptor. Using dynamic force spectroscopy we will measure the interactions at different time scale which will allow reconstructing the dynamic energy landscape of the kpOmpA before and after binding to other physiological relevant molecules. This will allow drawing a detailed picture of the mechanisms by which kpOmpA serves as a receptor for host cells.

  1. Pautsch A, et al. J Mol Biol, 2000, Arora A, et al., Nat Struct Biol, 2001.
  2. Smith SGJ, et al, FEMS Microbiol Lett, 2007.
  3. Jeannin P, et al., Nat Immunol, 2000; Immunity 2005.
  4. PhD thesis,  M. Sugawara, UPS Toulouse, France, 2003.
  5. Renault M, et al., in preparation for Nat Struct Biol, 2007.
  6. Renault M, et al., C.R. Chimie, 2006.
  7. Gervais V,et al., 2004. Nat Struct Mol Biol, 2004.
  8. Soubias O, et al., Chemistry 2004.
  9. Soubias O, et al., Magn Reson Chem 2004.
  10. Ravault S, et al., Protein Sci 2005.
  11. Soubias O, et al., Biophys J 2005.
  12. Murata K, et al., Nature, 2000.
  13. Engel A, et al., Nat Struct Biol 2000.
  14. Muller DJ, et al., Curr Opin Struc Biol, 2006; Kedrov A, et al., Annu Rev Biophys Biomol Struct, 2007.
  15. Hoenger A, et al., J Struct Biol, 1998.
  16. Giraud N, et al., JACS 2004, 2005.