PhD projects


Structural Biology of Membrane Proteins

PhD project:

Structure – dynamics - function of OmpF channel protein from E. coli;

A joint AFM – SMFS – ssNMR – EM approach

Principal Investigator:

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

In collaboration with:

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

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

Gram negative bacteria are protected by an outer membrane in which trimeric channels, the porins, facilitate the passage of small solutes1. The pores are formed by 16 or 18 antiparallel membrane spanning antiparallel -strands, which are connected by short turns on the periplasmic side and long loops on the extracellular side. In all known porin structures loop 3 is folded inside the channel and forms the channel constriction2. Conductance measurements have shown that porin OmpF trimers exist in either open or closed states, depending on the transmembrane3. For OmpF, the critical voltage for channels closure depends on ionic strength4, pH5, and the presence of oligosaccharides6, 7, or polycations8. For LamB, the maltose specific porin, channel closure occurs at a pH < 49. In spite of the rich structural knowledge on porins (e.g. OmpF10), the dependence of their activity on voltage and pH remains enigmatic. For OmpF loop L3 was shown not be involved in voltage dependent channel closure11. OmpF mutants where single extracellular loops, except L3, were deleted one at a time, exhibited wild-type voltage gating, whereas deletions of loops L1, L7 or L8 affected the tendency of channels to close at acidic pH12. Involvement of external loops in channel closure was indicated by a striking conformational change of the extracellular OmpF domains at membrane potentials > 200 mV or pH < 4 observed by atomic force microscopy (AFM)13.

Therefore, the structure and dynamics of these extracellular loops are of prime functional significance, and are subject of the proposed study. To express OmpF, strain BZB111014 will be employed and the protein will be purified by an established protocol. OmpF will be reconstituted into 2D crystals to perform electron crystallography at neutral pH and pH <4. Such membranes will also be imaged by AFM and the channel conductance determined by scanning electrochemistry microscopy (SECM15) (Engel lab). The dynamics of the extracellular loops will be assessed by solid state NMR methods (Milon lab). Furthermore, single molecule force spectroscopy (SMFS) on such 2D crystals of OmpF will be performed in the Müller lab at neutral and low pH to determine the forces that dictate the OmpF fold and to explore their dependence on pH. To gain further insights, deletion mutants of specific extracellular loops described previously will be prepared, expressed, reconstituted and assessed by the same methods.

The group of D. Muller has recently developed efficient approaches to characterise the unfolding and refolding pathways and energy landscape of membrane proteins by SMFS16. These techniques have been applied so far on various helical membrane proteins, and deserve to be applied on beta barrel membrane proteins such as OmpF membrane domains.

The group of A. Engel is a leading lab in electron microscopy (EM) and AFM analyses of the structure and dynamics of membrane proteins17-19.

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. The proteins have 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.

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

- Molecular biology – protein biochemistry (A. Engel’s group, 6 months)

Wild-type OmpF and different deletion mutants will be expressed, purified and reconstituted. All the technology for producing these proteins with high yield is already available in the group.

- EM and AFM characterisation of OmpF in membranes (A. Engel’s group, 12 months)

OmpF reconstituted in lipid bilayers will be analysed by electron diffraction and AFM. Electron crystallography will reveal pH induced conformational changes, while the loops dynamics will be monitored by AFM. Simultaneous conductance measurements by SECM will address the functional state of OmpF.

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

SMFS provides a quantitative view of the energetic landscape for the folding and unfolding of membrane proteins. In a first step we will detect and map molecular interactions within OmpF. Dynamic SMFS will then map details of the energy landscape and observe the energy minima, energy barriers and kinetic properties of the OmpF beta barrels and loops. In the next step we will analyse the unfolding and refolding of OmpF as a function of the environment (lipid bilayers of different composition, various detergents), and as a function of the sequence (e.g., comparison between various mutants).

- Solid state NMR of OmpF (A. Milon’s group, 6 months)

The loop dynamics will be analysed using amino acid specific labelling, designed to select a limited number of C’N bonds. 15N relaxation and 13C-15N REDOR experiments will be employed to characterise the internal dynamics. These experiments will be performed on a 700 MHz solid state NMR Bruker avance spectrometer, using a 3.2 mm 1H-13C-15N triple resonance MAS probe.


  1. Gervais V, Lamour V, Jawhari A, Frindel F, Wasielewski E, Dubaele S, Egly JM, Thierry JC, Kieffer B, Poterszman A. 2004.
    TFIIH contains a PH domain involved in DNA nucleotide excision repair. Nat Struct Mol Biol 11(7):616-22.
  2. Ravault S, Soubias O, Saurel O, Thomas A, Brasseur R, Milon A. 2005.
    Fusogenic Alzheimer's peptide fragment Abeta (29-42) in interaction with lipid bilayers: secondary structure, dynamics, and specific interaction with phosphatidyl ethanolamine polar heads as revealed by solid-state NMR. Protein Sci 14(5):1181-9.
  3. Soubias O, Jolibois F, Massou S, Milon A, Reat V. 2005.
    Determination of the orientation and dynamics of ergosterol in model membranes using uniform 13C labeling and dynamically averaged 13C chemical shift anisotropies as experimental restraints. Biophys J 89(2):1120-31.
  4. Soubias O, Jolibois F, Reat V, Milon A. 2004.
    Understanding Sterol-Membrane Interactions, Part II: Complete 1H and 13C Assignments by Solid-State NMR Spectroscopy and Determination of the Hydrogen-Bonding Partners of Cholesterol in a Lipid Bilayer. Chemistry 10:6005-6014.
  5. Soubias O, Reat V, Saurel O, Milon A. 2004.
    15N T2' relaxation times of bacteriorhodopsin transmembrane amide nitrogens. Magn Reson Chem 42(2):212-7.
  6. Müller DJ, et al.,
    Voltage and pH-induced channel closure of porin OmpF visualized by atomic force microscopy. J Mol Biol, 1999.
  7. Engel A, et al.,
    Observing single biomolecules at work with the atomic force microscope. Nat Struct Biol, 2000.
  8. Muller DJ, et al.,
    Single-molecule studies of membrane proteins. Curr Opin Struc Biol, 2006.
  9. Kedrov A, et al.,
    Deciphering molecular interactions of native membrane proteins by single-molecule force spectroscopy. Annu Rev Biophys Biomol Struct, 2007.