PhD projects

ITP FP7 - SBMPs

Structural Biology of Membrane Proteins

PhD project:

Production, structural and functional characterization of membrane transport proteins from Archaea




Principal Investigator:

Margarida Archer, (Membrane Protein Crystallography Group), Instituto Tecnologia Química Biológica, ITQB-UNL, Av. República, EAN, Oeiras, Portugal; archer|itqb.un.l.pt.


In collaboration with:

Peter Henderson, Institute of Membranes and Systems Biology, Astbury Centre for Structural Molecular Biology, University of Leeds, UK.

Arnulf Kletzin, Institute of Microbiology and Genetics, Darmstadt University of Technology, Germany.

Eva Pebay-Peyroula, Institute of Structural Biology, CNRS, Grenoble, France, eva.pebay-peyroula|ibs.fr.

Membrane transport systems play crucial roles in essential cellular processes of all organisms. The Major Facilitator Superfamily (MFS) is one of the two largest families of membrane transporters and is found in Bacteria, Archaea, and Eukarya(1). The aim of the proposed work is the overexpression of sufficient amounts of several archaeal membrane transport proteins to allow their functional and structural characterization. We will focus on transporters responsible for uptake of sugars, nucleosides and amino acids; and needed for efflux of antibiotics. A number of these proteins are homologues to those found in man, so Archaea can provide a highly convenient model for elucidating by proxy the molecular mechanisms of mammalian transporters (which are themselves impossible to isolate in required amounts). Furthermore, mutants and chimaeric proteins can also be examined to locate substrate/inhibitor binding sites and elucidate the translocation process. Additionally, multidrug efflux proteins may be related with antibiotic resistance, and are thus potential targets for the development of new antimicrobial compounds.


Many of the presently cultivated Archaea are extremophiles, growing at elevated temperature, low pH, high salt, or a combination of these conditions. The selected archaeal species were isolated from hydrothermal vents, solfataras and hot springs(2). Studying proteins from extremophiles provides the advantage that we can also learn mechanisms of adaptation, thermal stability, and transport with or against steep gradients of solute concentration(3). The identification of parameters involved in the thermostability of such proteins is an important issue with biotechnological implications for industry.


At Darmstadt, there is a vast collection of extremophilic organisms and the genome of several species is already sequenced, which enables the cell growth of selected extremophiles and genomic DNA extraction. At Leeds, the strategy has been to develop diverse recombinant DNA approaches, amplified expression in both prokaryotic and eukaryotic hosts, detergent extraction and purification of active proteins, and a wealth of biophysical techniques. The determination of three-dimensional structures will be carried out at ITQB (methodology briefly described in ref. 16).

However, membrane proteins are known to be more difficult to crystallize and subsequently crystals are more fragile to handle compared to soluble proteins. The difficulty in obtaining crystals suitable for X-ray diffraction is one of the major obstacle to 3D structure determination of membrane proteins. Crystal may be obtained either by the standard vapor diffusion method starting from proteins solubilized in detergent, or from lipidic phases such as cubic phases(5). Since the group at ITQB is not familiar with this technique, the crystallization screenings using the meso-lipidic phases will be performed at the laboratory headed by Eva Pebay-Peyroula, CNRS, Grenoble, France, which is developing crystal growth using this recent strategy.

References:

  1. Pao S.S., Paulsen I.T., Saier M.H. Jr. (1998).
    Microbiol Mol Biol Rev. 62: 1–34.
  2. Huber, H. & Prangishvili, D. (2005).
    In The Prokaryotes: An evolving electronic resource for the microbiological community. Edited by M. Dworkin: Springer-Verlag, New York
  3. Sterner, R. & Liebl, W. (2001).
    Crit Rev Biochem Mol Biol 36, 39-106
  4. Hickman A.B & Davies D.R. (1997).
    Principles of Macromolecular X-ray Crystallography. In Current Protocols in Protein Science (ed.) pp 17.3.1-15, John Wiley&Sons, Inc.
  5. Landau and Rosenbusch, PNAS, 93, 14532-14535, (1996).