MEMBMACS: Membrane machineries for protein and solute transport through the bacterial cell envelope

• Unité de recherche: PCRD EU
• Numéro de projet: 99.0752-1
• Titre du projet anglais: MEMBMACS: Membrane machineries for protein and solute transport through the bacterial cell envelope

Mots-clé (Anglais)

SecYEG; HrcN; ATPase; PulD; 2D-crystallization; electron crystallography; AFM

Autre Numéro de projet (Anglais)

EU project number: HPRN-2000-00075

Programme de recherche (Anglais)

EU-programme: 5. Frame Research Programme - 4.1.1 Research training networks

Description succincte (Anglais)

See abstract

Autres indications (Anglais)

Full name of research-institution/enterprise:
Universität Basel
Biozentrum, C-CINA, WRO-1058.P.24
Maurice E. Müller Institute for Structural Biology, c/o Syngenta AG

Partenaires et organisations internationales (Anglais)

Coordinator: Institut Pasteur, Paris (F)

Résumé des résultats (Abstract) (Anglais)

In addition to SecYEG translocase of E.coli we now also have SecYEG translocase of Thermotoga, of which 2D crystallization experiments are in progress. SecYEG of E.coli (including two SecYE mutants), and SecYEG translocase of Thermotoga have all been reconstituted into paracrystalline arrays using the monolayer technique. Unfortunately, the 2D crystals achieved are yet too small for electron crystallography experiments.
The Iraklion team has shown that HrcN is the putative type III protein secretion (TTS) ATPase of Pseudomonas syringae pathovar phaseolicola. Experiments using scanning transmission electron microscopy (STEM) of unstained and TEM of negatively stained samples demonstrate that HrcN has a pronounced tendency to from oligomers. These clearly fall into four ranges that can be described by Gauss peaks centred at 225±97kDa, 601±142kDa, 1.18±0.13MDa, and at 1.74±0.27Mda. The major peak in the unstained, freeze-dried preparation is centered at 1.18 MDa and represents a complex comprising 2 HrcN dodecamers. Although particles prepared by negative staining exhibited significant variability in size and shape, a cylindrical shape of the HrcN complex having a diameter of 13±1 nm is suggested by reference-free alignment and averaging of projections along the cylinder axis.
A novel spreading method using lipid monolayers has been applied to secretin PulD. This approach allows the complex to be prepared for analysis by cryo-electron microscopy and image processing. Promising preliminary images that revealed 12 peripheral petals after image averaging have recently been obtained. PulD-PulS complexes are now compared to PulD complexes to locate the PulS domain.
A major investment is the development of 2D crystallization methods that may be applied to delicate membrane protein complexes, which are sensitive to high-cmc detergents. A novel computer controlled dilution apparatus has been built, which allows 2D crystals to be grown by slow dilution. To prove this concept we have assembled 2D crystals from bacteriorhodopsin, porin and photosystem I. In addition, a machine has been developed that allows the detergent concentration to be measured accurately and fast.

Références bases de données (Anglais)

Swiss Database: Euro-DB of the
State Secretariat for Education and Research
Hallwylstrasse 4
CH-3003 Berne, Switzerland
Tel. [phone]
Swiss Project-Number: 99.0752-1