Perdeuteration of the mechanosensitive channel protein MscL

Watts, A. Yeagle, P. (University of Oxford, UK), Grage, S. (IFIA, Forschungszentrum Karlsruhe, Germany) and Martinac, B. (The University of Western Australia Crawley, Australia)

with Moulin M. and Hartlein M. (ILL, France)

The Mechanosensitive Channel Protein MscL

Bacterial cells have developed sophisticated defense systems against environmental stress such as osmotic shock. In the event of increased osmotic stress, cellular pressure is released by channel proteins. The most prominent of these membrane proteins is the mechanosensitive protein MscL. It allows the equilibration of osmotic pressure by forming a large, unspecific pore across the membrane. A crystal structure of the protein from M. tuberculosis in its closed state is known, and models for the structure in the open conformation have been proposed [1-3]. However, more direct experimental evidence is desirable, and neutron reflection and NMR in micelle solution present suitable methods to monitor differences in the overall shape of the protein as well as determine structural details on the atomic scale.

The pore of the homo-pentameric protein is formed by five transmembrane helices (TM1), which are surrounded by another circle of five helices. A hydropobic lock holds the channel in its closed state, and it is believed that membrane tension together with thinning of the lipid bilayer are responsible for opening the pore under osmotic stress. A large, unspecific pore is then achieved by tilting of the transmembrane helices so to match the reduced thickness of the stretched bilayer. This process leads to an increase in the protein's lateral diameter and a reduction of its thickness. To characterize these conformational changes upon opening of the pore is the major focus of the structural investigations within this project.

Perdeuteration of MscL

As a first step for efficient growth in a deuterated environment, a suitable plasmid construct with antibiotic resistance compatible with the growth to high cell densities in feedback controlled fermentor systems was developed in the Deuteration Laboratory by Martine Moulin and Michael Härtlein. The E.coli host strain JM109, with a plasmid vector based on pQE70 and encoding for the mscl gene product and kanamycin resistance (JM109pQE70MscLKan), was used in first labeling trials: Plasmid DNA isolated from M15pQE70 expressing MscL with a C-terminal His-tag (obtained from Christel Norma, Boris Martinac Lab, University of Western Australia, Crawlay, was transformed into JM109. Colonies were tested for ampicillin resistance and kanamycin sensitivity to avoid double transformation with the co-purified repressor plasmid pREP1. Plasmid DNA isolated from these cells was then in vitro transposed with a kanamycin casette using EZ::TN <KAN-2> insertion kit (Epicentre, Madison, USA) and kanamycin resistant and ampicillin sensitive clones were selected and tested for MscL expression by western blotting using anti-His antibodies. MscL expression appeared to be constitutive in these clones. A better control of expression was achieved with a modified expression construct: BL21(DE3) pQ70Kana co-transformed with placIts (tet) over-expression a temperature-sensitive lac-repressor (6).

These cells were adapted to D₂O minimal medium with deuterated (d-8) glycerol as only carbon source. A 100ml preculture was prepared to inoculate a 1l fermenter fed-batch culture at 30°C. Cells were grown to an OD₆₀₀=10 and protein expression was induced at 37°C for 4h. A high tetracyclin concentration (50mg/l) with light protection of the fermenter was necessary to stabilize the repressor plasmid. Controlled expression lead to higher cell density. An induction temperature of 37^oC instead of 42^oC was chosen to avoid cell lysis. In this way a significantly high bio-mass production (40g of perdeuterated cell paste) was obtained.

To purify the protein, the cells were lysed using a French press. The membrane fraction was isolated by ultracentrifugation, and solubilized in a Triton X100 solution. Finally, MscL was purified with the aid of Ni-binding of the C-terminal 6His-tag and 1mg of ²H labelled MscL was obtained.

SANS experiments at ILL on D22

With the aid of SANS experiments at ILL on D22 (project 8-02-336), changes in the dimensions of both, the protein and its membrane environment, were investigated. After first successful measurements on perdeuterated MscL, we plan to continue the SANS experiments as well as neutron reflection experiments at ISIS [4].

In neutron SANS and reflection experiments, measurements using deuterated MscL allow favorable possibilities for the contrast matching of the surrounding lipid matrix and aqueous subphase

[1] G. Chang, R. H. Spencer, A. T. Lee, M. T. Barclay, D. C. Rees: Structure of the MscL homolog from Mycobacterium tuberculosis: A gated mechanosensitive ion channel. Science 282, 2220-2226 (1998)

[2] E. Perozo, A. Kloda, D. M. Cortes, B. Martinac: Physical principles underlying the transduction of bilayer deformation forces during mechanosensitive channel gating. Nat Struct Biol 9, 696-703 (2002)

[3] E. Perozo, D. M. Cortes, P. Somponplsut, A. Kloda, B. Martinac: Open channel structure of MscL and the gating mechanism of mechanosensitive channels. Nature 418, 942-948 (2002)

[4] A. Watts, S. L. Grage, G. Mendes: ISIS project report RB14352 (2003)

[5] C. Fernandez, C. Hilty, S. Bonjour, K. Adeishvili, K. Pervushin, K. Wüthrich: Solution NMR studies of the integral proteins OmpX and OmpA from Escherichia coli. FEBS Lett. 504, 173-178 (2001).

[6] Noaman and Szybalski, (1995), Gene 163, 35-40.