The Outer membrane protein F of Escherichia coli K12 as a model for small angle X-ray scattering

3.7 The Outer membrane protein F of Escherichia coli K12 as a model for small angle X-ray scattering

3.7.1 Setting up small angle scattering experiments for membrane proteins

One of the main bottlenecks for structure determination of membrane proteins is to obtain diffracting crystals, in order to be able to determine the structure. However, solution scattering experiments can be done with protein in solution. A pilot experiment to use SAXS for obtaining low resolution structural information on membrane proteins was carried out by selecting a reference protein of known structure. Therefore the outer membrane protein F (OmpF) was expressed, isolated, purified and concentrated to 15 mg/ml (Phale et al., 2001). The concentration of the detergent C₈E₄ was chosen to be as low as possible (0.7%) to avoid micelles disturbing the measurements.

3.7.2 OmpF as a reference model

From the measurement an initial model could be built. The model corresponds well to the published 3-dimensional structure of OmpF (Phale et al., 2001). However, some rearrangements in solution might have taken place. Already in the initial model the pores were visible and the protein forming a trimer as its physiological nature implies. These results also agree with the crystal structure well. In contrast to the crystal structure the solution scattering model seems to have different dimensions at first sight. The overall superposition of the SAXS model and the crystal structure shows that the protein seems to have a larger diameter compared to the crystal structure.

Figures 3.52a-c: The pictures show from left to right the measurement of the OmpF, the SAXS model (yellow beads: detergent and grey beads: protein) and the crystal structure.

One possibility might be that the polyoxyethylene detergent was not taken into account when building the initial model. Later data evaluation with the detergent girdle around the protein trimer shows a much better agreement of both structures. In addition, the fact of such a protein as a dynamic system should be kept in mind. It might be possible that because of certain movements of the trimers the entire protein “system” seems to have a different diameter because of its movement.

For this reason the diameter might not be actually different of the crystal structures ones, but is in fact misleading. In conclusion, the overall experiment was set up successfully and SAXS proved to be a new potential method in structural analysis of membrane proteins.

-experimental data -fit ab initio model (mon04r.pdb) -fit atomic model (ompf.pdb)

3.7.3 Nramp

After the successful pilot project with OmpF, the procedure was tried for Nramp. The protein was kept in its solubilization detergent LDAO and tested in analogy to OmpF.

Figures 3.53a-b: Measurement of Nramp and scheme of discs (on top) and single particles (bottom).

The measurement of the Nramp sample was less successful. The maximum of the peak at higher angles supports the notion of detergent interference. It seems as if the detergent LDAO enhanced the formation of discs instead of keeping the protein molecules single and surrounded by their micelle.

This observation means that a wide range of different detergents might have to be tried for their feasibility in SAXS experiments. It is not yet clear whether the detergent LDAO itself is inappropriate for this technique or whether it is just the detergent in combination with this particular protein. Further measurements will answer these questions.

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4. Conclusions

This work was carried out with the aim of determining the 3 dimensional structures of proteins of the lysine biosynthetic pathway at high resolution. 3 target proteins were focused on, which were Rv0858c, Rv1201c and Rv1293.

The high resolution structure of Rv0858c was solved by X-ray crystallography at 2.0 Å and 3.2 Å resolution in 2 different crystal forms. The protein contained the co-factor PLP, one glycerol molecule in each subunit and one chloride ion bound to the protein. The glycerol molecules appear to occupy potential inhibitor binding sites. The chloride ions bind to helices, which expose a highly hydrophobic surface. These may be possible sites for complex formation. Both crystal forms are related to each other, with the orthorhombic one being a subgroup of the tetragonal. This tetragonal structure is identical to the orthorhombic model despite the fact that it is present as a monomer. The active site residues which are responsible for co-factor binding are highly conserved.

The second target protein Rv1201c was crystallised and first attempts towards structure determination were carried out. Although a 2.8 Å resolution data set was collected, the major attempt was to identify the derivative peak positions. From the symmetry of the space groupand the self rotation function, it could be deduced that Rv1201c crystallises as a 60 mer exhibiting icosahedral symmetry (point group 235).

The structure Rv1293 had been solved from the same organism previously. Nevertheless the structure was refined against a data set from a different crystal form and extended to a higher resolution. Aside from the different space group there is also a difference in the oligomeric state of both structures. This may have some implications with respect to the reaction mechanism.

All 3 molecules carry out major steps within the lysine biosynthetic pathway. Therefore these target proteins are potential candidates for an inhibitor screen in order to design new anti tuberculosis drugs.

The overall direction of the structural analysis of membrane proteins of Deinococcus radiodurans R1 were the first steps towards the structural analysis of outer membrane proteins and the inner membrane protein Nramp. The proteins of this organism were selected by their sequence annotation.

After all possible attempts of isolating an outer membrane protein failed, a highly conserved inner membrane protein was chosen. The steps for this target including PCR, cloning, expression, purification and crystallisation were established during this work. The obtained crystals could be improved to get first diffraction patterns by X-rays. These first diffracting crystals of the Nramp protein lead to the suggestion of testing more detergents in order to improve the diffraction quality.

In addition, a first SAXS experiment was successfully set up to broaden the range of techniques for structure determination of membrane proteins. This technique however could not be applied for Nramp yet. Nevertheless initial measurements of the OmpF are promising towards this application for inner membrane proteins.