Abstract
(Englisch)
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1. Crystallisation and structure determination of membrane proteins: the XcpQ as component of the Pseudomas secretion machinery in the outer membrane.
Due to still very limited expression and, consequently. purification of the transmembrane protein XcpQ (10 ug), its crystallisation has not been successful. To have a minimal chance of success. crystallization to large. well-ordered three-dimensional crystals requiring at least 10 mg (3 orders of magnitude difference!) is necessary. Consequently. it was discussed and decided at the last meeting in Derbyshire to be content with the resolution achievable by electron microscopy (25-10 A) for the time being.
Instead, we have studied structure of another outer membrane protein. since more knowledge about patterns can only assist in estimating the diversity of the foldings which, in turn. may help in designing approaches to the crystallization of proteins such as the XcpQ. In the Basel group, it was possible to crystallise and to determine the structure to 2.7 A, of a ligand-gated siderophore translocating protein in the outer membrane of E. coli. Two states could be crvstallized and solved independently: the ligand-free (apo) form. and the ligand-bound (complex) form. The overall structure consists of a porin-like barrel containing 22 anti parallel b-strands, and a plug which reveals both short a-helices and b-strands. This observation of mixed secondary structures is novel, as no homologue among integral membrane proteins has been observed. The finding is attractive, as the structure of XcpQ, as revealed by electron microscopy. appears to exhibit a domain structure. Apart from being able to trace the a-carbon backbone and the side chains, the allosteric chainges occurring upon binding could be show at atomic resolution.
CD-measurements performed on the XcpQ-protein. This revealed an overall b-structure of XcpQ. Analytical centrifugation revealed that the protein easily form aggregates, dimers and higher forms with only 5 % monomers present.
2. Planar bilayer measurements
XcpQ was reconstituted into lipid bilayers. Besides a number of clear conductance steps, much non-Ohmic noise has been observed. Steps were between 0.5 and l nS, independent of the N-terminal extension being present or not. It still cannot be ruled out rigorously whether a contaminating channel is co-purified with the XcpQ-protein. However, a very pure preparation of XcpQ, with two columns combined with a sucrose gradient purification step and subsequent excision of a band from a SDS-PAGE showed the same noise signal. Interestingly, a similar behaviour was reported for the PulD protein of Klebsella.
3. Crystallisation of the LipH-chaperon from Ps. aeruginosa
Several attempts were made to crystallise LipH. Different techniques such as sitting and hanging drop and capillary diffusion crystallisation were applied. All buffer conditions (a matrix of l50) from the commercially available Hampton crystallisation kit as well as 50 other conditions with protein concentrations ranging from 3, 4, 6, 8, 10 to 12 mg/ml have been used, but success was, unfortunately, not obtained. Co-crystallisation of the chaperone with the lipase might offer a solution: to this end, protein will be send to us, or we might purify the various components ourselves. An alternative might be an NMR approach. The corresponding contacts have been made by Mathias Winterhalter with James Ferretti at the NIH, Bethesda, MD.
4. Protein interactions between the different Xcp components.
Interaction between the different components of the Xcp-machinery are being studied in several laboratories of contributing partners to the EU-network project. Our contribution is to provide the biophysical methods to study the interactions.
A clear interaction between LipA and LipB of B. glumae has been shown by CD measurements. This was also confirmed by gel permeation using the SMART-system from Pharmacia. Furthermore, monitoring denaturation of LipA during a temperature gradient with CD revealed the importance of the calcium-ion for stability of the structure. While LipA was denatured in 8M urea up to heating at 45deg C, the secondary structure was released immediately in the presence of 8M urea and EDTA. Moreover. a folding intermediate has been detected. When LipA, after being denatured, was diluted in phosphate buffer (90 mM urea final concentration), the resulting CD- and tryptophanyl fluorescence spectra were indistinguishable from those obtained with native. Activity tests, performed in Utrecht, showed that the refolded LipA was not active.
Recently, interaction between LipH and XcpP of Ps. aeruginosa was studied. CD-measurements did not show interactions or major secondary structural changes, but Trp-fluorescence measurements did suggest interactions to occur. LipH contains 1 Trp-residue situated in the predicted coiled-coil region while XcpP is lacking Trp. The signal increase upon binding is rather small, indicating that there is no serious shift in environment (polar to apolar). A preliminary binding constant of 10 6 M-1 was determined at a 1:1 stoichiometry. Quenching experiments with cobalt-ions showed no significant difference between free LipH and LipH plus XcpP, whereas quenching using acrylamide showed that the Vip-residue in the free form was more easily accessible to collisions thin the one in complex with XcpP. Both these observations are in agreement with the Trp being part of a coiled-coil domain. However, interaction probably does not take place by formation of a coiled coil between LipH and XcpP since CD denaturation scans did not show a difference in transition temperature. Further experiments are needed to confirm this. Additional gel permeation on SMART will be performed as well as experiments with optical wave guide devices.
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