Background

Proteins from hyperthermophilic organisms are considered to be more stable and more rigid than their mesophilic counterparts. Therefore, the probability of obtaining stable, homogeneous and crystallizable membrane protein complexes should be higher with complexes from thermophilic organisms than with complexes from mesophilic ones. Thus, we have chosen the hyperthermophilic eubacterium Aquifex aeolicus as our target organism. The relatively small size of its genome (less than 1/3 of that of Escherichia coli) and the consequently lower number of protein complexes implies that it is comparatively easy to purify individual membrane protein complexes. Most genes in the Aquifex genome are found in separate operons, but genes belonging to one pathway or one protein complex are often dispersed throughout the genome. Several homologues of one gene often exist, leading to the necessity to identify which homologue of one protein subunit is present in the respective complex.

We isolate and characterize as many membrane proteins and membrane protein complexes from native membranes of Aquifex aeolicus as possible by conventional biochemical techniques. Because the complete genome sequence is known, the purified proteins are identified by mass spectrometry, subjected to crystallization trials, identification of function and mechanistic investigations. For crystallized proteins annotated as putative proteins, we use both bioinformatics tools and biochemical experiments for the identification of their function.

We have purified and characterized the NADH:quinone oxidoreductase, a possible respiratory supercomplex consisting of the cytochrome bc₁ complex and of the cytochrome c oxidase, the F₁F_o-ATP synthase, the sulfide-quinone oxidoreductase, and several complexes of hypothetical proteins. All protein complexes that we have purified to homogeneity in sufficient quantities, have yielded diffracting crystals (Fig. 2). Crystals of two membrane protein complexes diffract X-rays to resolutions better than 2 Å. One of them is sulfide-quinone oxidoreductase, its structure has been solved at 2 Å resolution.

Examples of purified membrane protein complexes

Aquifex respiratory complex I is highly stable and active, the specific activity for electron transfer from NADH to Q₁₀ of the isolated complex is 29 U/mg at 80 ºC with a half-life of about 10 h. Single particle electron microscopy revealed many details in its cytoplasmic arm (Fig 1., left-most panel, upper left portion of the protein complex), a pronounced invariant angle (90º) between the cytoplasmic arm and the membrane arm indicates a good preservation of the enzyme and a homogeneous preparation, a promising property for crystallization attempts. Recently, the structure of the enzyme hydrophilic domain has been determined using X-ray crystallography.

We have identified all subunits of the F₁F_o-ATP synthase. Interestingly, two versions of the b subunit, b₁ and b₂, with only low sequence homology to each other, were found. Electron microscopic single particle analysis displays the structural details with particular emphasis on the peripheral stalk, and the central stalk appears to be tilted and/or kinked shown in several orientations (Fig 1., second panel from left).

Sulfide-quinone oxidoreductase (SQR) is a monotopic membrane enzyme that belongs to the superfamily of flavoprotein disulfide reductases. It catalyzes sulfide oxidation to polysulfur through direct flavin-quinone electron transfer. The enzyme could be purified to a highly stable, homogeneous and active form from A. aeolicus. We could solve its 3-D X-ray structure at 2.0 Å by de novo phase determination. The protein is a homotrimer (Figure 3a ). The structure reveals how the SQR interacts with the membrane and how it selectively binds sulfide and quinone analogues. Most interestingly, it shows that FAD is bound to the protein through an unusual putative persulfide bridge and that the product of the reaction is covalently bound to the active site and cocrystallizes with the enzyme (Figure 3b). Through the structure, we obtained a detailed insight into A. aeolicus SQR and we formulated new hypothesis on the mechanism by which this interesting enzyme may catalyze sulfur-polymerization.

One of the best diffracting crystals (at 1.8 Å) was obtained with the hypothetical protein Aq_1862, an outer membrane porin protein. The protein is one of the most abundant proteins in Aquifex membranes. We have analyzed the function of this porin after incorporation into black lipid membranes (BLM) (Fig.3). A sequence comparison demonstrated homology to two classes of outer membrane porins, the phosphate-selective porin O and P family and the putative ammonium transporter FmdC. Secondary and tertiary structure predictions (using PRED-TMBB and 3D-PSSM) predicted an 18-stranded b-barrel as well as similarity to the fold of the outer membrane porin PhoE of E. coli.

AQ_1760 is another “hypothetical protein”. It is not predicted to be a membrane protein, but it is always present in the membrane fraction, neither in the cytosol, nor in the culture medium. A sequence comparison demonstrated that it contains the Linocin_M18 bacteriocin domain. Proteins sharing this domain show various functions including antimicrobial effects and a proteolytic activity. All form homo-oligomeric complexes.

In a collaboration with the Institute of Oceanology of the Chinese Academy of Sciences, Qingdao, we isolate and try to crystallize protein complexes from algae and sea animals. In a collaboration with Prof. V. Mueller (Frankfurt University, Frankfurt am Main), we are also crystallizing A₁A_o-ATP synthases from archaea.                                  

Selected publications

Peng, G.H., Fritzsch, G., Zickermann, V., Schägger, H., Mentele, R., Lottspeich, F., Bostina, M., Radermacher, M., Huber, R., Stetter K.O. and Michel, H. Isolation, Characterization and Electron Microscopic Single Particle Analysis of the NADH:Ubiquinone Oxidoreductase (Complex I) from the Hyperthermophilic Eubacterium Aquifex aeolicus. Biochemistry 42, 3032-3039 (2003)

Peng, G. , Bostina, M., Radermacher, M., Rais, I., Karas, M., Michel, H. Bichemical and electron microscopic characterization of the F₁F_o ATP synthase from the hyperthermophilic eubacterium Aquifex aeolicus. FEBS Letter 580, 5934-5940 (2006)

Wedemeyer, U., Peng, G., Michel, H., Hartung, K. The protein AQ_1862 from the hyperthermophilic bacterium Aquifex aeolicus is a porin and contains two simultaneous conductance pathways of different selectivity. Biophys.  J. 93, 2678-2687 (2007)

Marcia, M., U. Ermler, G. Peng and H. Michel: The structure of Aquifex aeolicus sulfide:quinone oxidoreductase, a basis to understand sulfide detoxification and respiration. Proc. Natl. Acad. Sci. U.S.A. 106, 9625-9630 (2009)

Funding

Max-Planck-Society

Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 628)

National Science Foundation of China (NSFC)