Synchrotron radiation diffraction from two-dimensional protein crystals at the air/water interface.

Protein structure determination by classical x-ray crystallography requires three-dimensional crystals that are difficult to obtain for most proteins and especially for membrane proteins. An alternative is to grow two-dimensional (2D) crystals by adsorbing proteins to ligand-lipid monolayers at the surface of water. This confined geometry requires only small amounts of material and offers numerous advantages: self-assembly and ordering over micrometer scales is easier to obtain in two dimensions; although fully hydrated, the crystals are sufficiently rigid to be investigated by various techniques, such as electron crystallography or micromechanical measurements. Here we report structural studies, using grazing incidence synchrotron x-ray diffraction, of three different 2D protein crystals at the air-water interface, namely streptavidine, annexin V, and the transcription factor HupR. Using a set-up of high angular resolution, we observe narrow Bragg reflections showing long-range crystalline order in two dimensions. In the case of streptavidin the angular range of the observed diffraction corresponds to a resolution of 10 A in plane and 14 A normal to the plane. We show that this approach is complementary to electron crystallography but without the need for transfer of the monolayer onto a grid. Moreover, as the 2D crystals are accessible from the buffer solution, the formation and structure of protein complexes can be investigated in situ.

Specific interaction and two-dimensional crystallization of histidine tagged yeast RNA polymerase I on nickel-chelating lipids.

Nickel-chelating lipid monolayers were used to generate two-dimensional crystals from yeast RNA polymerase I that was histidine-tagged on one of its subunits. The interaction of the enzyme with the spread lipid layers was found to be imidazole dependent, and the formation of two-dimensional crystals required small amounts of imidazole, probably to select the specific interaction of the engineered tag with the nickel. Two distinct preparations of RNA polymerase I tagged on different subunits yielded two different crystal forms, indicating that the position of the tag determines the crystallization process. The orientation of the enzyme in both crystal forms is correlated with the location of the tagged subunits in a three-dimensional model which shows that the tagged subunits are in contact with the lipid layer.

Structural study of the response regulator HupR from Rhodobacter capsulatus. Electron microscopy of two-dimensional crystals on a nickel-chelating lipid.

Two-dimensional crystals of the histidine-tagged-HupR protein, a transcriptional regulator from the photosynthetic bacterium Rhodobacter capsulatus, were obtained upon specific interaction with a Ni2+-chelated lipid monolayer. HupR is a response regulator of the NtrC family; it activates the transcription of the structural genes, hupSLC, of the [NiFe]hydrogenase. The lipid (Ni-NTA-DOGA) uses the metal chelator nitrilotriacetic group as the hydrophilic headgroup and contains unsaturated oleyl tails to provide the fluidity necessary for two-dimensional protein crystallization. A projection map of the full-length protein at 18 A resolution was generated by analysing electron microscopy micrographs of negatively stained crystals. The HupR protein appeared to be dimeric and revealed a characteristic "propeller-like" motif. Each monomer forms an L-shaped structure.

Lipoxygenase products mediate the attachment of rat macrophages to glomeruli in vitro.

Because there is an accumulation of macrophages in the Bowman's space during human and experimental glomerulonephritis, we have studied the binding of [3H]-uridine labeled macrophages to isolated glomeruli. Binding was related to the glomerular protein and macrophage concentrations, temperature, time of incubation, and was a saturable process. Macrophage adherence depended on glomerular lipoxygenase activity but not on glomerular cyclooxygenase activity since preincubation of glomeruli with nordihydroguaiaretic acid (NDGA) inhibited this phenomenon whereas preincubation with indomethacin was ineffective. Glomeruli interacted with macrophages in converting arachidonic acid (C20:4) to prostaglandins (PG) since productions of 6 keto-PGF1 alpha, TXB2, and PGD2 by glomeruli and macrophages incubated in combination were much greater than the sums of their respective productions by glomeruli and macrophages incubated separately. Macrophages were the source of the supplementary synthesis of PG which was abolished when these cells were pretreated with aspirin. Stimulation of macrophages by glomeruli was blunted by pretreatment of glomeruli with NDGA. Production of PG and of 12-HETE by macrophages was stimulated by a lipid extract of glomeruli containing the oxygenated metabolites of C20:4. Direct addition of 12-HPETE also stimulated macrophage functions. These data suggest that macrophage attachment to glomeruli and macrophage stimulation in the presence of glomeruli depend on glomerular lipoxygenase activity.