We chose crambin, a hydrophobic and water-insoluble protein originally isolated from the seeds of the plant Crambe abyssinica, as a model for NMR investigations of membrane-associated proteins. We produced isotopically labeled crambin(P22,L25) as a cleavable fusion with staphylococcal nuclease and refolded the protein by an approach that has proved successful for the production of proteins with multiple disulfide bonds. We used NMR spectroscopy to determine the threedimensional structure of the protein in two membrane-mimetic environments: in a mixed aqueousorganic solvent (75%/25%, acetone/water) and in DPC micelles. With the sample in the mixed solvent, it was possible to determine (>NH···OC<) hydrogen bonds directly by the detection of h3 J NC′ couplings. H-bonds determined in this manner were utilized in the refinement of the NMRderived protein structures. With the protein in DPC micelles, we used manganous ion as an aqueous paramagnetic probe to determine the surface of crambin that is shielded by the detergent. With the exception of the aqueous solvent exposed loop containing residues 20 and 21, the protein surface was protected by DPC. This suggests that the protein may be similarly embedded in physiological membranes. The strategy described here for the expression and structure determination of crambin should be applicable to structural and functional studies of membrane active toxins and small membrane proteins.

A structure-activity relationship study of hypoxia inducible factor-1α inhibitor 3-aminobenzoic acid-based chemical probes, which were previously identified to bind to mitochondrial malate dehydrogenase 2, was performed to provide a better understanding of the pharmacological effects of LW6 and its relation to hypoxia inducible factor-1α (HIF-1α) and malate dehydrogenase 2 (MDH2). A variety of multifunctional probes including the benzophenone or the trifluoromethyl diazirine for photoaffinity labeling and click reaction were prepared and evaluated for their biological activity using a cell-based HRE-luciferase assay as well as a MDH2 assay in human colorectal cancer HCT116 cells. Among them, the diazirine probe 4a showed strong inhibitory activity against both HIF-1α and MDH2. Significantly, the inhibitory effect of the probes on HIF-1α activity was consistent with that of the MDH2 enzyme assay, which was further confirmed by the effect on in vitro binding activity to recombinant human MDH2, oxygen consumption, ATP production, and AMP activated protein kinase (AMPK) activation. Competitive binding modes of LW6 and probe 4a to MDH2 were also demonstrated.

A new chemical inhibitor against severe acute respiratory syndrome (SARS) coronavirus helicase, 7-ethyl-8-mercapto-3-methyl-3,7-dihydro-1H-purine-2,6-dione, was identified. We investigated the inhibitory effect of the compound by conducting colorimetry-based ATP hydrolysis assay and fluorescence resonance energy transfer-based double-stranded DNA unwinding assay. The compound suppressed both ATP hydrolysis and double-stranded DNA unwinding activities of helicase with IC50 values of 8.66 ± 0.26 μM and 41.6 ± 2.3 μM, respectively. Moreover, we observed that the compound did not show cytotoxicity up to 80 µMconcentration. Our results suggest that the compound might serve as a SARS coronavirus inhibitor.

p38, which has been suggested to be a sca¡old protein for the assembly of a macromolecular tRNA synthetase complex, contains a leucine zipper-like motif. To understand the importance of the leucine zipper-like motif of p38 (p38LZ) in macromolecular assembly, the p38LZ solution structure was investigated by circular dichroism and nuclear magnetic resonance spectroscopy. The solution structure of p38LZ showed an amphipathic K K-helical structure and characteristics similar to a coiled-coil motif. The protein^protein interaction mediated by p38LZ was examined by an in vitro binding assay. The p43 protein, another non-synthetase component of the complex, could bind to p38LZ via its N-terminal domain, which is also predicted to have a potential coiled-coil motif. Thus, we propose that the p38^p43 complex would be formed by coiledcoil interactions, and the formation of the binary complex would facilitate the macromolecular assembly of aminoacyl-tRNA synthetases. ß