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MATERIALS AND METHODS and L-[2',4',5',6',7'-2H5]Escherichia coli K3300 F+ infected with fd bacteriophage was grown on chemically defined media containing the labeled amino acids. The virus was isolated from the growth media as described (4). After purification on cesium chloride gradients and exhaustive dialysis against distilled water, the NMR samples were prepared by pelleting the virus in an ultracentrifuge. The deuterium-labeled samples were repeatedly resuspended in deuterium-depleted water (Aldrich).The 13C (37.8-MHz) and 15N (15.2-MHz) NMR spectra were obtained by cross-polarization (5) on a home-built spectrometer with a 3.5-T magnet. Magic-angle sample spinning was carried out by using double air bearing cylindrical rotors of the design described by Veeman and coworkers (6). The 2H NMR spectra were obtained on a home-built spectrometer with a 5.8-T magnet, using the quadrupole-echo pulse sequence (7) to generate single-sideband free induction decays that were subject to complex Fourier transformation. RESULTS AND DISCUSSIONHigh-Resolution Solid-State NMR. Conventional high-resolution NMR spectroscopy, as it is routinely used to study small molecules in solution, is not applicable for solids or quasisolids such as biological supramolecular structures in solution. In the absence of rapid isotropic motion, the spin interactions are not averaged effectively and the observed resonances are broad. Fig. 1 A and C demonstrates the difficulties associated with natural-abundance '3C and 15N NMR of fd virus in solution. Because fd is a 900-nm by 9-nm filament of 16 X 106 daltons, the virus in concentrated solutions has overall reorientation rates slow compared to the NMR time scales, and thus only intramolecular motions influence lineshapes.Useful 13C and 15N NMR spectra of fd are obtained by removing the broadening effects of the unaveraged dipole-dipole and chemical shift interactions. High-power irradiation at the 'H resonance frequency decouples the heteronuclear dipolar interactions. Because homonuclear dipolar couplings are small for dilute spins, the resulting proton-decoupled spectra have their lineshapes determined by chemical shift anisotropy. The powder patterns are averaged to isotropic resonances by rapid mechanical rotation ofthe sample at the magic angle (54.70) with respect to the applied magnetic field (8). Specific nuclear spin labels biosynthetically incorporated into fd aid sensitivity and assignments. The success of the entire experimental protocol, in which Trp-26 offd coat protein is labeled and the spectra are obtained by cross-polarization with proton decoupling and magic-angle sample spinning, is demonstrated in Fig. 1 B and D. These spectra are analogous to those seen for the isolated coat protein in solution, where rapid rotational diffusion is effective in averaging the static spin interactions (4).Both Trp-26-labeled fd samples have a single resonance line. This is strong evidence that all 2700 coat protein subunits have the same conformation and environment around Trp-26. This may h...

Thyroid cancer 1 (TC-1) is a 106-residue naturally disordered protein that has been found to associate with thyroid, gastric, and breast cancers. Recent studies showed that the protein functions as a positive regulator in the Wnt/b-catenin signaling pathway, a pathway that is known to play essential roles in developmental processes and causes tumor formation when misregulated. By competing with b-catenin for binding to Chibby (Cby), a conserved nuclear protein that antagonizes the b-catenin-mediated transcriptions, TC-1 up-regulates a number of b-catenin target genes that are known to be involved in the aggressive behavior of cancers. In order to gain a molecular understanding of the role TC-1 plays in regulating the Wnt/b-catenin signaling pathway, detailed structural studies of the protein and its interaction with Cby are essential. In this work, we used nuclear magnetic resonance (NMR) spectroscopy to elucidate the structure of TC-1 and its interaction with Cby. Our results indicate that even though TC-1 is naturally disordered, the protein adopts fairly compact conformations under nondenaturing conditions. Chemical shift analysis and relaxation measurements show that three regions (D44-R53, K58-A64, and D73-T88) with high-helical propensity are present in the C-terminal portion of TC-1. Upon addition of Cby, significant broadening of resonance signals derived from these helical regions of TC-1 was observed. The result indicates that the intrinsically disordered TC-1 interacts with Cby via its transient helical structure.Keywords: thyroid cancer 1 (TC-1); Chibby (Cby); nuclear magnetic resonance; disordered protein; Wnt/ b-catenin signaling pathway; cancers Supplemental material: see www.proteinscience.org Since its first identification as a novel gene highly expressed in thyroid cancer (Chua et al. 2000), thyroid cancer 1 (TC-1) has subsequently been found to be involved also in gastric (Kim et al. 2003) and breast cancers. Recently, Lee and his coworkers discovered that TC-1 functions as a positive regulator in the Wnt/b-catenin signaling pathway ), a pathway that is known to play essential roles in developmental processes and lead to cancers when misregulated (Polakis 2000;Giles et al. 2003). In the absence of a Wnt signal, the cellular concentration of b-catenin is tightly regulated by a multi-protein destruction complex composed of Axin, adenomatous polyposis coli (APC), and glycogen synthase kinase 3b (GSK-3b). To maintain the cytoplasmic b-catenin at a low level, GSK-3b phosphorylates the protein and the product is subsequently ubiquitylated and targeted for degradation by the proteasome. The Wnt signal, on the other hand, Reprint requests to: Wing Yiu Choy, Department of Biochemistry, University of Western Ontario, London, Ontario N6A 5C1, Canada; e-mail: jchoy4@uwo.ca; fax: (519) 661-3175.Abbreviations: TC-1, thyroid cancer 1; CD, circular dichroism; Cby, Chibby; TCF, T-cell factor; LEF, lymphoid enhancer factor; NMR, nuclear magnetic resonance; R 1 , longitudinal relaxation rate; R 1r , rel...

The absorption spectra and MCD have been obtained for Mn(CO)5X (X=Cl, Br, and I) in the region of the 370 to 420 nm absorption band of these complexes. The MCD consists primarily of a positive A term, consistent with the assignment of (1A1 → 1E) for this band found in the literature. In addition, both the dipole strength (D), and the ratio (A/D), change regularly in going down the series Cl, Br, I, which provides evidence that the orbitals involved in this transition are e[π, x] → b1[σ*, 3dx2−y2] as given in the MO treatment of Fenske and De Kock [Inorg. Chem. 9, 1053 (1970)], rather than b2[π*, 3dxy] → e[π*, CO(5) suggested in an earlier work by Gray et al.

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