The 600-MHz 1H NMR spectrum of the des-Val-Val mutant of human transforming growth factor alpha (TGF-alpha) was reassigned at pH = 6.3. The conformation space of des-Val-Val TGF-alpha was explored by distance geometry embedding followed by restrained molecular dynamics refinement using NOE distance constraints and some torsion angle constraints derived from J-couplings. Over 80 long-range NOE constraints were found by completely assigning all resolved cross-peaks in the NOESY spectra. Low NOE constraint violations were observed in structures obtained with the following three different refinement procedures: interactive annealing in DSPACE, AMBER 3.0 restrained molecular dynamics, and dynamic simulated annealing in XPLOR. The segment from Phe15 to Asp47 was found to be conformationally well-defined. Back-calculations of NOESY spectra were used to evaluate the quality of the structures. Our calculated structures resemble the ribbon diagram presentations that were recently reported by other groups. Several side-chain conformations appear to be well-defined as does the relative orientation of the C loop to the N-terminal half of the protein.
The 31P NMR spectrum of dí-Pt(NH3)2|d(T1C2T3C4G5G6T7C8T9Cl0)-N7(5),N7(6)j has been reported to contain unusual signals with two signals downfield and two upfield of the normal range (ca. -4.0 to -4.4 ppm from trimethyl phosphate standard). This pattern suggested a distorted structure. Since we are interested in metal-induced distortions and only three of the 3IP signals have been assigned (G6pT7 normal shift, C4pG5 upfield, and G5pG6 downfield), we have used 170-labeling methods to identify four additional signals, including the other two unusual signals, as C2pT3 and T3pC4. 37H3-_P values were measured by 2D J and selective reverse chemical shift correlation spectroscopy. Three 31P signals exhibited unusually large coupling to H3' (C2p, 8.9 Hz; T3p, 8.0 Hz; G5p, 8.9 Hz). The C4'-C3'-03'-P torsional angle (e) values, which may reflect averaging of several conformers, correspond to 217°for C2p and Gsp and 211°for T3p, compared to ~206°for the normal range signals. Because all these unusual signals are for phosphate groups on the 5' end of the strand, we evaluated two models for the distortions as follows: (1) model I in which the 5' region T!C2T3C4 is looped around the Pt moiety, with at least one hydrogen bond with the Pt amine ligands, and (2) model II in which T,C2T3C4 bends away from the Pt moiety and the terminal T]C2 bases form base pairs with GG. Further evidence ( NMR spectroscopy, electrophoresis, and UV absorbance temperature-dependence studies) was consistent with model I. The lack of NMR imino signals observed in the 15-11 ppm region indicates that no base pairs are formed. Electrophoretic analysis demonstrates a decrease in mobility of the oligomer upon platination, evidence against a compact structure such as a hairpin. Staining of the electrophoresis band with acridine orange suggested a single-stranded structure. The UV absorbance showed no increase over the range 20-70 °C. The 195Pt NMR signal was observed at -2450 ppm, a value consistent with a normal coordination environment. Limited spectral studies performed on the analogous Pt(en) (en = ethylenediamine) and di-Pt(MeNH2)2 adducts gave further evidence for model I. The chemical shift of the C2pT3 signal and the temperature dependence of one of the upfield signals were sensitive to the nature of the amine ligand. The chemical shift of the downfield GpG 31P signal can be correlated with the potential hydrogen-bonding ability of the amine ligand. The 31P NMR spectrum of the duplex [m-Pt(NH3)2|d(TCTCGGTCTC)-N7( 5),N7( 6)|]-d(GAGACCGAGA) has two unusual 31P signals previously assigned as G5pG6 (-3.2 ppm) and C4pG5 (-4.9 ppm). The similarity in values of 3/hcop for G5p and C4p to those measured in the single strand indicates that the backbone structure of the platinated C4G5G6 does not change significantly upon duplex formation. Two sequential temperature-dependent structural transitions were evident for solutions of the duplex. The first transition (melting of the duplex) was characterized by sigmoidal changes in chemical shifts of the unusual 31P ...
Site-directed mutants of transforming growth factor-alpha (TGF-alpha) were expressed in an Escherichia coli outer membrane protein A (ompA) expression/secretion vector under the transcriptional control of the lambda PL promoter. TGF-alpha mutant proteins were isolated from cell pellets using alkaline extraction with 0.1 M-Tris (pH 10.5). The levels of protein expression of 23 TGF-alpha mutants were comparable with those of wild-type TGF-alpha, as determined by immunoblotting and radioimmunoassay. An analysis of biological activity using as assays radioreceptor binding competition and colony formation in soft agar showed that the following mutations destroy the activity of TGF-alpha: Gly-19 to Val, Val-33 to Pro and Gly-40 to Val. Mutations of Arg-42 to Lys, Leu-48 to Ala, Tyr-38 to Trp or Phe-17 to Tyr significantly decrease, but do not destroy, biological activity when compared with the wild-type. Mutations in 14 other residues did not significantly alter receptor binding or colony-forming activity. These studies suggest that two domains localized at the surface of TGF-alpha are important in receptor binding and colony-forming activity. Domain I involves amino acid residues which include Tyr-38 and Leu-48; domain II includes residues Phe-15, Phe-17 and Arg-42.
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