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.

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The alpha 1-adrenergic receptors (alpha 1-AR) belong to the G-protein coupled seven-transmembrane biogenic amine receptor family. Three subtypes have been successfully cloned in the alpha 1-adrenergic receptor family, and they share 50% identical amino acid sequences and 70% similarity. We have constructed seven chimeric receptors of the alpha 1A-AR. Each of the chimeras contains alpha 1D-subtype amino acid sequences within the membrane-spanning domains. Comparisons of ligand affinities with these chimeras has provided information on the importance of certain amino acid residues in determining receptor subtype specificity in the alpha 1A- and alpha 1D-ARs. With ligands in the dihydropyridine series, the niguldipine analog 1 was found to have respective pKi's of 9.32 +/- 0.17 for alpha 1A-AR; 6.84 +/- 0.24 for alpha 1D-AR; and 6.76 +/- 0.28 for alpha 1A/D(TM2), respectively. This trend was also exhibited by two other niguldipine analogs, 2 and 3, which had similar pKi's toward alpha 1D-AR and alpha 1A/D(TM2). This subtype selectivity was also maintained in the piperdine derivative, 4, and alpha 1A-AR selective ligand, which showed the same parallel trends in binding affinities with alpha 1A-AR and the six chimeras as the niguldipine analogs. Since in considering the second membrane-spanning domain, the alpha 1A- and alpha 1D-ARs only differ at positions 76, 77, 85, and 86, we were able to show through mutational studies that phenylalanine 86 is solely responsible for the selectivity found in the chimeric receptor alpha 1A/D(TM2) exhibited against the ligands 1-4 used in this study. A model based on the rhodopsin structure places the amino acid at position 86 in the final turn toward the extracellular region. This is four helical turns above aspartic acid-79, a conserved amino acid in the second membrane-spanning domain. This is the first report that suggests a significant involvement of the second membrane-spanning domain in antagonist binding in the biogenic amines class of the superfamily of seven-transmembrane receptors.

Burkholdines are cyclic lipopeptides with unusual antifungal potency, making them promising leads as a new class of antifungal agents. However, a recent report using knockout mutagenesis indicates that these and related compounds, such as occidiofungins, xylocandins, and cepacidines, may also be synonymous with the long-known hemolytic virulence factors found in diverse Burkholderia isolates. Because of their possible roles in causing Burkholderia infections or curing fungal infections, it is important to fully define their structures and biological activities using pure compounds. Here, we report the structures of three further burkholdines, Bk-1119, Bk-1213, and Bk-1215, which were elucidated using spectroscopic methods. The absolute configuration of this compound class was determined for the first time using a combination of spectroscopy and chemical degradation techniques. Antifungal and hemolytic activities were assessed for five pure burkholdines, representative of the structural diversity of this lipopeptide class. All of the burkholdines were potent antifungal and hemolytic agents, validating their probable role in virulence. However, one of the burkholdines (Bk-1119) exhibited a >30-fold selectivity for fungi versus sheep erythrocytes and was more than 25-fold more potent than amphotericin against some fungal strains. Therefore, burkholdines have potential to selectively target fungal infections.

The isolation and characterization of the major crystalline alkaloids of three Crinum species, C. defixum, C. scabrum, and C. latifolium, are reported. A new alkaloid, 5a-hydroxyhomolycorine (1) has been isolated from C. defixum. The latter plant also contains an alkaloid identified as 9-0-demethylhomolycorine which differs in physical properties from that previously reported for this compound. Evidence is provided for the structure of 9-0-demethylhomolycorine by ' H and 13C NMR studies. In the latter, the exploitation of long-range ' H coupling in the 13C spectra of lactones in this series is found to be diagnostically useful in assigning aromatic substitution patterns. A survey of the CD spectra of lactone alkaloids of the benzopyrano[3,4-g]indole system indicates that this technique can provide useful structural information.The Crinum genus of the amaryllidaceae has a wide geographical distribution in the temperate and subtropical regions. The species C. defixum Ker-Gawl, C. latifolium L., and C. scabrum Herb. are indigenous to the Indian subcontinent. Several preliminary reports2 have established the presence of lycorine in each of these species and a more definitive study3 of C. defixum has shown the presence of caranine, crinamine, crinine, galanthamine, galanthine, haemanthamine, and hippeastrine from C. defixum grown in Holland.

The DNA.DNA duplex d(CGCGTTSCH2OTTGCGC).d(GCGCAAAACGCG) (designated duplex III) containing a 3'-thioformacetal (3'-TFMA) linkage in the center of the sequence was characterized in detail by two- and three-dimensional homonuclear NMR spectroscopy. The NMR results were analyzed and compared with those of two duplexes of the same sequence: One is an unmodified reference sequence and the other contains a formacetal (OCH2O) linkage at the central T--T step (designated duplex I and duplex II, respectively). In general, the NMR spectra of duplex III closely resemble those of the analogous duplexes I and II, suggesting an overall B-type structure adopted by the 3'-TFMA-modified duplex III. Nonetheless, the detection of several distinct spectral features originating from the protons at the T6(3'-SCH2O)T7 modification site is indicative of a local conformation that is clearly different from the corresponding region in duplexes I and II. The 3'-thioformacetal linker, in contrast to the formacetal (FMA) linkage, cannot be accommodated in a conformation usually found in natural nucleic acid duplexes. As a consequence, the 3'-TFMA-modified T6 sugar adopts an O4'-endo form (an intermediate structure between the usual C2'-endo and C3'-endo forms). This change is accompanied by a change in the epsilon (C4'-C3'-S3'-CH2) dihedral angle and by subsequent adjustments of other torsion angles along the backbone. Notably, this conformational readjustment at the T6-T7 backbone linkage is localized; its collective result has negligible effect on base-base stacking of the T6 and T7 residues. A close examination of the COSY data in all three duplexes reveals a subtle variation in sugar geometry, with more S-type character adopted by the modified duplexes II and III. The results of this study illustrate that, although the difference between FMA and 3'-TFMA linkages is merely in the substitution of the T6(O3') in the former by a sulfur atom in the latter, the stereoelectronic difference in a single atom can induce significant local structural distortion in an otherwise well-structured oligonucleotide duplex.

Potent antifungal cyclic lipopeptides, burkholdines (Bk), were isolated from a culture of Burkholderia ambifaria 2.2N. Bk-1229 (1) and Bk-1097 (2) are octapeptides comprised of nonproteinogenic amino acids, including -hydroxytyrosine, -hydroxyasparagine, and a new fatty acyl amino acid. 1 and 2 are fungicidal against a panel of fungi with potencies 2-60-fold better than amphotericin B control.

The duplex formed by annealing the formacetal backbone modified dodecamer d-(CGCGTTOCH2OTTGCGC) to its complementary strand, d(GCGCAAAACGCG) (duplex I), has been studied by NMR techniques and analyzed with reference to its unmodified counterpart (duplex II). Comparison of parameters such as 2D cross-peak intensities, coupling constants, and spectral patterns indicates that structural perturbations caused by the incorporation of the formacetal linkage are minimal and localized to the central T4.A4 block. Duplex I adopts a B-type helical conformation with regular Watson-Crick base pairing and normal minor groove width. The methylene group is accommodated along the phosphate backbone in a conformation similar to that of the PO2 group found in the B-form DNA family. The central T6-T7 base pairs of duplex I melt simultaneously with the duplex, indicating a cooperative transition to single strands. Although the formacetal linkage affects global melting, as evidenced by a 3 degree C reduction in Tm for duplex I with respect to duplex II, the present study indicates that this is not the result of localized premelting at the formacetal site of duplex I but rather reflects the subtle interplay of several structural and energy factors which need to be further explored.