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Of the 650 marine species assayed during the 1978 Alpha Helix Caribbean Expedition (AHCE 1978)3 the colonial tunicate Eudistoma olivaceum4 was the most active against Herpes simplex virus, type 1 (HSV-1). In the present report we assign structures 1, 2, 5, and 6 (Table I), containing the previously unreported condensed oxathiazepine ring system, to eudistomins C, E, K, and L, respectively, which include the most active antiviral components of E. olivaceum. In the following communication5 *we assign the structures of eudistomins A, D, G, , I, J, , N, O, P, and Q, additional bioactive components isolated from E. olivaceum.The methanol-toluene (3:1) extract3 of E. olivaceum (IRCE l-VII-81-3-1, IFE 21-V-82-1-3) was partitioned with toluene and water. The toluene phase yielded eudistomins G, H, and I (see following communication),5 while extraction of the aqueous phase with chloroform yielded an oil, which was subjected to C18 reversed-phase medium-pressure liquid chromatography (MPLC) with methanol-water (7:3) then to silica gel MPLC

Human ubiquitin is a 76-residue protein that serves as a protein degradation signal when conjugated to another protein. Ubiquitin has been shown to exist in at least three states: native (N-state), unfolded (U-state), and, when dissolved in 60% methanol:40% water at pH 2.0, partially folded (A-state). If the A-state represents an intermediate in the folding pathway of ubiquitin, comparison of the known structure of the N-state with that of the A-state may lead to an understanding of the folding pathway. Insights into the structural basis for ubiquitin's role in protein degradation may also be obtained. To this end we determined the secondary structure of the A-state using heteronuclear three-dimensional NMR spectroscopy of uniformly 15N-enriched ubiquitin. Sequence-specific 1H and 15N resonance assignments were made for more than 90% of the residues in the A-state. The assignments were made by concerted analysis of three-dimensional 1H-15N NOESY-HMQC and TOCSY-HMQC data sets. Because of 1H chemical shift degeneracies, the increased resolution provided by the 15N dimension was critical. Analysis of short- and long-range NOEs indicated that only the first two strands of beta-sheet, comprising residues 2-17, remain in the A-state, compared to five strands in the N-state. NOEs indicative of an alpha-helix, comprising residues 25-33, were also identified. These residues were also helical in the N-state. In the N-state, residues in this helix were in contact with residues from the first two strands of beta-sheet. It is likely, therefore, that residues 1-33 comprise a folded domain in the A-state of ubiquitin. On the basis of 1H alpha chemical shifts and weak short-range NOEs, residues 34-76 do not adopt a rigid secondary structure but favor a helical conformation. This observation may be related to the helix-inducing effects of the methanol present. The secondary structure presented here differs from and is more thorough than that determined previously by two-dimensional 1H methods [Harding et al. (1991) Biochemistry, 30, 3120-3128].

No abstract

Interleukin-1 (IL-1) proteins, such as IL-1 beta, play a key role in immune and inflammatory responses. Interaction of these cytokines with the IL-1 receptor induces a variety of biological changes in neurologic, metabolic, hematologic, and endocrinologic systems. Interleukin-1 receptor antagonist protein (IRAP) is a naturally occurring inhibitor of the interleukin-1 receptor. The 153-residue protein binds to the receptor with an affinity similar to that of IL-1 beta but does not elicit any physiological responses. As a first step toward understanding IRAP's mode of action, we have used multidimensional, heteronuclear NMR spectroscopy to determine the antagonist's solution secondary structure and global fold. Using a combination of 3D 1H-15N NOESY-HMQC and TOCSY-HMQC and 3D 1H-15N-13C HNCA and HN(CO)CA experiments on uniformly 15N- or doubly 13C/15N-enriched IRAP, we have made resonance assignments for more than 90% of the main-chain atoms. Analysis of short- and long-range NOE's indicates that IRAP is predominantly beta-sheet, with the same overall topology as IL-1 beta but with different regions of the primary sequence comprising the beta-strands. Two short helical segments also were identified. The 14% sequence identity between IL-1 beta and IRAP increases to 25% when differences in the locations of secondary structure elements in the primary sequences are taken into account. Still, numerous differences in side chains, which ultimately play a major role in receptor interaction, exist.(ABSTRACT TRUNCATED AT 250 WORDS)

The binding of the antitumor drug CC-1065 has been studied with nuclear magnetic resonance (NMR) spectroscopy. This study involves two parts, the elucidation of the covalent binding site of the drug to DNA and a detailed investigation of the noncovalent interactions of CC-1065 with a DNA fragment through analysis of 2D NOE (NOESY) experiments. A CC-1065-DNA adduct was prepared, and an adenine adduct was released upon heating. NMR (1H and 13C) analysis of the adduct shows that the drug binds to N3 of adenine by reaction of its cyclopropyl group. The reaction pathway and product formed were determined by analysis of the 13C DEPT spectra. An octamer duplex, d(CGATTAGC.GCTAATCG), was synthesized and used in the interaction study of CC-1065 and the oligomer. The duplex and the drug-octamer complex were both analyzed by 2D spectroscopy (COSY, NOESY). The relative intensity of the NOEs observed between the drug (CC-1065) and the octamer duplex shows conclusively that the drug is located in the minor groove, covalently attached to N3 of adenine 6 and positioned from the 3'----5' end in relation to strand A [d(CGATTA6GC)]. A mechanism for drug binding and stabilization can be inferred from the NOE data and model-building studies.

Sequence-specific 1H and 15N resonance assignments have been made for all 145 non-prolyl residues and for the flavin cofactor in oxidized Desulfovibrio vulgaris flavodoxin. Assignments were obtained by recording and analyzing 1H-15N heteronuclear three-dimensional NMR experiments on uniformly 15N-enriched protein, pH 6.5, at 300 K. Many of the side-chain resonances have also been assigned. Observed medium-and long-range NOEs, in combination with 3JNH alpha coupling constants and 1HN exchange data, indicate that the secondary structure consists of a five-stranded parallel beta-sheet and four alpha-helices, with a topology identical to that determined previously by X-ray crystallographic methods. One helix, which is distorted in the X-ray structure, is non-regular in solution as well. Several protein-flavin NOEs, which serve to dock the flavin ligand to its binding site, have also been identified. Based on fast-exchange into 2H2O, the 1HN3 proton of the isoalloxazine ring is solvent accessible and not strongly hydrogen-bonded in the flavin binding site, in contrast to what has been observed in several other flavodoxins. The resonance assignments presented here can form the basis for assigning single-site mutant flavodoxins and for correlating structural differences between wild-type and mutant flavodoxins with altered redox potentials.

Unregulated or overexpressed matrix metalloproteinases (MMPs), including stromelysin, collagenase, and gelatinase, have been implicated in several pathological conditions including arthritis and cancer. Small-molecule MMP inhibitors may have therapeutic value in the treatment of these diseases. In this regard, the solution structures of two stromelysin/ inhibitor complexes have been investigated using 'H, I3C, and I5N NMR spectroscopy. Both inhibitors are members of a novel class of matrix metalloproteinase inhibitor that contain a thiadiazole group and that interact with stromelysin in a manner distinct from other classes of inhibitors. The inhibitors coordinate the catalytic zinc atom through their exocyclic sulfur atom, with the remainder of the ligand extending into the SI& side of the active site. The binding of inhibitor containing a protonated or fluorinated aromatic ring was investigated using 'H and "F NMR spectroscopy. The fluorinated ring was found to have a reduced ring-flip rate compared to the protonated version. A strong, coplanar interaction between the fluorinated ring of the inhibitor and the aromatic ring of Tyr155 is proposed to account for the reduced ring-flip rate and for the increase in binding affinity observed for the fluorinated inhibitor compared to the protonated inhibitor. Binding interactions observed for the thiadiazole class of ligands have implications for the design of matrix metalloproteinase inhibitors.