Victor Wray

The Solution Structure and Heme Binding of the Presequence of Murine 5-Aminolevulinate Synthase

The mitochondrial import of 5‐aminolevulinate synthase (ALAS), the first enzyme of the mammalian heme biosynthetic pathway, requires the N‐terminal presequence. The 49 amino acid presequence transit peptide (psALAS) for murine erythroid ALAS was chemically synthesized, and circular dichroism and 1H nuclear magnetic resonance (NMR) spectroscopies used to determine structural elements in trifluoroethanol/H2O solutions and micellar environments. A well defined amphipathic α‐helix, spanning L22 to F33, was present in psALAS in 50% trifluoroethanol. Further, a short α‐helix, defined by A5–L8, was also apparent in the 26 amino acid N‐terminus peptide, when its structure was determined in sodium dodecyl sulfate. Heme inhibition of ALAS mitochondrial import has been reported to be mediated through cysteine residues in presequence heme regulatory motifs (HRMs). A UV/visible and 1H NMR study of hemin and psALAS indicated that a heme–peptide interaction occurs and demonstrates, for the first time, that heme interacts with the HRMs of psALAS.

Triterpenoid saponins from Bellium bellidioides

A new acylated triterpenoid saponin carrying the rare 2,3-branched glucose at C-28 of the aglycone bayogenin (2 beta, 3 beta, 23-trihydroxyolean-12-en-28-oic acid) was isolated from the whole plants of Bellium bellidioides. The structure was elucidated mainly by high field NMR experiments (1H and 13C NMR, HMBC, HMQC, COSY-45 at 600/150 MHz) as 3-O-beta-D-glucopyranosyl-28-O-[2-O-alpha-L-rhamnopyranosyl-3-O-beta-D- glucopyranosyl-6-O-acetyl-beta-D-glucopyranosyl]-23-O-acetylbayogenin . Additionally, three acylated derivatives of the known bellissaponin BS1 were obtained.

Triterpenoid saponins from Bellium bellidioides. structures of the minor deacylsaponins

A new deacylsaponin of polygalacic acid, desacylbellidioside B4, was obtained from the whole plants of Bellium bellidioides L. The structure has been elucidated by a general strategy involving mass spectrometry (ESI-MS, including tandem MS, and GC-MS) and high-field one- and two-dimensional NMR spectroscopy (1H and 13C NMR, COSY-45, HMQC, HMBC) as 3-O-alpha-L-rhamnopyranosyl-2 beta, 3 beta, 16 alpha, 23-tetrahydroxyolean-12-en-28-oic acid 28-O-alpha-L-rhamnopyranosyl(1-->3)-beta-D-xylopyranosyl(1-->4)-alpha-L- rhamnopyranosyl(1-->2)-[alpha-L-arabinofuranosyl(1-->3)-beta-D-fucopyran oside. Moreover, bellissaponin BS2 and besysaponin C12 have also been isolated, demonstrating the close chemical relationship of B. bellidioides to the Bellis genus.

Detoxification of the macrolide toxin brefeldin A by Bacillus subtilis

The macrolide brefeldin toxin A is a determinant of Alternaria leaf blight disease in safflower, which causes severe economic losses worldwide. Soilborne bacteria, classified as Bacillus subtilis spp., were isolated and shown to readily metabolize brefeldin A in laboratory culture to one major product. This product was identified by high resolution 2D 1H NMR and FAB mass spectroscopies as the acid resulting from hydrolysis of the macrolide ring in brefeldin A. In contrast to brefeldin A, the acid completely lacked phytotoxic activity in the standard leaf bioassay. Detoxification of brefeldin A by the lactonase activity from Bacillus subtilis may be exploited in the future to introduce resistance to Alternaria leaf blight in sufflower.

Structure, Phosphorylation, and Biological Function of the HIV-1 Specific Virus Protein U (Vpu)

Knowledge describing the structure and function of the small regulatory human immunodeficiency virus type 1 (HIV-1) viral protein U (Vpu) has increased significantly over the last decade. Vpu is an 81 amino acid class I oligomeric integral-membrane phosphoprotein that is encoded exclusively by HIV-1. It can therefore be anticipated, that Vpu might contribute to the increased pathogenic potential of HIV-1 when compared with HIV-2 that has so far had a lower impact on the acquired immune deficiency syndrome (AIDS) pandemic. Various biological functions have been ascribed to Vpu: first, in the endoplasmic reticulum (ER) Vpu induces degradation of CD4 in a process involving the ubiquitin-proteasome pathway and phosphorylation of its cytoplasmic tail. In addition, there is also evidence that Vpu interferes with major histocompatibility complex (MHC) class I antigen presentation and regulates Fas mediated apoptosis. Second, Vpu augments virus release from a post ER compartment by a cation-selective ion channel activity mediated by its transmembrane (TM) anchor. The phosphorylation of the molecule is mediated by the ubiquitous protein kinase caseinkinase 2 (CK-2) within a central conserved dodecapeptide at positions Ser52 and Ser56 located in a flexible hinge region between two helical domains. Structural information, provided experimentally mainly by solution- and solid-state nuclear magnetic resonance (NMR) spectroscopy and made possible through the availability of synthetic and recombinant material, have shown that the biological activities of Vpu are localized in two distinct domains that are mainly confined to the C-terminal cytoplasmic and N-terminal TM domains, respectively. Similar to other small viral proteins that interact with membranes Vpu is a very flexible molecule whose structure is exceptionally environment dependent. It assumes it’s most structured form in the hydrophobic environment in or at the membrane. An initial 20–23 residue α-helix in the N-terminus adopts a TM alignment while the cytoplasmic tail forms an α-helix-flexible-α-helix-turn motif, of which at least a part is bound parallel to the membrane surface. Details of the arrangement of oligomeric forms of the molecule that are presumably required for the ion channel activity, are emerging from recent theoretical calculations, while this particular function is currently the area of pharmaceutical interest.

Noroleanane saponins from celmisia spectabilis

Two new saponins were isolated as the major components of the deacylated saponin extract from the underground parts of Celmisia spectabilis. Their structures were established by NMR and mass spectral data and derivative formation as 2 beta,3 beta,17,23- tetrahydroxy-28-norolean-12-en-16-one-3-O-alpha-L-arabinopyrano syl (1-->2)-alpha-L-arabinopyranosyl(1-->6)-beta-D-glucopyranoside and 2 beta,3 beta,17,23- tetrahydroxy-28-norolean-12-en-16-one-3-O-alpha-L-arabinopyrano syl (1-->2)-alpha-L-arabinopyranosyl(1-->6)-[alpha-L-rhamnopyranosyl (1-->2)]-beta-D-glucopyranoside.

Use of Synthetic Peptides for Structural and Functional Analyses of Viruses Like HIV

ABSTRACT Efficient solid-state synthesis now affords ready access, particularly in situations in which recombinant protocols usually fail, to high-quality synthetic peptides that are equivalent to many full-length biologically active systems or domains thereof, including posttranslational modifications such as phosphorylation and acetylation. Here we survey recent applications of such peptides to the investigations of human immunodeficiency viruses (HIV), with the focus on the regulatory peptides Vpu, Vpr, and Tat, as well as the protease, the Gag peptides nucleocapsid, matrix, and p6 from HIV-1. The importance of the full-length synthetic viral peptides for molecular characterization that involves both detailed biochemical and structural studies at the atomic level and their potential for therapeutic applications are emphasized.