Synthesis of functionalized rab GTPases by a combination of solution- or solid-phase lipopeptide synthesis with expressed protein ligation

Prenylated proteins with non-native functionalities are generally very difficult to obtain by recombinant or enzymatic means. The semisynthesis of preparative amounts of prenylated Rab guanosine triphosphatases (GTPases) from recombinant proteins and synthetic prenylated peptides depends largely on the availability of functionalised prenylated peptides corresponding to the proteins' native structure or modifications thereof. Here, we describe and compare solution-phase and solid-phase strategies for the generation of peptides corresponding to the prenylated C terminus of Rab7 GTPase. The solid-phase with utilisation of a hydrazide linker emerges as the more favourable approach. It allows a fast and practical synthesis of pure peptides and gives a high degree of flexibility in their modification. To facilitate the analysis of semisynthetic proteins, the synthesised peptides were equipped with a fluorescent group. Using the described approach, we introduced fluorophores at several different positions of the Rab7 C terminus. The position of the incorporated fluorescent groups in the peptides did not influence the protein-ligation reaction, as the generated peptides could be ligated onto thioester-tagged Rab7. However, it was found that the positioning of the fluorescent group had an influence on the functionality of the Rab7 proteins; analysis of the interaction of the semisynthetic Rab7 proteins with REP (Rab escort protein) and GDI (guanosine diphosphate dissociation inhibitor) molecules revealed that modification of the peptide side chains or of the C-terminal isoprenoid did not significantly interfere with complex formation. However, functionalisation of the C terminus was found to have an adverse effect on complex formation and stability, possibly reflecting low structural flexibility of the Rab GDI/REP molecules in the vicinity of the lipid-binding site.     

Thiol- and disulfide-modified oligonucleotide monolayer structures on polycrystalline and single-crystal Au(111) surfaces

We provide a comprehensive study of single- (ss) and double-strand (ds) oligonucleotides with either 25 or 10 bases or base pairs (bp) immobilized on polycrystalline and single-crystal Au(111) surfaces. The study is based on X-ray photoelectron spectroscopy, cyclic and differential pulse voltammetry, interfacial capacitance data, and electrochemical scanning tunnelling microscopy (in situ STM). The sequences used were the 25-bp sequence from the BRCA1 gene (25-mer), while the 10-bp oligonucleotides contained solely linear adenine and thymine sequences. The oligonucleotides were modified by the dimethoxytrityl group (DMT) via a disulfide group [DMT-S-S-ss25-mer and DMT-S-S-ds(AT)₁₀], a pure disulfide group (A₁₀-S-S-T₁₀), or a thiol group [HS-ss25-mer and HS-ds-(AT)₁₀], all via a hexamethylene linker. The overall pattern suggests strategies for controlled adsorption of DNA-based molecules and recognition of complementary strands or other molecules.     

Synthesis and properties of allenic natural products and pharmaceuticals

Nowadays, about 150 natural products comprising an allenic or cumulenic structure are known. The chemistry of these compounds has turned out to be a very attractive and prolific area of interest: advances in the isolation and characterization of new allenic natural products have led to the establishment of efficient synthetic procedures which in many cases also open up an access to enantiomerically pure target molecules. Inspired by the intriguing biological activities of many allenic natural products, allene moieties are now systematically introduced in pharmacologically active classes of compounds (steroids, prostaglandins, amino acids, nucleosides). The functionalized allenes thus obtained often exhibit impressive activities as mechanism-based enzyme inhibitors, cytotoxic, or antiviral agents. A prerequisite for further developments in this field is the efficient stereoselective synthesis of allene derivatives.     

EFFECTS OF BOUND MONOCLONAL ANTIBODIES ON THE DECAY OF THE PHOTOTRANSFORMATION INTERMEDIATES I1,21700 FROM NATIVE Avena PHYTOCHROME*

Abstract–Thc kinetics of the microsecond phototransformation intermediates of 124 kDa Avena phytochrome (1700^1,2) were studied in the prcsence of bound monoclonal antibodies at various temperatures. A global analysis was applied to the decays at all wavelengths at each temperature in order to derive the rate constants and the decay-associated spectra of the three decay components. Monoclonal antibodies bound to specific epitopes altered the Arrhenius parameters of both 1700^1,2 decay components. The strongest influence on these parameters was observed with OAT 8 (epitope between residues 624 and 686), which decreased by more than 50% the activation parameters of both components. This decrease is interpreted to result from an increased flexibility induced by this antibody in the ground state or in the transition state of bonds changing during the decay of both 1700 transients. Thus, the OAT 8 cpitope appears to be functionally important during the decay of the 1700^1,2 intermediates. For the case of 1100¹ bound OAT 23 and OAT 25 (epitopes between residues 1 and 66) reduced even further the relatively small flexibility of these bonds in the red light-absorbing form of phytochrome (P1) without antibodies, as reflected by the high preex-ponential factors for its decay. This resulted also in higher activation energies for this decay in the presence of the antibodies. Thus, the amino-terminus should act as a rigid spacer of the chromophore cavity without affecting it during the microsecond transformation, because the Arrhenius parameters for these decays are similar to those for small phytochrome. The possible implications of the influence of the various antibodies on the bleaching remaining after the decay of 1700^1,2 are discussed.     

Laser desorption mass spectrometry on thin liquid jets

A recently developed soft desorption method for mass spectrometry is presented, which is called Laser Induced Liquid Beam Ionization/Desorption (LILBID). Analyte ions are desorbed from a thin jet of analyte solution directly into vacuum by means of an IR laser pulse, which has been tuned to a vibrational resonance of the solvent. A comparative experiment with ammonium chloride and aniline hydrochloride shows that ion formation via proton transfer takes place in the solution. Thermally unstable compounds, as well as supra- and biomolecular complexes, can be detected intact and mass analyzed in a reflectron time-of-flight (Re-TOF) mass spectrometer. During the desorption process, noncovalent interactions and some solvation characteristics are preserved. Three examples for the capacity of LILBID are given in this short overview: (a) ion-solvent interactions with the formation of a clathrate structure Cs⁺(H₂O)₂₀, (b) host-guest interactions with the K⁺ selectivity of valinomycin, and (c) noncovalent interactions with the dimerization of gramicidin. Received: 29 July 1997 / Revised: 4 September 1997 / Accepted: 12 September 1997     

Resolution enhancement in multidimensional solid-state NMR spectroscopy of proteins using spin-state selection

We show that the resolution of homonuclear multidimensional solid-state NMR correlation experiments can be significantly improved using transition selection and spin-state-selective polarization transfer techniques. The selection and transfer of single states allow the removal of the J-coupling contribution from the line width in both the direct and indirect spectral dimensions. This is demonstrated with a new spin-state-selective CO-Calpha correlation experiment, applied to a microcrystalline 85-residue protein. With this new sequence, all four components of the CO-Calpha cross-peaks are separated into different spectra, obtained by linear combination of four recorded data sets. Line narrowing of up to 44% was obtained on the protein sample for the spin-state-selective CO-Calpha spectrum compared to a standard spin-diffusion experiment. The new technique also allows an easy distinction between "direct" and "relayed" transfer cross-peaks.     

Measurement of γp→K + Λ and γp→K + Σ 0 at photon energies up to 1.47 GeV

The reactions pK ⁺ and pK ^{+ 0} have been measured with the multiparticle detector system SAPHIR at ELSA in Bonn. Besides the differential cross sections the polarization and, for the first time, the ⁰ polarization have been determined in a photon induced reaction. All data are presented as functions of the photon energy (from threshold up to 1.47 GeV) and of the kaon production angle (0°–180°). The polarization of both and ⁰ is substantial at all energies and varies strongly with the production angle.This work is supported by the Bundesminister für Forschung und Technologie (BMFT), FK 06 BN 621 I     

Un substrat pour le dosage chimique de la rénine

A fluorogenic renin substrate, N-CBO-L -prolyl-L -phenylalanyl-L -histidyl-L -leucyl-L -leucyl-L -valyl-L -tyrosyl-L -seryl-β-naphthylamide, has been synthesized. Upon incubation at pH 5,6 with renin and an excess of the auxiliary enzyme aminopeptidase M, it gives rise to β-naphthylamine at a rate related to the quantity of renin.     

Strong Attraction of Caffeine to the Mercurous Dumbbell in the Salt [Hg2(Caf)2](ClO4)2(H2O)2

Colourless single crystals of the caffeine adduct of mercurous perchlorate dihydrate, [Hg₂(Caf)₂](ClO₄)₂(H₂O)₂, were grown from aqueous solutions of mercurous perchlorate and caffeine by isothermal evaporation at ambient temperature. The crystal structure (monoclinic, P2₁/n, Z = 4, a = 1628.0(2), b = 780.4(1), c = 2229.6(3) pm, β = 99.84(1)°, R₁(all data) = 0.0894) contains [trans‐Caf‐Hg‐Hg‐Caf]²⁺ cations with a Hg‐Hg distance of 250.88(6) pm, Hg‐N (bond) distances of 214.4(6) and 215.1(6) pm and Hg‐Hg‐N angles of 176.9(2) and 165.1(2)°, respectively. These cations are attached via weak Hg‐O contacts to dimers which are further arranged to leave large channels into which one crystal water molecule is included. The second water molecule and the two perchlorate anions are weakly attracted to one Hg atom.