Synthesis of Multiphosphorylated Peptide from a C-Terminal Bovine Rhodopsin
Sequence. Pp. 361-368.
Anatol Arendt, J. Hugh McDowell, Galina Abdulaeva and Paul A. Hargrave.
[Abstract]
Synthesis of HSV Epitope Peptides with Acid Sensitive ASP-PRO Bond. Pp. 369-376.
Gábor Mezo, Marianna Mák, Szilvia Bosze and Ferenc Hudecz.
[Abstract]
Identification of a Peptide Binding to the HIV-1 Nucleocapsid Protein (NCP7). Pp. 377-384.
Raffaele A. Calogero, Daniela Lener, Gianni Antico, Rosanna del Guadio, Anna Aulicino and Guiseppe Geraci.
[Abstract]
The Use of Maldi-TOF Mass Spectrometry in Quantification of the Stability of Prolyl
Endopeptidase Inhibitors. Pp. 385-392.
Jörn Schmidt, Michael Wermann, Fred Rosche and Hans-Ulrich Demuth.
[Abstract]
Nonendocytic, Amphipathicity Dependent Cellular Uptake of Helical Model Peptides. Pp. 393-398.
Johannes Oehlke, Eberhard Krause, Burkhard Wiesner, Michael Beyermann and
Michael Bienert.
[Abstract]
Structural Analysis of the Native and Drug-Resistant HIV-1 Proteinases Complexed
with an Aminodiol Inhibitor. Pp. 399-406.
Jukka Kervinen, Normada Thanki Alexander Zdanov, Joseph Tino, Joel Barrish,
Pin Fang Lin, Richard Colonno, Keith Riccardi, Himadri Samanta and
Alexander Wlodawer.
[Abstract]
Molecular Surface Morphology Studies of beta-Amyloid Self-Assembly: Effect of pH on Fibril Formation. Pp. 407-414.
A.P. Shivji, M.C. Davies, C.J. Roberts, S.J.B. Tendler and M.J. Wilkinson.
[Abstract]
Bacteriophage Display of Chymotrypsin Inhibitor 2. Pp. 415-422.
David L.R. Holmes, Dimitris Mantafounis, Walter H.J. Ward and
Robin J. Leatherbarrow.
[Abstract]
Crystallisation Reports
Crystallization of the Secondary Alcohol Dehydrogenase from Thermoanaerobacter ethanolicus 39E. Pp. 423-426.
R.K. Arni, L. Watanabe, M. Fontes, D.S. Burdette and J.G. Zeikus.
[Abstract]
Crystal Structure of Porcine Aldehyde Reductase at 2.0 A Resolution: Modeling an
Inhibitor in the Active Site of the Enzyme. Pp. 427-434.
Ossama El-Kabbani, Deborah A. Carper, Michelle H. McGowan and Stephan L. Ginell.
[Abstract]
[Back to top] S-(1,2-Dicarboxyethyl) Glutathione, Intrinsic Tripeptide in Liver, Heart and Lens, Elevates gamma-Glutamylcysteine Synthetase Activity. Naoko Nakagawa, Seiji Tsuboi, Naoki Kaneko, Takahiro Sakaue, Kazumi Ogata and Shinji Ohmori.
The effect of S-(1,2-dicarboxyethyl)glutathione (DCB-GS) on glutathione (GSH) synthesis, especially gamma-glutamylcysteine synthetase (gamma-GC S), was studied in rat liver homogenate. It was found that the feedback inhibition of the y-GCS by GSH was released by the addition of DCE-GS in the dose dependent manner. Namely, the activity of gamma-GCS decreased to 17 % of the normal level when GSH at the concentration of 10 mM was added to the reaction mixture. However, the addition of DCB-GS offset the inhibition at 20 uM and increased the gamma-GCS activity to 1.7 and 3 times from the normal level at 50 and 100 uM, respectively. These results suggest that DCB-GS plays a role in stimulating the GSH synthesis.
[Back to top] Synthesis of Multiphosphorylated Peptide from a C-Terminal Bovine Rhodopsin Sequence. Anatol Arendt, J. Hugh McDowell, Galina Abdulaeva and Paul A. Hargrave.
We have synthesized and purified a 19-amino acid peptide, containing 4 phosphothreonines and 3 phosphoserines, of sequence derived from the C-terminal region of bovine rhodopsin. The peptide has been synthesized on Pam resin using Boc-O-(diphenylphosphono)-serine and -threonine. Cleavage and deblocking have been performed in a single step using catalytic hydrogenolysis in the presence of palladium (II) and platinum (IV) with anhydrous trifluoroacetic acid as solvent.
[Back to top] Synthesis of HSV Epitope Peptides with Acid Sensitive ASP-PRO Bond. Pp. 369-376. Gábor Mezo, Marianna Mák, Szilvia Bosze and Ferenc Hudecz.
Peptides from Herpes Simplex virus type I glycoprotein-D, containing the Asp-Pro bond were synthesized by SPPS using benzyl or cyclohexyl (cHex) protection at the Asp/Glu side chains. Increased stability of the cHex group was demonstrated during the treatment with TMSOTf-thioanisole/TFA. This observation was successfully utilised to prepare partly protected 9-mer peptides and their 18-mer derivatives by fragment condensation both in solution and on solid support.
[Back to top] Identification of a Peptide Binding to the HIV-1 Nucleocapsid Protein (NCP7). Raffaele A. Calogero, Daniela Lener, Gianni Antico, Rosanna del Guadio, Anna Aulicino and Guiseppe Geraci.
In HIV-1 the nucleocapsid protein (NC) is essential in the virjon assembly process. The discovery of substances able to interfere with the HIV-1 NC functions could be a starting point for the design of new drugs that might inhibit the HIV-1 viral infectivity. A single peptide sequence binding to the NC was isolated using a conformationally homogeneous phage displayed-peptide library. The minimal requirement for the NCp7/peptide interaction was the NCp7 region encompassing the second zinc finger.
[Back to top] The Use of Maldi-TOF Mass Spectrometry in Quantification of the Stability of Prolyl
Endopeptidase Inhibitors. Jörn Schmidt, Michael Wermann, Fred Rosche and Hans-Ulrich Demuth.
Z-Phe-Pro-(isonicotinic acid methylester) methylketone is a highly potent inhibitor (Ki* = 18 nM) of prolyl endopeptidase from Flavobacterium meningosepticum. It acts as a slow-binding inhibitor and decomposes to compounds still possessing inhibitory activity. The pathway of decomposition was analyzed qualitatively and quantitatively applying MALDI-TOF mass spectrometry.
[Back to top] Nonendocytic, Amphipathicity Dependent Cellular Uptake of Helical Model Peptides. Johannes Oehlke, Eberhard Krause, Burkhard Wiesner, Michael Beyermann and Michael Bienert.
For N-terminally fluoresceine labelled KLALKLALKALKAALKLA-NH2 (I) extensive incorporation into aortic endothelial cells (AEC) was found, resulting in an about 500-fold enrichment within the cell interior. The cellular uptake was not influenced by energy depletion and (only partly by lowering the temperature to 0 degrees C. Using double-D-amino acid analogs of this peptide, possessing a graduate propensity to adopt an alpha-helical structure, clear dependency of the peptide incorporation upon amphipathicity could be demonstrated.
[Back to top] Structural Analysis of the Native and Drug-Resistant HIV-1 Proteinases Complexed with an Aminodiol Inhibitor. Jukka Kervinen, Normada Thanki Alexander Zdanov, Joseph Tino, Joel Barrish, Pin Fang Lin, Richard Colonno, Keith Riccardi, Himadri Samanta and Alexander Wlodawer.
Crystal structures of the native HIV-1 proteinase and an A71T/V82A mutant have been solved in complexes with a symmetric aminodiol inhibitor, BMS-182193. Some of the conformational differences between these two complexes can be traced to their non-isomorphous crystal forms, but numerous rearrangements due to mutations are clearly visible. The V82A mutation creates more space at the periphery of the active site cleft, allowing distal parts of the inhibitor to move more freely, whereas the non-active-site mutation A7IT stabilizes the enzyme backbone. The role of mutations in eliciting drug resistance is discussed.
[Back to top] Molecular Surface Morphology Studies of beta-Amyloid Self-Assembly: Effect of pH on Fibril Formation. A.P. Shivji, M.C. Davies, C.J. Roberts, S.J.B. Tendler and M.J. Wilkinson.
Deposition of beta-amyloid fibrils within senile plaques in the brain is a major hallmark of Alzheimer's disease. In this study, atomic force microscopy (AFM) has been used to provide surface topographical data of beta-amyloid fibrillization as a function of pH. Alteration of pH has led to observed changes in fibril self-assembly rate, diameter and branching. These results suggest that pH has a profound affect on beta-amyloid self-assembly and surface morphology.
[Back to top] Bacteriophage Display of Chymotrypsin Inhibitor 2. David L.R. Holmes, Dimitris Mantafounis, Walter H.J. Ward and
Robin J. Leatherbarrow.
A chymotrypsin inhibitor 2 variant, C12(G83D), was expressed as a fusion with fd minor coat protein, pIll, via a flexible linker (Gly-Ala3) and displayed on the surface of bacteriophage. Biopanning with immobilised subtuisin BPN' demonstrated that the inhibitor~displaying phage could be selectively enriched 13-fold over control phage in one round of affinity purification. The inhibitor (incorporating the linker sequence) was also expressed as soluble protein in E. coli and purified to homogeneity. Kinetic analysis revealed the displayed inhibitor possesses inhibition characteristics that approximate those of this soluble inhibitor. The results demonstrate that functionally active inhibitor can be displayed on the phage surface. Further use of this technology should facilitate the selection of remodelled inhibitor variants exhibiting novel protease binding specificities.
[Back to top] Crystallization of the Secondary Alcohol Dehydrogenase from Thermoanaerobacter ethanolicus 39E. R.K. Arni, L. Watanabe, M. Fontes, D.S. Burdette and J.G. Zeikus.
The secondary alcohol dehydrogenase from the thermophile Thermoanaerobacter ethanolicus 39E has been crystallized at 40 degrees C by vapour ditussion using polyethelene glycol as a precipitant. The orthorhombic crystals belong to the space group P 2(1)2(1)2 with cell constants of a=170.0 A, b=125.7 A and c=80.5 A. A native X-ray diffraction data set has been collected to 2.7 A resolution.
[Back to top] Crystal Structure of Porcine Aldehyde Reductase at 2.0 A Resolution: Modeling an Inhibitor in the Active Site of the Enzyme. Ossama El-Kabbani, Deborah A. Carper, Michelle H. McGowan and Stephan L. Ginell.
Aldehyde reductase is an enzyme capable of metabolizing a wide variety of aldehydes to their corresponding alcohols. The X-ray crystal structure of porcine aldehyde reductase holoenzyme has been refined to a crystallographic R-factor of 0.20 at 2.0 A resolution. We have modeled the inhibitor zopolrestat in the active site of porcine aldehyde reductase in order to obtain a picture of the binding conformation of inhibitors to the enzyme.