Chemical characteristics and gelling properties of agar from two Philippine Gracilaria spp. (Gracilariales, Rhodophyta)

The chemical structure of agars extracted from Philippine Gracilaria arcuata and G. tenuistipitata were determined by NMR and infrared spectroscopy. Agar with alternating 3-linked 6-O-methyl-β-D-galactopyranosyl and 4-linked 3,6-anhydro-2- O-methyl-α-L-galactopyranosyl units was isolated from G. arcuata, while the agar from G. tenuistipitata possesses the regular agarobiose repeating unit with partial methylation at the 6-position of the D-galactosyl residues. Both agars exhibit sulphate substitution at varying positions in the polymer. Chemical analyses reveal higher 3,6-anhydrogalactose and lower sulphate contents in alkali-modified than in native agar from both samples. Also, alkali modification enhanced agar gel strength and syneresis. Native G. arcuata agar produces a viscous solution (2000 cP at 75 °C) with a high gelling point (>60 °C) that forms a soft gel even after alkali modification (gel strength: <300 g cm⁻²). On the other hand, the agar from G. tenuistipitata exhibits gel qualities typical of most Gracilaria agars. This revised version was published online in June 2006 with corrections to the Cover Date.     

Determination of drug levels in human serum by circular dichroism

A study of the applicability of circular dichroism (CD) for the determination of drug levels in human serum is described and a new method for the quantitative determination of optically active absorbing drugs having Cotton effects at wavelengths above 250 nm in human serum and/or plasma is proposed. The principal advantages of this method are speed, economy, and simplicity, no derivatization or chromatographic separation steps being needed. The validity of the CD determination was confirmed by analysis of variance, β-lactam antibiotics being chosen as model drugs. In addition, the validation studies performed confirm the accuracy and precision of the proposed method. For β-lactam antibiotics lacking Cotton effects above 250 nm, an alternative method based on the extraction of the drug from serum is considered. Chirality 10:507–512, 1998. © 1998 Wiley-Liss, Inc.     

Monitoring of solid phase peptide synthesis by FT-IR spectroscopy

Aggregation phenomena of growing peptides on the resin have seldom been investigated. We report here how conformations are determined by FT-IR spectroscopy. Therefore the sequence 80–99 of HIV 1-protease was synthesized. After every coupling a resin sample was taken out of the reaction column and a FT-IR spectrum recorded. The results were compared with the UV monitoring obtained from another synthesis of the same peptide. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

Flexible glycine rich motif of Escherichia coli deoxyuridine triphosphate nucleotidohydrolase is important for functional but not for structural integrity of the enzyme

Deoxyuridine triphosphate nucleotidohydrolase (dUTPase), a ubiquitous enzyme of DNA metabolism, has been implicated as a novel target of anticancer and antiviral drug design. This task is most efficiently accomplished by X-ray crystallography of the relevant protein–inhibitor complexes. However, the topic of the present investigation, a glycine-rich strictly conserved structural motif of dUTPases, could not be located in the crystal structure of the Escherichia coli enzyme, probably due to its increased flexibility. The present work shows that removal of a C-terminal 11-residue fragment, including this motif, by limited trypsinolysis strongly impairs catalytic activity. Kinetic analysis of the intact and digested variants showed that k_cat decreases 40-fold, while K_M increases less than twofold upon digestion. The tryptic site was identified by mass spectrometry, amino acid analysis and N-terminal sequencing. The shortened enzyme variant retains the secondary, tertiary, and quaternary (trimeric) structure of the intact species as suggested by UV absorption, fluorescence and circular dichroism spectroscopy, and analytical gel filtration. Moreover, binding affinity of the shortened variant toward the substrate analogue MgdUDP is identical to the one displayed by the intact enzyme. I conclude that the glycine-rich motif is functionally relevant for E. coli dUTPase. It may play a role in enzymatic catalysis by contributing to the formation of the catalytically potent enzyme–substrate complex. Proteins 28:568–579, 1997. © 1997 Wiley-Liss, Inc.     

The analysis of merino wool cuticle and cortical cells by Fourier transform Raman spectroscopy

Wool fibers are comprised of proteins known as α-keratins and have a complex morphological structure. The major components of this structure, the cuticle and cortical cells, differ in the conformations of their peptide chains as well as their amino acid compositions. High quality Fourier transform Raman spectra of cortical and cuticle cells isolated from fine Merino wool fibers have been obtained. Raman spectroscopy has been shown to be sensitive to the differences in both secondary structure and amino acid composition. The cortical cells were found to be higher in α-helical content as compared to the cuticle cells, which had an increased disordered content. Specific information, consistent with amino acid analysis results, regarding cystine, tyrosine, tryptophan, and phenylalanine residues, were obtained for both the cortical and cuticle cells. In addition, the Raman spectra provided information about free thiol groups, amino acids residues with amide group side chains, and residues with protonated carboxyl group side chains. Middle ir transmission spectra of these isolated cells were also obtained. In comparison to the Raman data, the middle ir spectra were found to be not as rich in information. © 1997 John Wiley & Sons, Inc. Biopoly 42: 7–17, 1997     

Crystallization and preliminary diffraction analysis of Ca2+-calmodulin-drug and apocalmodulin-drug complexes

Ca²⁺-calmodulin is crystallized with two new and potent drugs: a bisindol derivative (KAR-2, 3”-(β-chloroethyl)-2”,4”-dioxo-3,5”-spiro-oxazolidino-4-deacetoxy-vinblastine) with antitumor activity and an arylalkylamine fendiline analogue (N-(3,3-diphenylpropyl)-N'-[1-(3,4-di-n-butoxy-phenyl)-ethyl]-1,3-diaminopropane) with anticalmodulin activity. The crystals diffract beyond 2.8 Å and differ in unit cell parameters from each other as well as from crystals of Ca²⁺-calmodulin or Ca²⁺-calmodulin-ligand complexes, as reported thus far. Attempts to crystallize Ca²⁺-free calmodulin without drugs failed, in consonance with earlier results; however, single Ca²⁺-free calmodulin crystals diffracting beyond 2.5 Å resolution were grown in the presence of KAR-2. Results indicate that binding of the two drugs to apocalmodulin or Ca²⁺-calmodulin may induce unique novel protein conformers, targets of further detailed X-ray studies. © 1997 Wiley-Liss Inc.     

Peptide-derived self-assembled monolayers: Assorption of N-stearoyl L-systeine methyl ester on gold

The work reported herein concerns the assembly of N-stearoyl L -cysteine methyl ester [CH₃(CH₂)₁₆COCysOMe, 1] on the surface of gold. This compound serves as a simple model of a related polypeptide, which has been designed to adopt a β-sheet architecture on metallic and oxide surfaces. We describe the preparation of monolayers of 1, and characterization of these layers via ellipsometry, vibrational spectroscopy and X-ray photoelectron spectroscopy. The results are most consistent with a disordered array of the alkyl chains, in which close packing is frustated by a mismatch in the cross-sectional areas of the cysteinyl ester head group and the stearoyl chains of the thiol. Despite the disorder, the alkyl chains form a hydrophobic surface layer, with an advancing contact angle for water comparable to that observed for octadecanethiol on gold © 1997 John Wiley & Sons, Ltd.     

Total synthesis of zervamicin IIB and its deuterium-labelled analogues

For the first time the total synthesis of the peptaibol antibiotic zervamicin IIB is described. Synthesis of this peptaibol was achieved by the Fmoc/tert-butyl strategy in solution using a fragment condensation approach. Three fragments of zervamicin IIB were obtained by stepwise elongation with Fmoc amino acids using BOP as a coupling reagent. For the introduction of the highly sterically hindered α-aminoisobutyric acid residues BOP/DMAP activation was applied. The Fmoc group was removed by reaction with 0.1 M NaOH in dioxane/methanol/water (30/9/1, v/v/v). Peptide fragments were coupled by means of a new coupling reagent, CF₃-PyBOP. Using the strategy developed, zervamicin IIB and two analogues specifically deuterium-labelled at different positions of the glutamine-11 residue have been synthesized in 40% overall yield based on the isotopically labelled amino acid and with 98±2% of isotope enrichment. FAB mass spectroscopy, 600 MHz ¹H-NMR spectroscopy and high-performance liquid chromatography provided convincing evidence that the synthetic products, zervamicin IIB and its deuterium-labelled analogues, fully correspond to the naturally occurring zervamicin IIB. © 1997 European Peptide Society and John Wiley & Sons, Ltd.     

Rescuing activity of oxygen-damaged pyruvate formate-lyase by a spare part protein

Pyruvate formate-lyase (PFL) is a glycyl radical enzyme (GRE) that converts pyruvate and coenzyme A into acetyl-CoA and formate in a reaction that is crucial to the primary metabolism of many anaerobic bacteria. The glycyl radical cofactor, which is posttranslationally installed by a radical S-adenosyl-L-methionine (SAM) activase, is a simple and effective catalyst, but is also susceptible to oxidative damage in microaerobic environments. Such damage occurs at the glycyl radical cofactor, resulting in cleaved PFL (cPFL). Bacteria have evolved a spare part protein termed YfiD that can be used to repair cPFL. Previously, we obtained a structure of YfiD by NMR spectroscopy and found that the N-terminus of YfiD was disordered and that the C-terminus of YfiD duplicates the structure of the C-terminus of PFL, including a β-strand that is not removed by the oxygen-induced cleavage. We also showed that cPFL is highly susceptible to proteolysis, suggesting that YfiD rescue of cPFL competes with protein degradation. Here, we probe the mechanism by which YfiD can bind and restore activity to cPFL through enzymatic and spectroscopic studies. Our data show that the disordered N-terminal region of YfiD is important for YfiD glycyl radical installation but not for catalysis, and that the duplicate β-strand does not need to be cleaved from cPFL for YfiD to bind. In fact, truncation of this PFL region prevents YfiD rescue. Collectively our data suggest the molecular mechanisms by which YfiD activation is precluded both when PFL is not damaged and when it is highly damaged.