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PubMed2004

Rational and combinatorial design of peptide capping ligands for gold nanoparticles.

Based on protein folding considerations, a pentapeptide ligand, CALNN, which converts citrate-stabilized gold nanoparticles into extremely stable, water-soluble gold nanoparticles with some chemical properties analogous to those of proteins, has been designed. These peptide-capped gold nanoparticles can be freeze-dried and stored as powders that can be subsequently redissolved to yield stable aqueous dispersions. Filtration, size-exclusion chromatography, ion-exchange chromatography, electrophoresis, and centrifugation can be applied to these particles. The effect of 58 different peptide sequences on the electrolyte-induced aggregation of the nanoparticles was studied. The stabilities conferred by these peptide ligands depended on their length, hydrophobicity, and charge and in some cases resulted in further improved stability compared with CALNN, yielding detailed design criteria for peptide capping ligands. A simple strategy for the introduction of recognition groups is proposed and demonstrated with biotin and Strep-tag II.

Amino Acid Sequence↗
PubMed1998

Surface salt bridges stabilize the GCN4 leucine zipper.

We present a study of the role of salt bridges in stabilizing a simplified tertiary structural motif, the coiled-coil. Changes in GCN4 sequence have been engineered that introduce trial patterns of single and multiple salt bridges at solvent exposed sites. At the same sites, a set of alanine mutants was generated to provide a reference for thermodynamic analysis of the salt bridges. Introduction of three alanines stabilizes the dimer by 1.1 kcal/mol relative to the wild-type. An arrangement corresponding to a complex type of salt bridge involving three groups stabilizes the dimer by 1.7 kcal/ mol, an apparent elevation of the melting temperature relative to wild type of about 22 degrees C. While identifying local from nonlocal contributions to protein stability is difficult, stabilizing interactions can be identified by use of cycles. Introduction of alanines for side chains of lower helix propensity and complex salt bridges both stabilize the coiled-coil, so that combining the two should yield melting temperatures substantially higher than the starting species, approaching those of thermophilic sequences.

Alanine↗
PubMed2003

RNA cleaving '10-23' DNAzymes with enhanced stability and activity.

'10-23' DNAzymes can be used to cleave any target RNA in a sequence-specific manner. For applications in vivo, they have to be stabilised against nucleolytic attack by the introduction of modified nucleotides without obstructing cleavage activity. In this study, we optimise the design of a DNAzyme targeting the 5'-non-translated region of the human rhinovirus 14, a common cold virus, with regard to its kinetic properties and its stability against nucleases. We compare a large number of DNAzymes against the same target site that are stabilised by the use of a 3'-3'-inverted thymidine, phosphorothioate linkages, 2'-O-methyl RNA and locked nucleic acids, respectively. Both cleavage activity and nuclease stability were significantly enhanced by optimisation of arm length and content of modified nucleotides. Furthermore, we introduced modified nucleotides into the catalytic core to enhance stability against endonucleolytic degradation without abolishing catalytic activity. Our findings enabled us to establish a design for DNAzymes containing nucleotide modifications both in the binding arms and in the catalytic core, yielding a species with up to 10-fold enhanced activity and significantly elevated stability against nucleolytic cleavage. When transferring the design to a DNAzyme against a different target, only a slight modification was necessary to retain activity.

Binding Sites↗
PubMed2002

Intensification of lipase performance for long-term operation by immobilization on controlled pore silica in presence of polyethylene glycol.

In agreement with previous studies, promising results were obtained when lipase was immobilized on controlled pore silica (CPS) in the presence of polyethylene glycol (PEG 1500). This methodology rendered immobilized derivatives with higher operational stability than those lacking PEG 1500. This article extends the scope of this approach by evaluating the combined effects of PEG concentration and lipase loading employing a multivariate statistical approach. A 2(2) factorial design with center point was adopted for a full understanding of these effects and their interactions. Conditions that maximize the immobilization yield were different from those attained for the biocatalyst's operational stability. Possible reasons for the increase in both activity and stability of lipase immobilized on CPS in the presence of PEG 1500 are discussed in light of the influence of surface hydrophilic/hydrophobic balance.

Candida↗
PubMed2004

Early prediction of pharmaceutical oxidation pathways by computational chemistry and forced degradation.

PURPOSE: To show, using a model study, how electronic structure theory can be applied in combination with LC/UV/MS/MS for the prediction and identification of oxidative degradants. METHODS: The benzyloxazole 1, was used to represent an active pharmaceutical ingredient for oxidative forced degradation studies. Bond dissociation energies (BDEs) calculated at the B3LYP/6-311+G(d,p)//B3LYP/6-31G(d) level with isodesmic corrections were used to predict sites of autoxidation. In addition, frontier molecular orbital (FMO) theory at the Hartree-Fock level was used to predict sites of peroxide oxidation and electron transfer. Compound 1 was then subjected to autoxidation and H2O2 forced degradation as well as formal stability conditions. Samples were analyzed by LC/UV/MS/MS and degradation products proposed. RESULTS: The computational BDEs and FMO analysis of 1 was consistent with the LC/UV/MS/MS data and allowed for structural proposals, which were confirmed by LC/MS/NMR. The autoxidation conditions yielded a number of degradants not observed under peroxide degradation while formal stability conditions gave both peroxide and autoxidation degradants. CONCLUSIONS: Electronic structure methods were successfully applied in combination with LC/UV/MS/MS to predict degradation pathways and assist in spectral identification. The degradation and excipient stability studies highlight the importance of including both peroxide and autoxidation conditions in forced degradation studies.

Chromatography, High Pressure Liquid↗
PubMed1999

Rice bran lipase: extraction, activity, and stability

A simple procedure for the extraction of the lipolytic activity from rice bran has been developed. Various conditions of extraction have been optimized so as to obtain maximum yield of the lipase. It was found that high enzyme activity could be obtained by first defatting the rice bran to remove the lipid component. This was followed by five cycles of aqueous extraction (potassium phosphate buffer, 50 mM and pH 7, containing 0.5 mM of CaCl(2)). The stability of the rice bran lipase under storage and operative conditions was investigated. Further, the influence of glycerol as a stabilizer has been assessed. It was found that further purification using micro- and ultrafiltration yielded an enzyme preparation with higher activity and specific activity and better stability.

Journal Article↗
PubMed2002

Crystal structures and increased stabilization of the protein G variants with switched folding pathways NuG1 and NuG2.

We recently described two protein G variants (NuG1 and NuG2) with redesigned first hairpins that were almost twice as stable, folded 100-fold faster, and had a switched folding mechanism relative to the wild-type protein. To test the structural accuracy of our design algorithm and to provide insights to the dramatic changes in the kinetics and thermodynamics of folding, we have now determined the crystal structures of NuG1 and NuG2 to 1.8 A and 1.85 A, respectively. We find that they adopt hairpin structures that are closer to the computational models than to wild-type protein G; the RMSD of the NuG1 hairpin to the design model and the wild-type structure are 1.7 A and 5.1 A, respectively. The crystallographic B factor in the redesigned first hairpin of NuG1 is systematically higher than the second hairpin, suggesting that the redesigned region is somewhat less rigid. A second round of structure-based design yielded new variants of NuG1 and NuG2, which are further stabilized by 0.5 kcal/mole and 0.9 kcal/mole.

Crystallography, X-Ray↗
PubMed2001

Pyrrolidyl polyamines: branched, chiral polyamine analogues that stabilize DNA duplexes and triplexes.

[figure: see text] Pyrrolidyl polyamines (III-VI) are conformationally restricted, chiral analogues of linear spermine elaborated by the addition of aminopropyl chains to yield branched diastereomers. It is demonstrated that in concentrations as low as 0.01 mM, these compounds remarkably stabilize DNA duplexes and triplexes through strong electrostatic interactions. The synthesized compounds are potential dendrons with a chiral pyrrolidine core, and such molecules may have potential as DNA delivery and transfection agents.

DNA↗
PubMed1990

Haemoglobin pyridoxalation in phosphate buffer: comparison of results with those obtained with Tris-HCl buffer.

Most workers studying haemoglobin solutions re-establish a P50 close to physiological values by covalent fixation of pyridoxal phosphate (PLP) using the method first described by Benesch. This is performed with deoxyhaemoglobin in a Tris-HCl buffer, considered to be necessary for transimination. To simplify the chemical procedure of the conjugation of a macromolecule to Hb-PLP we have performed the pyridoxalation in a phosphate buffer. Physico-chemical analysis of pyridoxalated haemoglobin solutions show a slight degradation of the protein, a amount of fixed PLP to the haemoglobin and a right-shift of the dissociation curve similar whether the coupling is performed in the phosphate or the Tris buffer. The pharmacological evaluation by total and isovolumic exchange transfusion in rats, of solutions prepared from both methods of pyridoxalation show identical survival times, vascular persistence and stability of the conjugates. Thus the pyridoxalation takes place with a comparable yield whatever reagent is used, proving that Tris is not essential to haemoglobin pyridoxalation.

Animals↗
PubMed1995

Some properties of acid-reassembled tropomyosin.

The preference for parallelism of the two chains in tropomyosin coiled coils is thought to result from interchain salt bridges. To examine this idea, studies are presented of tropomyosin molecules reassembled from chaotropic solvents in acid solution, where cross-links cannot exist. The acid-reassembled molecules are appreciably less disulfide cross-linkable in acid than native molecules, a result explainable if some antiparallel dimers indeed form at low pH. Physical studies (backbone- and tyrosine-region CD and intrinsic viscosity) indicate that refolding in acid yields a molecular population demonstrably different in tyrosine-region CD from native, but having comparable (but not identical) helix content, thermal stability, and dimensions. Moreover, the refolding in acid after either thermal or chaotropic-solvent denaturation yields the same final state, arguing that it is an equilibrium state. All these results are consistent with, but do not prove, that the acid-reassembled population includes an appreciable fraction (2/3) of antiparallel coiled-coil dimers.

Animals↗
PubMed2005

Siderophore-manganese(III) interactions. I. Air-oxidation of manganese(ll) promoted by desferrioxamine B.

Recent studies suggest that aqueous Mn(ll) complexes, particularly those with non-carboxylated ligands such as microbial siderophores, may be stable in soil and aquatic environments. In this paper, we determine the stability constants for Mn(ll) and Mn(lll) complexes with the common trihydroxamate siderophore, desferrioxamine B (DFOB). Base and redox titrations were conducted to determine DFOB conditional protonation constants and conditional stability constants for 1:1 DFOB complexes with Mn(ll) and Mn(lll). The conditional protonation constants agree well with literature values. We determined stability constants for three Mn(ll)-DFOB species and one Mn(lll)-DFOB species at 25 degrees C in 0.1 M NaCl. The Mn(lll) HDFOB+ complex can be formed readily by air-oxidation of Mn(ll)-DFOB. This reaction exhibits pseudo first-order kinetics with a rate coefficient that can be characterized as the product of oxygen concentration with a linear combination of the concentrations of the three Mn(ll)-DFOB complexes. The second-order rate coefficients appearing in this linear combination are 1 to 2 orders of magnitude smaller than that associated with oxidation of the hydrolytic species Mn(OH)(0)2. The Mn(lll)HDFOB+ complex is stable for pH in the range of 7.0-11.3; but, at pH < 7.0 it decomposes by internal electron transfer, yielding oxidized DFOB products and Mn(ll). For p[H+] > 11.3, the complex degrades by disproportionation, yielding Mn(ll) and solid MnO2. This range of pH stability supports the hypothesis that aqueous Mn(lll) may play a vital role in the biogeochemical cycling of not only manganese, but also other elements, such as carbon, sulfur, nitrogen, oxygen, and redox-active metals.

Deferoxamine↗
PubMed2005

Biased mutagenesis in the N-terminal region by degenerate oligonucleotide gene shuffling enhances secretory expression of barley alpha-amylase 2 in yeast.

Recombinant barley alpha-amylase 1 (rAMY1) and 2 (rAMY2), despite 80% sequence identity, are produced in very different amounts of 1.1 and <0.05 mg/l, respectively, by Saccharomyces cerevisiae strain S150-2B. The low yield of AMY2 practically excludes mutational analysis of structure-function relationships and protein engineering. Since different secretion levels of AMY1/AMY2 chimeras were previously ascribed to the N-terminal sequence, AMY1 residues were combinatorially introduced at the 10 non-conserved positions in His14-Gln49 of AMY2 using degenerate oligonucleotide gene shuffling (DOGS) coupled with homologous recombination in S.cerevisiae strain INVSc1. Activity screening of a partial library of 843 clones selected six having a large halo size on starch plates. Three mutants, F21M/Q44H, A42P/A47S and A42P rAMY2, also gave higher activity than wild-type in liquid culture. Only A42P showed wild-type stability and enzymatic properties. The replacement is located to a beta-->alpha loop 2 that interacts with domain B (beta-->alpha loop 3) protruding from the catalytic (beta/alpha)(8)-barrel. Most remarkably Pichia pastoris strain GS115 secreted 60 mg/l A42P compared with 3 mg/l of wild-type rAMY2. The crystal structure of A42P rAMY2 was solved and found to differ marginally from the AMY2 structure, suggesting that the high A42P yield stems from stabilization of the mature and/or intermediate form owing to the introduced proline residue. Moreover, the G to C substitution for the A42P mutation might have a positive impact on protein translation.

Amino Acid Sequence↗
PubMed1975

Purification of the subunits of transcarboxylase by affinity chromatography on avidin-sepharose.

Transcarboxylase consists of a central 12 SH subunits each of which is linked to the central subunit by two similar to 1.3 SE biotin carboxyl carrier proteins. The subunits from dissociated transcarboxylase have been difficult to isolate because conditions which stabilize them also promote their reassociation to the intact enzyme. In this paper, we describe the use of avidin-Sepharose to adsorb the enzyme from crude extracts or partially purified transcarboxylase of propionibacteria. After removing impurities by washing the column with phosphate buffer at pH 6.5, in which the transcarboxylase is stable, the enzyme is dissociated first by elution at pH 8 yielding a fraction containing mostly 12 SH subunit which can be rapidly stabilized against dissociation to 6 SH without the problem of reconstitution because the 1.3 SE and most of the 5 SE subunits are not eluted. The second elution is at pH 9 which yields the 5 SE subunit by dissociation from the 1.3 SE biotin subunit and the 1.3 SE subunit remains bound to the avidin. The 12 SH and 5 SE subunits are further purified by glycerol density gradient centrifugation or by chromatography on Bio-Gel. Very active enzyme can be reconstituted from these subunits upon the addition of the 1.3 SE subunit.

Avidin↗
PubMed2003

Identification of QTL for growth- and grain yield-related traits in rice across nine locations of Asia.

Rice double-haploid (DH) lines of an indica and japonica cross were grown at nine different locations across four countries in Asia. Genotype-by-environment (G x E) interaction analysis for 11 growth- and grain yield-related traits in nine locations was estimated by AMMI analysis. Maximum G x E interaction was exhibited for fertility percentage number of spikelets and grain yield. Plant height was least affected by environment, and the AMMI model explained a total of 76.2% of the interaction effect. Mean environment was computed by averaging the nine environments and subsequently analyzed with other environments to map quantitative trait loci (QTL). QTL controlling the 11 traits were detected by interval analysis using mapmaker/qtl. A threshold LOD of >/=3.20 was used to identify significant QTL. A total of 126 QTL were identified for the 11 traits across nine locations. Thirty-four QTL common in more than one environment were identified on ten chromosomes. A maximum of 44 QTL were detected for panicle length, and the maximum number of common QTL were detected for days to heading detected. A single locus for plant height (RZ730-RG810) had QTL common in all ten environments, confirming AMMI results that QTL for plant height were affected the least by environment, indicating the stability of the trait. Two QTL were detected for grain yield and 19 for thousand-grain weight in all DH lines. The number of QTL per trait per location ranged from zero to four. Clustering of the QTL for different traits at the same marker intervals was observed for plant height, panicle number, panicle length and spikelet number suggesting that pleiotropism and or tight linkage of different traits could be the possible reason for the congruence of several QTL. The many QTL detected by the same marker interval across environments indicate that QTL for most traits are stable and not essentially affected by environmental factors.

Asia↗
PubMed2002

Atomic force microscopy study of the effects of Mg(2+) and other divalent cations on the end-to-end DNA interactions.

Atomic force microscopy (AFM) was applied to directly visualize the end-to-end DNA interaction mediated by magnesium cations. We took advantage of the APS-mica, allowing the preparation of samples in a broad range of monovalent and divalent cations to separate the effects of Mg(2+) and Na(+) cations on the interaction of restriction DNA fragments with cohesive end. The AFM data clearly show that DNA restriction fragments with cohesive ends form substantial amount of circles in the presence of Mg(2+) cations, suggesting that Mg(2+) cations stabilize the interaction of cohesive ends. This effect depends on the MgCl(2) concentration, so that the yield of circles approaches 18% in the presence of 50 mM MgCl(2). Furthermore, we demonstrate that this conferred cohesive end stability is specific for divalent cations, as substitution of MgCl(2) with NaCl leads to a near complete loss of cohesive end stability. We further demonstrate that cohesive end stabilization is achieved by substituting Mg(2+) with Ca(2+), Mn(2+), or Zn(2+). The data obtained suggest that the end stabilization mediated by divalent cations is primarily the result of inter-base interactions rather than bridging of phosphate moieties.

Cations, Divalent↗
PubMed2002

Optimizing the heterologous production of epothilone D in Myxococcus xanthus.

The heterologous production of epothilone D in Myxococcus xanthus was improved by 140-fold from an initial titer of 0.16 mg/L with the incorporation of an adsorber resin, the identification of a suitable carbon source, and the implementation of a fed-batch process. To reduce the degradation of epothilone D in the basal medium, XAD-16 (20 g/L) was added to stabilize the secreted product. This greatly facilitated its recovery and enhanced the yield by three-fold. The potential of using oils as a carbon source for cell growth and product formation was also evaluated. From a screen of various oils, methyl oleate was shown to have the greatest impact. At the optimal concentration of 7 mL/L in a batch process, the maximum cell density was increased from 0.4 g dry cell weight (DCW)/L to 2 g DCW/L. Product yield, however, depended on the presence of trace elements in the production medium. With an exogenous supplement of trace metals to the basal medium, the peak epothilone D titer was enhanced eight-fold. This finding demonstrates the significant role of metal ions in cell metabolism and in epothilone biosynthesis. To further increase the product yield, a continuous fed-batch process was used to promote a higher cell density and to maintain an extended production period. The optimized fed-batch cultures consistently yielded a cell density of 7 g DCW/L and an average production titer of 23 mg/L.

Drug Stability↗
PubMed2005

Impact of carbon and nitrogen nutrition on the quality, yield and composition of blastospores of the bioinsecticidal fungus Paecilomyces fumosoroseus.

The impact of growing cultures of Paecilomyces fumosoroseus in liquid media containing four combinations of glucose and casamino acids (8 g l(-1) or 80 g l(-1) glucose, 1.32 g l(-1) or 13.2 g l(-1) casamino acids) was evaluated, based on blastospore production, germination rate, viability after freeze-drying and short-term storage stability. When blastospores were produced using a high casamino acid concentration, blastospore yields and germination rates were significantly higher (13.2-18.5 x 10(7) blastospores ml(-1), 50-60% germination after 4 h), compared to cultures grown in media containing lower casamino acid concentrations (0.4-2.3 x 10(7) blastospores ml(-1), 10-20% germination after 4 h). Chemical analyses of blastospore composition showed that accelerated blastospore germination may be related to increased proteinaceous reserves rather than to glycogen or lipid accumulation. Tolerance to freeze-drying by blastospores suspended in spent medium was enhanced by a high initial casamino acid concentration in the culture medium (75% survival) and by the residual glucose concentrations in the spent medium. Under the conditions of this study, the storage stability of blastospores of P. fumosoroseus was unaffected by the nutritional condition in which they were produced.

Amino Acids↗