An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol

Background: Mutagenesis plays an essential role in molecular biology and biochemistry. It has also been used in enzymology and protein science to generate proteins which are more tractable for biophysical techniques. The ability to quickly and specifically mutate a residue(s) in protein is important for mechanistic and functional studies. Although many site-directed mutagenesis methods have been developed, a simple, quick and multi-applicable method is still desirable. Results: We have developed a site-directed plasmid mutagenesis protocol that preserved the simple one step procedure of the QuikChange (TM) site-directed mutagenesis but enhanced its efficiency and extended its capability for multi-site mutagenesis. This modified protocol used a new primer design that promoted primer-template annealing by eliminating primer dimerization and also permitted the newly synthesized DNA to be used as the template in subsequent amplification cycles. These two factors we believe are the main reasons for the enhanced amplification efficiency and for its applications in multi-site mutagenesis. Conclusion: Our modified protocol significantly increased the efficiency of single mutation and also allowed facile large single insertions, deletions/truncations and multiple mutations in a single experiment, an option incompatible with the standard QuikChange (TM). Furthermore the new protocol required significantly less parental DNA which facilitated the DpnI digestion after the PCR amplification and enhanced the overall efficiency and reliability. Using our protocol, we generated single site, multiple single-site mutations and a combined insertion/deletion mutations. The results demonstrated that this new protocol imposed no additional reagent costs (beyond basic QuikChange T) but increased the overall success rates.Publisher PDFPeer reviewe

Enhanced imaging of lipid rich nanoparticles embedded in methylcellulose films for transmission electron microscopy using mixtures of heavy metals

This work was supported by funds from the St Andrews EM facility and the University of St Andrews. CP is a Royal Society of Edinburgh (RSE) Personal Research Fellow, funded by the Scottish Government. CP is also supported by a Tenovus Scotland Award (T15/41). JML was supported by a Programme grant from the Wellcome Trust (Number 045404).Synthetic and naturally occurring lipid-rich nanoparticles are of wide ranging importance in biomedicine. They include liposomes, bicelles, nanodiscs, exosomes and virus particles. The quantitative study of these particles requires methods for high-resolution visualization of the whole population. One powerful imaging method is cryo-EM of vitrified samples, but this is technically demanding, requires specialized equipment, provides low contrast and does not reveal all particles present in a population. Another approach is classical negative stain-EM, which is more accessible but is difficult to standardize for larger lipidic structures, which are prone to artifacts of structure collapse and contrast variability. A third method uses embedment in methylcellulose films containing uranyl acetate as a contrasting agent. Methylcellulose embedment has been widely used for contrasting and supporting cryosections but only sporadically for visualizing lipid rich vesicular structures such as endosomes and exosomes. Here we present a simple methylcellulose-based method for routine and comprehensive visualization of synthetic lipid rich nanoparticles preparations, such as liposomes, bicelles and nanodiscs. It combines a novel double-staining mixture of uranyl acetate (UA) and tungsten-based electron stains (namely phosphotungstic acid (PTA) or sodium silicotungstate (STA)) with methylcellulose embedment. While the methylcellulose supports the delicate lipid structures during drying, the addition of PTA or STA to UA provides significant enhancement in lipid structure display and contrast as compared to UA alone. This double staining method should aid routine structural evaluation and quantification of lipid rich nanoparticles structures.Publisher PDFPeer reviewe

Pironetin reacts covalently with cysteine-316 of α-tubulin to destabilize microtubule

This work was funded by grants to C.L. from the National Natural Science Foundation of China (81373283 and U1402222). J.H.N. is supported as an award holder of Chinese National Thousand Talents Program, Wellcome Trust Senior Investigator Award (WT100209MA) and Royal Society Wolfson Merit Award. X.Z. is supported by Sichuan Province Thousand Talents Scheme in China and the State Key Program of National Natural Science of China (21534008).Molecules which alter the normal dynamics of microtubule assembly and disassembly include many anticancer drugs in clinical use. So far all such therapeutics target β-tubulin and structural biology has explained the basis of their action and permitted design of new drugs. However by shifting the profile of β-tubulin isoforms, cancer cells become resistant to treatment. Compounds that bind to α-tubulin are less well characterized and unexploited. The natural product pironetin is known to bind to α-tubulin and is a potent inhibitor of microtubule polymerization. Previous reports had identified that pironetin reacts with lysine-352 residue however analogues designed on this model had much lower potency which was difficult to explain, hindering further development. We report crystallographic and mass spectrometric data that reveal that pironetin forms a covalent bond to cysteine-316 in α-tubulin via a Michael addition reaction. These data provide a basis for the rational design of α-tubulin targeting chemotherapeutics.Publisher PDFPeer reviewe

UNE SCULPTURE D\u27IVAN DUKNOVlĆ

L\u27auteur clecrit la sculpture en pierre representant Saint Jean I\u27Evangeliste — I\u27un des chefs cl\u27oeuvre de I\u27artiste, bien connu, c le la Renaissance: Ivan Duknović ( lohannes Dalmata) qui se trouve clans une chapelle cle la cathedrale cle la ville croate de Tlogl I . Dans la grande exposition cl\u27Art yougoslave qui s\u27est tenue a Paris en 1971 cette statue a et e exposee; a cette occasion, et pour la premiere fois, en ont ćte photographiee les cotes, le dos et la base sur lacluelle est gravee la signature du sculpteur, que I\u27auteur cle cet ar ticle avait cleja remarquee et publiće. Grace a ces cliches cles cleux profils et cl u clos, cette oeuvre a r evele I\u27habilete cl\u27Ivan Duknović qui, en plein volume, moclele un personnage humain grancleur naturelle, de merne qu\u27i l I \u27avait fai t lors de I\u27execution du peti t »put to« a i l ć clui a et e recemment trouvć a Trogir, l icu cle naissance de I\u27artiste, et qui a et e public clans le dernier numero cle cette revue

Inferring the chemical mechanism from structures of enzymes

Chemistry has once again embraced the study of enzyme mechanism as a core discipline. Chemists are uniquely able to contribute to the analysis of enzymes through their understanding of the reactivity of atoms. In this tutorial review for the Corday-Morgan medal, I will concentrate on the work from my lab over the past six years. I discuss enzymes which transform carbohydrates and incorporate halogens. The tutorial review will emphasise the strengths and limitations of structural biology as a means to deducing the chemical mechanism.</p

Chance-Constrained Linear Programming with Chi-Square Type Deviates

The implications of replacing the assumption of normal distribution of the parameters (A, b, c) by chi-square and other related nonnegative distributions are discussed here in the framework of chance-constrained linear programming.

TNFa and the TNF Receptor Superfamily: Structure-Function Relationship(s)

Tumour Necrosis Factor alpha (TNF alpha), is an inflammatory cytokine produced by macrophages/monocytes during acute inflammation and is responsible for a diverse range of signalling events within cells, leading to necrosis or apoptosis. The protein is also important for resistance to infection and cancers. TNF alpha exerts many of its effects by binding, as a trimer, to either a 55 kDa cell membrane receptor termed TNFR-1 or a 75 kDa cell membrane receptor termed TNFR-2. Both these receptors belong to the so-called TNF receptor superfamily. The superfamily includes FAS, CD40, CD27, and RANK. The defining trait of these receptors is an extra cellular domain comprised of two to six repeats of cysteine rich motifs. Additionally, a number of structurally related "decoy receptors" exist that act to sequester TNF molecules, thereby rescuing cells from apoptosis. The crystal structures of TNF alpha, TNF beta, the extracellular domain of TNFR-1 (denoted sTNFR-1), and the TNF beta sTNFR-1 complex have been defined by crystallography. This article will review the structure/function relationships of the TNF alpha and the TNF receptor superfamily. It will also discuss insights as to how structural features play a role in the pleiotropic effects of TNF alpha. (C) 2000 Wiley-Liss, Inc.</p

Mechanisms of silicate polymerisation, carbohydrate epimerisation and metalloprotease inhibition

In biotechnology and drug delivery silica materials are of interest but the controlled generation of silicic acid is difficult. To get more insight into the molecular mechanisms that control biosilification, it is important to study the proteins involved in this process. The sponge protein silicatein α synthesises part of the axial filament in the spicules which in situ polymerises silicic acid. It has been demonstrated that the polymerisation of siloxanes such as for example tetraethoxysilane (TEOS) can be carried out by both wild type and recombinant silicatein α. Unfortunately, it has not been possible yet to get reasonable amounts of wild type or recombinant silicatein α to perform biophysical studies. The human cysteine protease cathepsin L has almost 50 % identical residues with silicatein α. To get more insight into the mechanism of silica polymerisation, cathepsin L mutants were generated by our collaborators. Those mutants show sequence features and activity specific for silicatein α. The X-ray structure of one of those mutants (mutant 4SER) to 1.5 Å has allowed us to propose a new chemical mechanism for the catalysis of silicic acid polymerisation. ADP-β-D-glycero-D-mannoheptose and ADP-β-L-glycero-D-mannoheptose are interconverted by the SDR-enzyme ADP-β-L-glycero-D-mannoheptose 6-epimerase (AGME). This epimerisation reaction is the final reaction in the biosynthetic route of the precursor of heptose. Heptose is a part of the inner core of the lipopolysaccharide in Gram-negative bacteria. In mutants which do not have heptose, nonpolar compounds can penetrate more easily through the outer membrane. These mutants also show less pathogenicity. As a consequence the lipopolysaccharide biosynthetic pathway represents an interesting target for antimicrobial compounds. The crystal structure of AGME in complex with ADP-α-glucose has already been solved. From this structure a catalytic mechanism for this enzyme has been proposed with Tyr140 and Lys178 operating as acid/base residues. The disordered nature of the nucleotide sugar’s glucose moiety in the previous structure due to the wrong configuration of the sugar has hindered assignment of a mechanism. The determination of the X-ray structure of AGME Y140F in complex with a substrate in the β-manno configuration (ADP-β-mannose) to 2.4 Å resolution has given new insight into the mechanism of this SDR enzyme. A mechanism is proposed with only Tyr140 operating as catalytic acid/base. Initially it was thought that MMP-3 participates in the synovitis cascade. Glycoproteins, several parts of the ECM such as fibronectin and laminin and also collagens and procollagens are targets of this matrixin. Furthermore MMP-3 can undergo autocatalysis and is also able to cleave a range of other members of the matrixin family. Matrixins also play an important role in diseases such as cancer, rheumatoid arthritis and osteoporosis. This makes them targets for inhibitor design. Many structures of matrix metalloproteinases, such as stromelysin-1, in complex with various inhibitors have already been solved. The structures of the catalytic domain of MMP-3 in complex with two nonpeptide inhibitors are discussed.EThOS - Electronic Theses Online ServiceGBUnited Kingdo