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Biomedical subjects

Elaine Stephens

Publications and source records attributed to Elaine Stephens.

10 recordsLinked to original sources
PubMed2004

Understanding noncovalent interactions: ligand binding energy and catalytic efficiency from ligand-induced reductions in motion within receptors and enzymes.

Noncovalent interactions are sometimes treated as additive and this enables useful average binding energies for common interactions in aqueous solution to be derived. However, the additive approach is often not applicable, since noncovalent interactions are often either mutually reinforcing (positively cooperative) or mutually weakening (negatively cooperative). Ligand binding energy is derived (positively cooperative binding) when a ligand reduces motion within a receptor. Similarly, transition-state binding energy is derived in enzyme-catalyzed reactions when the substrate transition state reduces the motions within an enzyme. Ligands and substrates can in this way improve their affinities for these proteins. The further organization occurs with a benefit in bonding (enthalpy) and a limitation in dynamics (cost in entropy), but does not demand the making of new noncovalent interactions, simply the strengthening of existing ones. Negative cooperativity induces converse effects: less efficient packing, a cost in enthalpy, and a benefit in entropy.

Binding Sites↗
PubMed2004

Identification of 4-amino-4-deoxychorismate synthase as the molecular target for the antimicrobial action of (6s)-6-fluoroshikimate.

(6S)-6-Fluoroshikimate has antimicrobial activity. The molecular basis of this effect had not been identified, but there was speculation that (6S)-6-fluoroshikimate is first converted in vivo into 2-fluorochorismate, which then could inhibit 4-amino-4-deoxychorismate synthase (ADCS). 2-Fluorochorismate was prepared from E-fluorophosphoenolpyruvate and erythose-4-phosphate by the sequential reactions of DAHP synthase, dehydroquinate synthase, dehydroquinase, shikimate dehydrogenase, EPSP synthase, and chorismate synthase. Inhibition studies on ADCS showed that it was inhibited rapidly and irreversibly by 2-fluorochorismate. Electrospray mass spectrometry of the inactivated enzyme showed an additional mass of 198 +/- 10 Da. A novel peptide of 1087.6 Da was identified in the HPLC trace for the tryptic digest of 2-fluorochorismate-inactivated ADCS. Sequencing of this peptide by MS/MS showed that the peptide corresponded to residues 272-279 with a modification of 206.1 Da on Lys-274. This observation is particularly exciting in the context of a recent proposal for the catalytic mechanism of ADCS.

Anti-Infective Agents↗
PubMed2004

Order changes within receptor systems upon ligand binding: receptor tightening/oligomerisation and the interpretation of binding parameters.

Recent hydrogen-deuterium exchange experiments have highlighted tightening and loosening of protein structures upon ligand binding, with changes in bonding (DeltaH) and order (DeltaS) which contribute to the overall thermodynamics of ligand binding. Tightening and loosening show that ligand binding respectively stabilises or destabilises the internal structure of the protein, i.e. it shows positive or negative cooperativity between ligand binding and the receptor structure. In the case of membrane-bound receptors, such as G protein-coupled receptors (GPCRs) and ligand gated ion channel receptors (LGICRs), most binding studies have focussed on association/dissociation constants. Where these have been broken down into enthalpic and entropic contributions, the phenomenon of "thermodynamic discrimination" between antagonists and agonists has often been noted; e.g. for a receptor where agonist binding is predominantly enthalpy driven, antagonist binding is predominantly entropy driven and vice versa. These data have not previously been considered in terms of the tightening, or loosening, of receptor structures that respectively occurs upon positively, or negatively, cooperative binding of ligand. Nor have they been considered in light of the homo- and hetero-oligomerisation of GPCRs and the possibility of ligand-induced changes in oligomerisation. Here, we argue that analysis of the DeltaH and DeltaS of ligand binding may give useful information on ligand-induced changes in membrane-bound receptor oligomers, relevant to the differing effects of agonists and antagonists.

Biopolymers↗
PubMed2004

A PNA4 quadruplex.

A tetrameric PNA, TGGG, has been shown to form an intermolecular G-quadruplex. Nanoelectrospray mass spectrometry, combined with solution-phase H/D exchange, established formation of a specific tetramolecular complex. UV melting studies show that this complex undergoes a quadruplex melting transition. This is a novel four-stranded structure that offers the gross structural features of a DNA quadruplex, but without the negatively charged backbone.

Chemical Phenomena↗
PubMed2004

Fragmentation characteristics of neutral N-linked glycans using a MALDI-TOF/TOF tandem mass spectrometer.

The fragmentation characteristics of native and permethylated oligosaccharides using a matrix-assisted laser desorption/ionization (MALDI) time-of-flight/time-of-flight tandem mass spectrometer are described. The use of two MALDI matrixes, alpha-cyano-4-hydroxycinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB), is shown to control the nature and extent of fragmentation observed in collision-induced dissociation experiments on synthetic oligosaccharides. CHCA promotes the occurrence of glycosidic cleavages, whereas DHB promotes a wide range of fragmentations. These latter fragmentations include glycosidic cleavages, cross-ring cleavages, and the formation of "internal" cleavage ions, which are derived from elimination of substituents from around the pyranose ring. This extensive fragmentation is shown to facilitate the detailed structural characterization of high-mannose and hybrid-type N-glycans purified from avidin. Importantly, the cross-ring fragments reveal linkage information, unambiguously define antennae substitutions, and differentiate isomeric glycoforms.

Avidin↗
PubMed2004

The N-linked oligosaccharides of aminopeptidase N from Manduca sexta: site localization and identification of novel N-glycan structures.

Mass spectrometric studies on the N-linked glycans of aminopeptidase 1 from Manduca sexta have revealed unusual structures not previously observed on any insect glycoprotein. Structure elucidation of these oligosaccharides was carried out by high-energy collision-induced dissociation (CID) using a matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) tandem mass spectrometer. These key experiments revealed that three out of the four N-linked glycosylation sites in this protein (Asn295, Asn623 and Asn752) are occupied with highly fucosylated N-glycans that possess unusual difucosylated cores. Cross-ring fragment ions and 'internal' fragment ions observed in the CID spectra, showed that these fucoses are found at the 3-position of proximal GlcNAc and at the 3-position of distal GlcNAc in the chitobiose unit. The latter substitution has only been previously observed in nematodes. In addition, these core structures can be decorated with novel fucosylated antennae composed of Fucalpha(1-3)GlcNAc. Key fragment ions revealed that these antennae are predominantly found on the upper 6-arm of the core mannose. The paucimannosidic N-glycan (Man(3)GlcNAc(2)), commonly found on other insect glycoproteins, is the predominant oligosaccharide found at the remaining N-glycosylation site (Asn609).

Amino Acid Sequence↗
PubMed2003

How can enzymes be so efficient?

Dudley Williams and his colleagues discuss how ligands can gain binding energy to their receptors, and substrate transition states to their enzymes, by tightening the protein structures, with a decrease in their dynamic behaviour.

Binding Sites↗
PubMed2003

Ligand binding energy and catalytic efficiency from improved packing within receptors and enzymes.

Some small molecules bind to their receptors, and transition states to enzymes, so strongly as to defy current understanding. We show that in the binding of biotin to streptavidin, the streptavidin structure becomes better packed. We conclude that this contraction of the streptavidin structure promotes biotin binding. The improved packing is associated with positively cooperative binding, occurring with a benefit in enthalpy and a cost in entropy. Evidence indicating that catalytic efficiency can also originate via improved packing in some enzyme transition states, derived from the work of others, is presented. Negatively cooperative ligand binding is concluded to induce converse effects (less efficient packing, a cost in enthalpy, and a benefit in entropy). It applies to the binding of O(2) to haemoglobin, which indeed occurs with a hitherto unreported loosening of the amide backbones of the haemoglobin monomers.

Animals↗