PubMed Health⌕ Search

Biomedical subjects

Dudley H Williams

Publications and source records attributed to Dudley H Williams.

15 recordsLinked to original sources
PubMed2005

Ligand binding energy and enzyme efficiency from reductions in protein dynamics.

Tetrameric rabbit muscle glyceraldehyde 3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) binds successively four molecules of its cofactor (NAD+) with affinities of ca 10(11) M(-1), 10(9) M(-1), 10(7) M(-1), and 10(5) M(-1). The reduction in the dynamics of the protein is greatest upon binding the first NAD+ molecule. Smaller reductions then occur upon binding the second and third NAD+ molecules, and the fourth NAD+ molecule binds without dynamic change. Reduction of the GAPDH dynamics, with consequent improvements in its internal bonding, can account for the increase in NAD+ binding affinity from 10(5) M(-1) to 10(11) M(-1). Evidence is provided that comparable fractions of the binding energy of other ligands, and of the catalytic efficiency of enzymes, may be derived in the same way.

Animals↗
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

Importance of structural tightening, as opposed to partially bound States, in the determination of chemical shift changes at noncovalently bonded interfaces.

Two models (A and B) have been proposed to account for decreased downfield chemical shifts of a proton bound by noncovalent interactions at a ligand/antibiotic interface as the number of ligand/antibiotic interactions is decreased. In model A, the proton involved in the noncovalent bond suffers a smaller downfield shift because the bond is, with a relatively large probability, broken, and not because it is longer. In model B, the proton involved in the noncovalent bond suffers a smaller downfield shift because the bond is longer, and not because it is, with a relatively large probability, broken. We show that model A cannot account for the chemical shift changes. Model B accounts for the process of positively cooperative binding, in which noncovalent bonds are reduced in length and thereby increase the stability of the organized state.

Binding Sites↗
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

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

Noncovalent interactions: defining cooperativity. Ligand binding aided by reduced dynamic behavior of receptors. Binding of bacterial cell wall analogues to ristocetin A.

Changes in the relative populations of the monomer and asymmetric dimer forms of ristocetin A, upon binding of two molecules of ligand, suggest that ligand binding is negatively cooperative with respect to dimerization. However, strong hydrogen bonds formed in the binding sites of the ligands are reinforced in the dimer relative to the monomer, and the barrier to dissociation of the dimer is increased upon binding of the ligands. It is concluded that the interactions which are common in the binding of both ligands are made with positive cooperativity with respect to those involved in dimerization. The conclusions are relevant to the binding of ligands to proteins, where ligand binding energy can be derived from stabilization of the protein in its ligand-bound form.

Bacteria↗
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↗
PubMed2003

Kinetic barriers and ordering of non-covalently bound states.

Binding of a dimer of a glycopeptide antibiotic to two molecules of a ligand that are bound to a membrane surface (by a hydrocarbon anchor) has been investigated. This binding on a surface is cooperatively enhanced (surface enhancement) relative to the binding in solution, because the former occurs intramolecularly on a template. Previously a correlation between surface enhancement and thermodynamic stability of the dimer in free solution (Kdimsol) was hypothesised. However, we found that two weakly dimerising antibiotics (vancomycin and ristocetin A) with similar Kdimsol give very different surface enhancements. We propose a model to explain the data correlating surface enhancement to the kinetic barrier to dissociation of the dimer. The surface enhancement of binding can be expected to increase with increasing tightness of the non-covalent interactions formed at the dimer interface. The effect should be found in general where cooperativity is exercised within an organised template (e.g., DNA duplexes and proteins).

Anti-Bacterial Agents↗
PubMed2002

Changes in motion vs. bonding in positively vs. negatively cooperative interactions.

From a consideration of the interactions between non-covalent bonds, it is concluded that positively cooperative binding will occur with a benefit in enthalpy and a cost in entropy, and that negatively cooperative binding will occur with a cost in enthalpy and a benefit in entropy; experimental data support these conclusions.

Journal Article↗
PubMed2002

Cooperative binding interactions of glycopeptide antibiotics.

Glycopeptide antibiotics of the vancomycin group bind to bacterial cell wall analogue precursors, and typically also form dimers. We have studied the interplay between these two sets of noncovalent bonds formed at separate interfaces. Indole-2-carboxylic acid (L) forms a set of hydrogen bonds to the glycopeptide antibiotic chloroeremomycin (CE) that are analogous to those formed by N-Ac-D-Ala. The ligand/CE dimer interactions (in L/CE/CE/L) are shown to occur with positive cooperativity and structural tightening at the dimer interface. From theoretical considerations and from other data, it is inferred, but not proven, that in the exercise of positive cooperativity, the interface that will be tightened to the greatest degree is the one that lies in the shallowest free energy well.

Anti-Bacterial Agents↗
PubMed2002

Mechanism of the regulation of type IB phosphoinositide 3OH-kinase byG-protein betagamma subunits.

Type IB phosphoinositide 3OH-kinase (PI3K) is activated by G-protein betagamma subunits (Gbetagammas). The enzyme is soluble and largely cytosolic in vivo. Its substrate, PtdIns(4,5)P(2), and the Gbetagammas are localized at the plasma membrane. We have addressed the mechanism by which Gbetagammas regulate the PI3K using an in vitro approach. We used sedimentation assays and surface plasmon resonance to determine association of type IB PI3K with lipid monolayers and vesicles of varying compositions, some of which had Gbetagammas incorporated. Association and dissociation rate constants were determined. Our results indicated that in an assay situation in vitro the majority of PI3K will be associated with lipid vesicles, irrespective of the presence or absence of Gbetagammas. In line with this, a constitutively active membrane-targeted PI3K construct could still be activated substantially by Gbetagammas in vitro. We conclude that Gbetagammas activate type IB PI3K by a mechanism other than translocation to the plasma membrane.

Animals↗