Dudley H. Williams

Surface plasmon resonance analysis at a supported lipid monolayer

Methods for the formation of supported lipid monolayers on top of a hydrophobic self assembled monolayer in a surface plasmon resonance instrument are described. Small unilamellar vesicles absorb spontaneously to the surface of the hydrophobic self-assembled monolayer to form a surface which resembles the surface of a cellular membrane. Lipophilic ligands, such as small acylated peptides or glycosylphosphatidylinositol-anchored proteins, were inserted into the absorbed lipid and binding of analytes to these ligands was analysed by surface plasmon resonance. Conditions for the formation of lipid monolayers have been optimised with respect to lipid type, chemical and buffer compatibility, ligand stability and reproducibility.

Binding of glycopeptide antibiotics to a model of a vancomycin-resistant bacterium.

BACKGROUNDnThe vancomycin group of glycopeptide antibiotics is active against a wide range of gram-positive bacteria. The increasing resistance to vancomycin is the result of a change of an amide linkage (D-Ala-D-Ala) to an ester linkage (D-Ala-D-Lactate) in the bacterial cell-wall precursors.nnnRESULTSnWe have used a peptide terminating in the sequence -Lys-D-Ala-D-Lactate linked by its amino terminus to a docosanoyl (C22) acyl chain and anchored in a supported lipid monolayer to mimic the surface of vancomycin-resistant enterococci. Surface plasmon resonance analysis was then used to investigate the binding of glycopeptide group antibiotics to this surface. Vancomycin, which dimerises weakly, bound with low affinity, whereas strongly dimerising antibiotics, such as chloroeremomycin, bound with higher affinities. Antibiotics that have attached hydrophobic groups, such as teicoplanin and biphenylchloroeremomycin (LY307599), bound to the lipid monolayer. This resulted in an enhanced affinity for the lipid-anchored peptide at the surface relative to affinities for an analogous non-anchored peptide in solution.nnnCONCLUSIONSnWe have shown that the affinities of glycopeptide antibiotics for a model of the surface of a vancomycin-resistant bacterium are enhanced relative to affinities determined in free solution. We have also shown that antibiotics that have membrane anchors bind tightly to the model surface and that this feature is an important determinant of the ability of an antibiotic to kill vancomycin-resistant enterococci.

Depolarization after resonance energy transfer (DARET): A sensitive fluorescence-based assay for botulinum neurotoxin protease activity

The DARET (depolarization after resonance energy transfer) assay is a coupled Förster resonance energy transfer (FRET)-fluorescence polarization assay for botulinum neurotoxin type A or E (BoNT/A or BoNT/E) proteolytic activity that relies on a fully recombinant substrate. The substrate consists of blue fluorescent protein (BFP) and green fluorescent protein (GFP) flanking SNAP-25 (synaptosome-associated protein of 25 kDa) residues 134-206. In this assay, the substrate is excited with polarized light at 387 nm, which primarily excites the BFP, whereas emission from the GFP is monitored at 509 nm. Energy transfer from the BFP to the GFP in the intact substrate results in a substantial depolarization of the GFP emission. The energy transfer is eliminated when the fluorescent domains separate on cleavage by the endopeptidase, and emission from the directly excited GFP product fragment is then highly polarized, resulting in an overall increase in polarization. This increase in polarization can be monitored to assay the proteolytic activity of BoNT/A and BoNT/E in real time. It allows determination of the turnover rate of the substrate and the kinetic constants (V(max) and k(cat)) based on the concentration of cleaved substrate determined directly from the measurements using the additivity properties of polarization. The assay is amenable to high-throughput applications.

A mass spectrometric assay for novel peptides: Application to Xenopus laevis skin secretions

The peptides secreted by the South African frog Xenopus laevis were screened systematically using a strategy based on fast atom bombardment mass spectrometry (FAB-MS). High performance liquid chromatography (HPLC) of crude and Sephadex G-10 chromatographed secretions showed that many more peptides were present in these secretions than those previously identified, i.e., xenopsin, caerulein, TRH and PGLa. Fractions from the HPLC were analyzed directly by FAB-MS to determine the molecular weights of these novel peptides. Subsequent analyses, using a combination of FAB-MS, manual Edman degradation, enzymatic digestions and amino acid analyses, identified the partial and sometimes complete sequences of these peptides which had molecular weights ranging from 700-2,700. Many peptides with structural features that are often indicative of biological activity, e.g., C-terminal amides and pyroglutamic acid, were readily identified by FAB-MS. In some cases, molecular weight data combined with partial sequence data was sufficient to identify peptides as originating from PGLa and the spacer regions in the precursors to xenopsin and caerulein.

Amino acid sequence around the active serine in the acyl transferase domain of rabbit mammary fatty acid synthase

Rabbit mammary fatty acid synthase was labelled in the acyl transferase domain(s) by the formation of the O‐ester intermediates after incubation with [14C]acetyl‐ or malonyl‐CoA. Elastase peptides containing the labelled acyl groups were isolated using high performance liquid chromatography and sequenced by fast atom bombardment mass spectrometry. An identical peptide (acyl‐Ser‐Leu‐Gly‐Glu‐Val‐Ala) was obtained after labelling with acetyl‐ or malonyl‐CoA. This confirms the hypothesis that, unlike Escherichia coli or yeast, a single transferase catalyses the transfer of both acetyl‐ and malonyl‐groups in the mammalian complex. The sequence at this site is compared with that around the active serine in other acyl transferases and hydrolases.

Biosynthesis of the vancomycin group of antibiotics: characterisation of a type III polyketide synthase in the pathway to (S)-3,5-dihydroxyphenylglycine

3,5-dihydroxyphenylacetate, a precursor for the non-proteinogenic amino acid 3,5-dihydroxyphenylglycine occurring in glycopeptide antibiotics, is determined to be catalysed by a type III polyketide synthase using malonyl-CoA as a starter unit.

Characterization of Förster resonance energy transfer in a botulinum neurotoxin protease assay

Our previous article described a fluorescence-based assay for monitoring the proteolytic activity of botulinum neurotoxin types A and E (BoNT/A and BoNT/E). As detailed in that article, the assay is based on depolarization due to Förster resonance energy transfer between blue fluorescent protein (BFP) and green fluorescent protein (GFP) moieties linked via residues 134-206 of SNAP-25 (synaptosome-associated protein of 25kDa), the protein substrate for BoNT/A and BoNT/E. Before cleavage of this recombinant substrate, the polarization observed for the GFP emission, excited near the absorption maximum of the BFP, is very low due to depolarization following energy transfer from BFP to GFP. After substrate cleavage and diffusion of the fluorescent proteins beyond the energy transfer distance, the polarization is high due to observation of the emission only from directly excited GFP. This change in fluorescence polarization allows an assay, termed DARET (depolarization after resonance energy transfer), that is robust and sensitive. In this article, we characterize the spectroscopic parameters of the system before and after substrate cleavage, including excitation and emission spectra, polarizations, and lifetimes.

Structural elucidation of N-terminal post-translational modifications by mass spectrometry: application to chicken enolase and the α- and β-subunits of bovine mitochondrial F1-ATPase

Peptides generated from enzymatic hydrolysis of chicken enolase and the alpha- and beta-subunits of bovine F1-ATPase were analyzed by mass spectrometry to determine the nature of their modified N-termini. In the case of chicken enolase, a peptide was isolated from a Staphylococcus aureus proteinase digest by HPLC and analyzed directly by fast atom bombardment mass spectrometry (FABMS). In conjunction with mass spectral evidence obtained from the methyl ester derivative and a secondary tryptic peptide, a structure is proposed containing an N-acetyl serine at the N-terminus. The alpha-subunit of bovine mitochondrial ATPase was chromatographed by HPLC after S. aureus proteinase digestion and a single peak was analyzed on the basis of predicted retention times. A Mr 716 was determined by FABMS and pyrrolidone carboxylic acid was deduced on the basis of its amino acid composition and partial Edman sequence data. The beta-subunit of ATPase produced a series of closely eluting peaks on HPLC after limited digestion with trypsin of the alpha 2 beta 2 complex. These peptides were analyzed by both Edman degradation and FABMS. These data showed the N-terminus to be frayed with N-terminal sequences beginning in pyro-Glu-Ala-Ser, Gln-Ala-Ser, Glu-Ala-Ser, Ala-Ser, and Ser but with no N-acetyl-Ser as was previously thought.

Structure of the antibiotic ristocetin A

Chemical and n.m.r. experiments, which define the last details of the structure of ristocetin A, are described.

Identification of the C-terminus of rabbit skeletal muscle glycogen synthase

The primary structure of a tryptic peptide containing one of the phosphorylation sites on rabbit skeletal muscle glycogen synthase (site 1b) has been redetermined and shown to correspond to the C-terminus of the protein. The sequence is: -SNSVDTSSLSTPSEPLSSAPSLGEERN.