Molecular Recognition of a Salmonella Trisaccharide Epitope by Monoclonal Antibody Se155-4

Bundle, David R.; Eichler, Eva; Gidney, M. A. J.; Meldal, Morten; Ragauskas, Arthur J.; Sigurskjold, Bent W.; Sinnott, B.; Watson, David; Yaguchi, Makoto; Young, N. Martin

doi:10.1021/bi00183a022

Cited by 101 publications

(110 citation statements)

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“…Analysis of the ES-MS data using the approach described above leads to an average K a value of (1.4 Ϯ 0.3) ϫ 10 3 M Ϫ1 for the (scFv ϩ 1) complex, Table 1. This value is indistinguishable, within experimental error, from the ITC-derived value of (1.5 Ϯ 0.4) ϫ 10 3 M Ϫ1 [17]. Following the same approach, a K a value of (1.7 Ϯ 0.9) ϫ 10 2 M Ϫ1 was determined for the (scFv ϩ 2) complex, which is also in good agreement with the ITC value of (1.2 Ϯ 0.5) ϫ 10 2 M Ϫ1 [17].…”

Section: Scfv-monosaccharide Bindingmentioning

confidence: 59%

“…This value is indistinguishable, within experimental error, from the ITC-derived value of (1.5 Ϯ 0.4) ϫ 10 3 M Ϫ1 [17]. Following the same approach, a K a value of (1.7 Ϯ 0.9) ϫ 10 2 M Ϫ1 was determined for the (scFv ϩ 2) complex, which is also in good agreement with the ITC value of (1.2 Ϯ 0.5) ϫ 10 2 M Ϫ1 [17]. In contrast, the assay did not detect any binding between the scFv and 3, consistent with the results of ITC measurements [Bundle, D.R., Unpublished results].…”

Section: Scfv-monosaccharide Bindingmentioning

confidence: 59%

“…Association constants have been determined by ITC for the binding of Se155-4 scFv to the monosaccharides 1 (1.5 ϫ 10 3 M Ϫ1 ) and 2 (1.2 ϫ 10 2 M Ϫ1 ) at pH 7 and 25°C [17]. The monosaccharide 3, which was shown by ITC not to bind to the scFv [Bundle, D.R., Unpublished results], served as a negative control.…”

Section: Determination Of Protein-ligand Affinitiesmentioning

confidence: 99%

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Quantifying labile protein—Ligand interactions using electrospray ionization mass spectrometry

El-Hawiet

Kitova

Liu

et al. 2010

J. Am. Soc. Mass Spectrom.

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A new electrospray ionization mass spectrometry (ES-MS) approach for quantifying proteinligand complexes that are prone to in-source (gas-phase) dissociation is described. The method, referred to here as the reference ligand ES-MS method, is based on the direct ES-MS assay and competitive ligand binding. A reference ligand (L ref ), which binds specifically to the protein (P), at the same binding site as the ligand (L) of interest, with known affinity and forms a stable protein-ligand complex in the gas phase, is added to the solution. The fraction of P bound to L ref , which is determined directly from the ES mass spectrum, is sensitive to the fraction of P bound to L in solution and enables the affinity of P for L to be determined. A mathematical framework for the implementation of the method in cases where P has one or two specific ligand binding sites is given. The assay is based on the direct detection and quantification of the abundance (Ab) of ligand-bound and unbound protein ions in the gas phase, e.g., PL nϩ and P nϩ , respectively. A key assumption is that the measured abundance ratio (R) is equivalent to the equilibrium concentration ratio of ligand-bound and free protein in solution, eq 1: The direct ES-MS assay has been used to measure affinities for a range of protein-ligand complexes, including antibody-antigen, lectin-carbohydrate, enzymesubstrate/inhibitor complexes and, in many instances, the K a values agree well with constants obtained by other analytical methods, including isothermal titration calorimetry (ITC), surface plasmon resonance, and frontal affinity chromatography MS [4 -10]. However, there have also been reports of protein-ligand complexes that could not be detected by ES-MS or, if detected, the relative abundance of ligand-bound and unbound protein ions did not match the distribution expected in solution, with less binding observed in the gas phase [11][12][13][14]. These anomalous results are often due the occurrence of in-source dissociation, whereby the gaseous complexes undergo partial or complete dissociation during ES-MS analysis. If the gas-phase PL ions are kinetically labile and undergo dissociation during analysis, the magnitude of the measured R value and, correspondingly, the K a value will be artificially low. In the extreme case, where no PL ions survive, in-source dissociation will result in a false negative. Recently, it was shown that solution or gas-phase additives can, in some instances, protect complexes from in-source dissociation [12,15]. However, this approach does have its limitations and the detection of very labile gas-phase complexes, which rapidly dissociate at ambient temperature, by ES-MS remains problematic. Here, we describe an indirect ES-MS approach to quantify protein-ligand interactions that are highly Address reprint requests to Dr.

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Section: Scfv-monosaccharide Bindingmentioning

confidence: 59%

Section: Scfv-monosaccharide Bindingmentioning

confidence: 59%

Section: Determination Of Protein-ligand Affinitiesmentioning

confidence: 99%

See 1 more Smart Citation

Quantifying labile protein—Ligand interactions using electrospray ionization mass spectrometry

El-Hawiet

Kitova

Liu

et al. 2010

J. Am. Soc. Mass Spectrom.

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“…Inclusion bodies containing ds-scFv were harvested from the bacterial cells and the antibody fragment was extracted, refolded and oxidized by the procedure of Buchner et al [12], except that the oxidation was carried out at 4°C for 3 days. The products were concentrated and the ds-scFv was purified by affinity chromatography on an antigen matrix [6]. The monomer forms of both scFv and ds-scFv were purified by size-exclusion HPLC on Superdex 75 (Pharmacia Biotech).…”

Section: Expression and Purification Of Antibody Fragmentsmentioning

confidence: 99%

“…We have identified another site for introduction of a disulphide bond in the antibody Se 155~,, an IgG121 anticarbohydrate antibody specific for the Salmonella serotype B polysaccharide [6] whose Fab and scFv structures have been determined at high resolution [7,8]. The site is intermediate between those of the above designs.…”

Section: Introductionmentioning

confidence: 99%

Thermal stabilization of a single‐chain Fv antibody fragment by introduction of a disulphide bond

et al. 1995

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A disulphide bond was introduced into a single-chain Fv form of the anticarbohydrate antibody, Se155-4 by replacing Ala-L57 of the light chain and Asp-H106 of the heavy chain with cysteines, by site-directed mutagenesis. To maintain the saltbridge from the latter residue to Arg-H98, Tyr-107 was also altered to Asp. The resulting ds-scFv was shown to retain full antigen-binding activity, by enzyme immunoassay and surface plasmon resonance analysis of binding kinetics. Compared with the parent scFv, the disulphide bonded form was shown to have enhanced thermal stability, by Fourier transform IR spectroscopy. The Tm was raised from 60°C to 69°C. The ds-scFv form thus combines the stable monomeric form of the disulphide form wifh the expression advantages of the scFv.

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Characterization of protein-glycolipid recognition at the membrane bilayer

Evans

MacKenzie

1999

J. Mol. Recognit.

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A growing number of important molecular recognition events are being shown to involve the interactions between proteins and glycolipids. Glycolipids are molecules in which one or more monosaccharides are glycosidically linked to a lipid moiety. The lipid moiety is generally buried in the cell membrane or other bilayer, leaving the oligosaccharide moiety exposed but in close proximity to the bilayer surface. This presents a unique environment for protein-carbohydrate interactions, and studies to determine the influence of the bilayer on these phenomena are in their infancy. One important property of the bilayer is the ability to orient and cluster glycolipid species, as strong interactions in biological systems are often achieved through multivalency arising from the simultaneous association of two or more proteins and receptors. This is especially true of protein-carbohydrate binding because of the unusually low affinities that characterize the monovalent interactions. More recent studies have also shown that the composition of the lipid bilayer is a critical parameter in protein-glycolipid recognition. The fluidity of the bilayer allows for correct geometric positioning of the oligosaccharide head group relative to the binding sites on the protein. In addition, there are activity-based and structural data demonstrating the impact of the bilayer microenvironment on the modulation of oligosaccharide presentation. The use of model membranes in biosensor-based methods has supplied decisive evidence of the importance of the membrane in receptor presentation. These data can be correlated with three-dimensional structural information from X-ray crystallography, NMR, and molecular mechanics to provide insight into specific protein-carbohydrate inter--actions at the bilayer.

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Molecular Recognition of a Salmonella Trisaccharide Epitope by Monoclonal Antibody Se155-4

Cited by 101 publications

References 51 publications

Quantifying labile protein—Ligand interactions using electrospray ionization mass spectrometry

Quantifying labile protein—Ligand interactions using electrospray ionization mass spectrometry

Thermal stabilization of a single‐chain Fv antibody fragment by introduction of a disulphide bond

Characterization of protein-glycolipid recognition at the membrane bilayer

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