Determination of the binding constants for three HPr-specific monoclonal antibodies and their Fab fragments.
Jel42, Jel44 and Jel323 are mouse monoclonal antibodies specific for HPr, the histidine-containing phosphocarrier protein, of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system. The binding constants, Kd, of the three antibodies and their Fab fragments have been determined: Jel42, 2.8+/-1.6 nM; Jel42Fab, 3. 7+/-0.3 nM; Jel44, 5.1+/-0.4 nM; Jel44Fab, 6.3+/-1.1 nM; Jel323, 5. 7+/-0.5 nM; Jel323Fab, 5.1+/-0.9 nM. The binding constants were determined by a fluorescence polarization assay that used the mutants Arg17Cys HPr and Phe2Cys HPr specifically labeled with fluorescein-5-maleimide. The latter was used for Jel323 as interaction with fluorescein-5-maleimide-labeled Arg17Cys HPr gave quenching of the fluorescence intensity. The specificity of each antibody and the Fab fragments for binding to many HPr mutants was determined by this solution assay. The Fab fragments had the same specificity or cross-reactivity as the antibodies. Comparison of relative binding specificity determined by a solid phase assay showed that the results from both types of assay are comparable. Neither Jel42 nor Jel323 binding was affected by ionic strength (approximately 45 to 245 mM salt), but Jel44 varied about two- to threefold. Charged residues are prominent in the Jel44 epitope and paratope. Initial thermodynamic characterization was investigated by temperature-dependent determinations of the Kd. The binding of Jel42 and Jel323 to HPr was entropic at low temperatures and enthalpic at physiological temperatures. Jel44 showed no change in the contributions of entropy and enthalpy over the temperature range 3 to 37 degreesC. The 2.5 A resolution structure of the complex of Jel42 Fab fragment bound to HPr described in the accompanying paper provides some structural intepretation for the mutational effects.
Department of Biochemistry Health Science Building, University of Saskatchewan, 107 Wiggins Road, Saskatoon, Saskatchewan, S7N 5E5, Canada.
J E
JE
Smallshaw
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S
Brokx
J S
JS
Lee
E B
EB
Waygood
D000911:Antibodies, Monoclonal / Q000737:chemistry*; D000915:Antibody Affinity; D000918:Antibody Specificity; D001426:Bacterial Proteins; D019277:Entropy; D000939:Epitopes; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D009994:Osmolar Concentration; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000235:genetics / Q000276:immunology*; D013816:Thermodynamics
D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D000939:Epitopes; D007140:Immunoglobulin Fab Fragments; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C030182:phosphocarrier protein HPr