Abstract
The Fv fragment of the 0.5beta monoclonal antibody has recently been constructed, expressed, and purified. It binds with nanomolar affinity to the immunogenic RP135 peptide that is derived from the principal neutralizing determinant of HIV-1 in the third hypervariable region of gp120. Here, we analyzed the temperature-dependence of binding of the 0.5beta Fv fragment to the RP135 peptide and a series of mutants thereof. Our results show that there is almost complete enthalpy-entropy compensation in the effects of mutations in the peptide on binding to the Fv, indicating that the mutations do not change the binding mechanism. There is good correlation, for residues within the antigenic epitope, between mutational effects on DeltaCp and calculated values of DeltaDeltaCp based on the extent of burial of polar and non-polar surface areas of amino acids. The value of DeltaCp for the binding of the 0.5beta Fv fragment to the wild-type RP135 peptide is found to be -5.0 (+/- 0.9) kcal K-1 mol-1 in the presence of 0.1% Tween-20 but only -0.1 (+/- 0.9) kcal K-1 mol-1 in its absence. This result has important implications for the successful application of the structural parameterization approach to predicting changes in heat capacity that accompany binding reactions carried out in the presence of detergent or protein-stabilizing agents.
Highlights
human immunodeficiency virus type 1 (HIV-1) found in the third hypervariable region of gp120
The thermodynamic parameters of the interaction between the 0.5 Fv fragment and the RP135 peptide at different temperatures between 10 and 35 °C were determined in the presence of different concentrations of Tween-20 by monitoring fluorescence enhancement upon binding
Heat capacity changes associated with peptide binding are usually in the range of Ϫ0.1 to Ϫ0.5 kcal molϪ1 KϪ1 some exceptions have been reported
Summary
Materials—Expression and purification of the 0.5 Fv fragment was achieved as before [6]. Interaction between the 0.5 Fv Fragment and RP135 Peptide obtained from Chembridge Corp., Illinois. Fluorescence Enhancement Titrations—Fluorescence measurements of the 0.5 Fv fragment in the presence of different peptide concentrations were made as described previously [6]. All the reactions were carried out in 50 mM sodium phosphate buffer (pH 7.5) containing different fixed amounts of the non-ionic detergent Tween-20. The titrations were carried out at least in duplicate over a temperature range from 10 to 35 °C (Ϯ 0.1 °C) in a thermostatted cuvette. The concentration of the Fv fragment was 26 M in 50 mM phosphate buffer (pH 7.5) without or with 0.05% Tween-20. Data analysis—Determination of dissociation constants (Kd) between the Fv fragment and the different peptides from the fluorescence enhancement titrations was achieved as described previously [6]. The data were fitted using Kaleidagraph (Version 2.1, Synergy Software (PCS Inc.)) to the following equation,
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