Browsing by Author "Smith-Gill, Sandra J."

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Association energetics of cross-reactive and specific antibodies
(Biochemistry, 2010-02) Mohan, S.; Kourentzi, Katerina D.; Schick, Kari A.; Uehara, Christian; Lipschultz, Claudia A.; Acchione, Mauro; DeSantis, Morgan E.; Smith-Gill, Sandra J.; Willson, Richard C.
HyHEL-8, HyHEL-10, and HyHEL-26 (HH8, HH10, and HH26, respectively) are murine monoclonal IgG1 antibodies which share over 90% variable-region amino acid sequence identity and recognize identical structurally characterized epitopes on hen egg white lysozyme (HEL). Previous immunochemical and surface plasmon resonance-based studies have shown that these antibodies differ widely in their tolerance of mutations in the epitope. While HH8 is the most cross-reactive, HH26 is rigidified by a more extensive network of intramolecular salt links and is highly specific, with both association and dissociation rates strongly affected by epitope mutations. HH10 is of intermediate specificity, and epitope mutations produce changes primarily in the dissociation rate. Calorimetric characterization of the association energetics of these three antibodies with the native antigen HEL and with Japanese quail egg white lysozyme (JQL), a naturally occurring avian variant, shows that the energetics of interaction correlate with cross-reactivity and specificity. These results suggest that the greater cross-reactivity of HH8 may be mediated by a combination of conformational flexibility and less specific intermolecular interactions. Thermodynamic calculations suggest that upon association HH8 incurs the largest configurational entropic penalty and also the smallest loss of enthalpic driving force with variant antigen. Much smaller structural perturbations are expected in the formation of the less flexible HH26 complex, and the large loss of enthalpic driving force observed with variant antigen reflects its specificity. The observed thermodynamic parameters correlate well with the observed functional behavior of the antibodies and illustrate fundamental differences in thermodynamic characteristics between cross-reactive and specific molecular recognition.
Salt Links Dominate Affinity of Antibody HyHEL-5 for Lysozyme through Enthalpic Contributions*
(Journal of Biological Chemistry, 1999-09) Wibbenmeyer, Jamie A.; Schuck, Peter; Smith-Gill, Sandra J.; Willson, Richard C.
The binding of murine monoclonal antibody HyHEL-5 to lysozyme has been the subject of extensive crystallographic, computational, and experimental investigations. The complex of HyHEL-5 with hen egg lysozyme (HEL) features salt bridges between Fab heavy chain residue Glu50, and Arg45 and Arg68 of HEL. This interaction has been predicted to play a dominant role in the association on the basis of molecular electrostatics calculations. The association of aspartic acid and glutamine mutants at position 50H of the cloned HyHEL-5 Fab with HEL and bobwhite quail lysozyme (BQL), an avian variant bearing an Arg68 ? Lys substitution in the epitope, was characterized by isothermal titration calorimetry and sedimentation equilibrium. Affinities for HEL were reduced by 400-fold (E50HD) and 40,000-fold (E50HQ) (??G� estimated at 4.0 and 6.4 kcal mol?1, respectively). The same mutations reduce affinity for BQL by only 7- and 55-fold, respectively, indicating a reduced specificity for HEL. The loss of affinity upon mutation is in each case primarily due to an unfavorable change in the enthalpy of the interaction; the entropic contribution is virtually unchanged. An enthalpy-entropy compensation exists for each interaction; ?H� decreases, while ?S� increases with temperature. The ?Cp for each mutant interaction is less negative than the wild-type. Mutant-cycle analysis suggests the mutations present in the HyHEL-5 Fab mutants are linked to those present in the BQL with coupling energies between 3 and 4 kcal mol?1.