Browsing by Author "Wibbenmeyer, Jamie A."

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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.
Water molecules in the antibody朼ntigen interface of the structure of the Fab HyHEL-5杔ysozyme complex at 1.7 A? resolution: comparison with results from isothermal titration calorimetry
(Acta Crystallographica Section D: Biological Crystallography, 2005) Cohen, Gerson H.; Silverton, Enid W.; Padlan, Eduardo A.; Dyda, Fred; Wibbenmeyer, Jamie A.; Willson, Richard C.; Davies, David R.
The structure of the complex between hen egg-white lysozyme and the Fab HyHEL-5 at 2.7 A? resolution has previously been reported [Cohen et al. (1996), Acta Cryst. D52, 315�6]. With the availability of recombinant Fab, the X-ray structure of the complex has been re-evaluated at 1.7 A? resolution. The refined structure has yielded a detailed picture of the Fab� lysozyme interface, showing the high complementarity of the protein surfaces as well as several water molecules within the interface that complete the good fit. The model of the full complex has improved significantly, yielding an Rwork of 19.5%. With this model, the structural results can be compared with the results of isothermal titration calorimetry. An attempt has been made to estimate the changes in bound waters that accompany complex formation and the difficulties inherent in using the crystal structures to provide the information necessary to make this calculation are discussed.