Article

Association Energetics of Cross-Reactive and Specific Antibodies

Department of Chemical and Biomolecular Engineering, University of Houston, Houston, Texas 77204-4004, USA.
Biochemistry (Impact Factor: 3.19). 02/2009; 48(6):1390-8. DOI: 10.1021/bi801901d
Source: PubMed

ABSTRACT HyHEL-8, HyHEL-10, and HyHEL-26 (HH8, HH10, and HH26, respectively) are murine monoclonal IgG(1) 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.

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    • "st in the complexes of HH8 with both HEL and JQL , smallest in those formed by HH26 , and intermediate in those of HH10 . This is not possible unless HH8 exhibits an ability to undergo the greatest conforma - tional rearrangement during association , albeit at a cost of large configurational entropic penalty , reflecting its inherent flexibility ( Mohan et al . , 2000 ) . The opposite of this situation is true of HH26 . Both these experimental results support the hypothesis that the three antibodies could exhibit different degrees of flexibilities . The retention ( as in HH26 ) or elimination ( as in HH8 ) of sequences capable of mediating salt bridges during the process of affinity maturation may we"
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