[摘要]：Human tumor necrosis factor alpha (TNF-alpha) exists in its functional state as a homotrimeric protein and is involved in inflammation processes and immune response of a human organism. Overproduction of TNF-alpha results in the development of chronic autoimmune diseases that can be successfully treated by inhibitors such as monoclonal antibodies. However, the nature of antibody-TNF-alpha recognition remains elusive due to insufficient understanding of its molecular driving forces. Therefore, we studied the energetics of binding of a therapeutic antibody fragment (Fab) to the native and non-native forms of TNF-alpha by employing calorimetric and spectroscopic methods. Global thermodynamic analysis of data obtained from the corresponding binding and urea-induced denaturation experiments has been supported by structural modeling. We demonstrate that the observed high affinity binding of Fab to TNF-alpha is an enthalpy-driven process due mainly to specific noncovalent interactions taking place at the TNF-alpha-Fab binding interface. It is coupled to entropically unfavorable conformational changes and accompanied by entropically favorable solvation contributions. Moreover, the three-state model analysis of TNF-alpha unfolding shows that at physiological concentrations, TNF-alpha may exist not only as a biologically active trimer but also as an inactive monomer. It further suggests that even small changes of TNF-alpha concentration could have a considerable effect on the TNF-alpha activity. We believe that this study sets the energetic basis for understanding of TNF-alpha inhibition by antibodies and its unfolding linked with the concentration-dependent activity regulation.
