Thermodynamics and kinetics of the interaction between T cell receptor specific for cytomegalovirus peptide (TCRCMV) and its specific ligand, pp65-HLA-A*0201 complex, were studied by surface plasmon resonance and stopped-flow methods. In the latter measurements, fluorescence resonance energy transfer (FRET) between fluorescently labeled reactants was used. Thermodynamic data derived from surface plasmon resonance measurements suggest that the complex formation is driven by both favorable enthalpy and entropy. Two reaction phases were resolved by the stopped-flow measurements. The rate constant of the first step was calculated to be close to the diffusion-controlled limit rate (3⅐10 5 to 10 6 M ؊1 ⅐s ؊1 ), whereas the second step's reaction rate was found to be concentration independent and relatively slow (2-4 s ؊1 at 25°C). These findings strongly suggest that the interactions between the TCR and its ligand, the peptide-MHC complex, proceed by a two-step mechanism, in which the second step is an induced-fit process, rate determining for antigen recognition by TCR.conformational changes ͉ T cell receptor-ligand interactions ͉ FRET ͉ stopped flow ͉ induced fit T cell receptor (TCR) association with its ligand, a peptide bound to molecules encoded by class I or II of the major histocompatibility complexes (pMHC), is the initial process in T cell activation (1-3). This process can induce different types of immune responses depending on the nature of the pMHC and the involved T cell (4-6). It is well established that despite the prevalent micromolar affinity, this interaction is characterized by such an exquisite sensitivity that even just a few antigenic pMHC molecules can be recognized on an antigen-presenting cell (APC) (7). At the same time, a TCR was shown to cross-react with different antigenic pMHC ligands (8 -10). Threedimensional structures determined for more than two dozen different TCR-pMHC complexes revealed similar diagonal orientations of TCR molecules with respect to their pMHC ligands (11,12). It was also found that two of the complementaritydetermining regions (CDRs) of the TCR, CDR1 and CDR2, interact predominantly with the MHC molecules, whereas the CDR3 interacts with exposed peptide residues. Importantly, significant conformational changes in the TCR, mainly in its CDR3, have been observed in crystallographic studies (10,13,14). These structural studies provide a static picture and require time-resolved analysis of the process to deepen the understanding of the molecular mechanism of antigen recognition by the TCR.Kinetic studies carried out to date have used the surface plasmon resonance (SPR) method, which has several deficiencies. First and foremost, its time resolution is limited. Essentially, results of all such studies were interpreted as bimolecular processes with a relatively slow rate constant of complex formation and a fast rate of its dissociation (3). This slow association rate was assigned to a conformational transition in the TCR binding site (15). This notion provided the basis f...