Am. chromophore via T-stacking, and the side chain of TrpL91, which interacts with the chromophore via parallel stacking. The T-stacking conversation appears to mediate relaxation around the picosecond timescale, while the parallel stacking appears to mediate relaxation on an ultrafast, femtosecond timescale, which dominates the response. The anti-MPTS Abs thus not only demonstrate the simultaneous use of the two limiting modes of stacking for molecular acknowledgement, but also provide a unique opportunity to characterize how dynamics might contribute to molecular acknowledgement. Both types of Kenpaullone stacking are common in proteins and protein complexes where they may similarly contribute to dynamics and molecular acknowledgement. Graphical Abstract INTRODUCTION Molecular acknowledgement underlies virtually all of the activities a protein can possess. Perhaps the most CSF3R remarkable example of molecular acknowledgement, and its evolution, is the adaptive immune system, in which antibodies (Abdominal muscles) specific for virtually any foreign molecule (antigen, Ag) can be produced via somatic mutation from a germline precursor (affinity maturation) within weeks of their first encounter. Since the earliest days of structural biology, Abdominal muscles have served as a paradigm for understanding how structure contributes to molecular acknowledgement.1 However, dynamics must also contribute, because the limiting models of Kenpaullone induced fit, conformational selection, and lock-and-key acknowledgement are differentiated by the level of flexibility attributed to the protein. In recent years, increasing attention has been paid to the study of conformational dynamics and its link to Ab specificity.2,3 Structural studies have provided evidence that affinity maturation can pre-order the Ab combining site for specific Ag acknowledgement,2,4C11 while other studies have demonstrated that mature Abs retain the ability to adopt multiple conformations that facilitate the recognition of different Ags.12,13 The effects of affinity maturation around the entropy of binding have also provided evidence of conformational restriction.14,15 Finally, computational studies have provided evidence that Ab rigidity is linked to specificity.16C19 However, our understanding of Ab dynamics, how it contributes to Ag recognition, and if it is tailored during affinity maturation remains incomplete, at least in part due to the experimental challenges associated with the direct characterization of protein dynamics. In addition to their unprecedented level of molecular acknowledgement, the use of Ab-Ag complexes to study protein dynamics is attractive because Abs can be developed to bind specific chromophores that facilitate characterization of dynamics, for example through the use of ultrafast nonlinear optical methods such as 3-pulse photon echo peak shift (3PEPS) spectroscopy.20C22 Unlike the conjugation of chromophores to a protein of interest, which is likely to be perturbative if the chromophore is buried within the protein or insensitive to the protein environment if surface exposed, the use of a chromophoric Ag, to which the Ab evolved to bind, is by definition non-perturbative and likely to be sensitive to biologically relevant dynamics. The response of the environment to the pressure exerted by a photoinduced switch in a chromophores electronic structure is typically discussed in terms of impulsive and diffusive motions, in analogy to solvation dynamics.22 However, inspired by the field of materials science, we have adopted the language of elasticity, anelasticity, and plasticity.23C25 Anelasticity corresponds to motions associated with barrier crossings that occur during the timescale of an experiment, while plasticity corresponds to the population of multiple deep minima separated by barriers that are sufficiently large to prevent interconversion around the timescale of the experiment and thus result in static inhomogeneity. In contrast, elasticity corresponds to motion within Kenpaullone a single potential energy minimum. During our efforts to develop Abs as models for the study of protein dynamics, we have.