Background. Proteins have evolved over billions of years to possess the properties that enable them to perform their specific biological functions. As products of the evolutionary process, these molecules are very different from abiological molecules - biological molecules may have in a sense, learned how to perform specific functions. In order to study how evolution affects a protein, a detailed structural and dynamic understanding of the protein before, during, and after its evolution is required. Unfortunately, this is generally not possible due to the time scale over which evolution occurs. As a result, we can only speculate about the evolutionary precursors of the proteins found in nature today.

A single exception, where the evolutionary time scale is amenable to the laboratory time scale, is offered by the immune system. Within weeks of the introduction of virtually any foreign molecule, the immune system uses mutation and selection to transform a polyspecific progenitor antibody (Ab) into a mature Ab that recognizes the foreign molecule (or antigen, Ag) with high affinity and specificity. To perform this remarkable feat of molecular recognition, it has been suggested that the immune system takes flexible Ab, which are capable of the conformational changes required to recognize different Ag, and evolves them into rigid Ab that are conformationally restricted in order to bind a specific Ag.

Testing the model that Ab evolution involves the tailoring of flexibility requires the isolation of Ab at various stages of evolution and the determination of both their structure and their flexibility. The flexibility of any material, including a protein, may be quantified by measuring its response to an applied force. For example, a flexible protein will respond to an applied force with low frequency, large amplitude motions, while a more rigid protein will respond to the same force with higher frequency, smaller amplitude motions. We have isolated, at various stages of their evolution, Ab that bind suitably chosen chromophoric Ag. Photoexcitation of the bound chromophore induces electronic and structural reorganization, resulting in a complex with the Ab that is out of equilibrium. In this manner, an applied force induces protein motions. Amplitudes and frequencies of these motions can be described by the spectral density, ρ(ω). Thus, ρ(ω) provides a quantitative description of flexibility. ρ(ω) may be determined using three pulse photon echo peak shift (3PEPS) spectroscopy, where the experimentally observable decay of the photon echo peak shift reflects the time scales and amplitudes of induced protein motions. We have also initiated the characterization of protein dynamics using NMR spectroscopy to understand the evolution of protein motions on all biologically relevant timescales. We recently characterized the evolution of molecular recognition and protein dynamics in the fluorescein binding Ab 4-4-20 (see image above). Click here to see how this Ab evolved.