Life is made possible by an astounding array of interactions between highly-evolved biomolecules that serve a variety of specific functions. Their functions are dictated by structure and flexibility or “dynamics,” both of which can change upon interaction with a partner molecule. Understanding these changes and the pathways that link them have revealed key insights into both disease and the foundations of life. However, because these interactions are often “allosteric,” wherein changes are propagated far from the site of contact, the subsequent changes are difficult to predict and sometimes counter-intuitive to understand, thus motivating directed, basic research.
Here, we aim to understand allostery by characterizing the activation mechanism of the paradigmatic, allosteric, ligand-responsive protein trp RNA-binding Attenuation Protein (TRAP), which regulates genes under control of the trp operon in Bacilli. The ring-shaped 11-mer TRAP is activated to recognize a specific trp-mRNA target upon binding up to 11 tryptophan (Trp) molecules; in contrast to the Trp-bound “holo” state of TRAP, the Trp-free “apo” state is inactive. In study 1, to understand how the apo and holo states of TRAP differ with respect to structure and dynamics, we used state of the art nuclear magnetic resonance (NMR) experiments. We learn that apo TRAP is flexible in the μs-ms time window, and that Trp binding yields local changes in structure, and a global reduction in μs-ms flexibility. In study 2, to understand the time-resolved pathway of the apo to holo activation process, we monitored Trp fluorescence upon rapid-mixing of Trp and apo TRAP in solution. We demonstrate that Trp encounters its binding site on TRAP a remarkable 2,000 times before entry is permitted, that activation occurs within one sec via step-wise increases in bondedness, order and compression, and we characterize two Trp-bound states reflecting holo TRAP flexibility on the sec timescale, putatively responsible for Trp release. In study 3, to characterize partially-bound TRAP donuts (i.e., between apo and holo states), we titrated Trp into TRAP and monitored structure via NMR and binding energetics via isothermal titration calorimetry (ITC). NMR data reveal that TRAP samples conformations in the supra-ms time window that are neither the apo nor holo states, and that the structure and dynamics of a given protomer can be altered even if Trp is not bound at that site. The ITC data reveal allostery in Trp binding that, if described via nearest-neighbor interactions between TRAP protomers, must invoke negative binding cooperativity (i.e., binding a second Trp molecule is less favorable than binding the first). Finally, because this research relies heavily on NMR experiments, we wrote a comprehensive methodological review paper, and developed an innovative analytical software package, “GUARDD.”