The information flow between distal elements of a protein may rely on allosteric communication trajectories lying along the protein's tertiary or quaternary structure. To unravel the underlying features of energy parsing along allosteric pathways in voltagegated K ؉ channels, high-order thermodynamic coupling analysis was performed. We report that such allosteric trajectories are functionally conserved and delineated by well defined boundaries. Moreover, allosteric trajectories assume a hierarchical organization whereby increasingly stronger layers of cooperative residue interactions act to ensure efficient and cooperative long-range coupling between distal channel regions. Such long-range communication is brought about by a coupling of local and global conformational changes, suggesting that the allosteric trajectory also corresponds to a pathway of physical deformation. Supported by theoretical analyses and analogy to studies analyzing the contribution of long-range residue coupling to protein stability, we propose that such experimentally derived trajectory features are a general property of allosterically regulated proteins.is a fundamental property of many allosteric proteins. Information transfer between such elements may be achieved by propagation of conformational changes through a protein structure, induced by changes in chemical or electrical potential. However, while the structures of stable conformational states of several allosteric proteins are known, the mechanism(s) by which ligand-induced structural changes propagate through the molecule remains elusive. In the absence of extensive experimental data accurately describing such allosteric networks, computational analyses have attempted to provide mechanistic explanations for long-range coupling in proteins. Structure-based computer simulations suggest that conformational changes may propagate signal transmission by a redistribution of native-state conformational ensembles (1-3). Others suggest that conformational changes may propagate by simple mechanical deformation of the protein structure along pathways of energetic connectivity, comprising adjacent amino acid positions in the tertiary structure (4, 5). Conformational changes are central to the function of voltage-activated potassium channels (Kv), poreforming proteins that open and close in response to changes in membrane potential. Such conformational transitions regulate the flow of potassium ions across the membrane (6-9), a process underlying many fundamental biological processes, in particular the generation of nerve and muscle action potentials (10). These conformational changes, moreover, play a fundamental role in mediating coupling between the voltage-sensor, activation gate and selectivity filter elements of Kv channels (6)(7)(8)(9)(11)(12)(13)(14).Amenable to rapid and highly accurate functional characterization without the need for protein purification, the Kv channel represents an excellent model for studying the features underlying allosteric communication networks in proteins. R...