ESR Resolves the C Terminus Structure of the Ligand-free Human Glutathione S-Transferase A1-1

Nitroxide-and Cu2+-based electron spin resonance (ESR) are combined to provide insight into the conformational states of the functionally important alpha-helix of the human glutathione S-transferase A1. Distance measurements on various spinlabeled dimeric human glutathione S-transferase A1-1 all result in bimodal distance distributions, indicating that the C-terminus exists in two distinct conformations in solution, one of which closely matches that found in the crystal structure of the ligand-bound enzyme. These measurements permit the generation of a model of the unliganded conformation. Room temperature ESR indicates that the second conformation has high mobility, potentially enabling the enzyme's high degree of substrate promiscuity. This model is then validated using computational modeling and further Cu2+-based ESR distance measurements. Cu2+-based ESR also provides evidence that the secondary structure of the second conformation is of helical nature. Addition of S-hexyl glutathione results in a shift in relative populations, favoring the state that is similar to the previously known structure of the ligand-bound enzyme.