Capturing the hemoglobin allosteric transition in a single crystal form.

Allostery in many oligomeric proteins has been postulated to occur via a ligand-binding-driven conformational transition from the tense (T) to relaxed (R) state, largely on the basis of the knowledge of the structure and function of hemoglobin, the most thoroughly studied of all allosteric proteins. However, a growing body of evidence suggests that hemoglobin possesses a variety of intermediates between the two end states. As such intermediate forms coexist with the end states in dynamic equilibrium and cannot be individually characterized by conventional techniques, very little is known about their properties and functions. Here we present complete structural and functional snapshots of nine equilibrium conformers of human hemoglobin in the half-liganded and fully liganded states by using a novel combination of X-ray diffraction analysis and microspectrophotometric O2 equilibrium measurements on three isomorphous crystals, each capturing three distinct equilibrium conformers. Notably, the conformational set of this crystal form varies according to shifts in the allosteric equilibrium, reflecting the differences in hemoglobin ligation state and crystallization solution conditions. We find that nine snapshot structures cover the complete conformational space of hemoglobin, ranging from T to R2 (the second relaxed quaternary structure) through R, with various relaxed intermediate forms between R and R2. Moreover, we find a previously unidentified intermediate conformer, between T and R, with an intermediate O2 affinity, sought by many research groups over a period of decades. These findings reveal a comprehensive picture of the equilibrium conformers and transition pathway for human hemoglobin.