Analysis of β-C-terminal salt bridges in T-state hemoglobin by trapping tertiary conformations in silica gels.

Encapsulation of a protein in wet silica gels greatly slows its large-scale motions, allowing the characterization of defined protein conformations during functional and spectroscopic measurements. This technique has revealed the coexistence of two or more functionally distinct tertiary conformations in T-state human hemoglobin, but their structural basis remains unclear. Here, we extend this approach to analyze the β-C-terminal salt bridges in the T-state hemoglobin populations. To this end, we first compare three representative sol-gel trapping protocols by transient absorption characterization of both directions of the hemoglobin allosteric transition (R-to-T and T-to-R) and by O equilibrium measurements. Results reveal protocol-dependent variations in slow-down factor for tertiary and quaternary conformational changes and demonstrate that our method most effectively traps tertiary hemoglobin conformations. Using this optimized protocol, we show that the kinetics of Cys93β sulfhydryl reactivity of T-state Ni(II)-substituted hemoglobin in silica gels is markedly biphasic under low-salt conditions but monophasic in the presence of inositol hexakisphosphate. These findings provide direct evidence for the coexistence of high- and low-affinity tertiary conformations with broken and unbroken β-C-terminal salt bridges, respectively, in the anion-free T quaternary structure of hemoglobin and elucidate why the O affinity of T-state deoxyhemoglobin varies dramatically with solution conditions.