Energetic components of the allosteric machinery in hemoglobin measured by hydrogen exchange

[摘要] A hydrogen exchange (HX) functional labeling method was used to study allosterically active segments in human hemoglobin (Hb) at the alpha-chain N terminus and the beta-chain C terminus. Allosterically important interactions that contact these segments were removed one or more at a time by mutation (Hbs Cowtown, Bunbury, Barcelona, Kariya), proteolysis (desArg141 alpha, desHis146 beta), chemical modification (N-ethylsuccinimidyl-Cys93 beta), and the withdrawal of extrinsic effecters (phosphate groups, chloride). The effects of each modification on HX rate at the local and the remote position were measured in the deoxy Hb T-state and translated into change in structural free energy at each position. The removal of individual salt links destabilizes local structure by 0.4 to 0.75 kcal/mol (pH 7.4, 0 degrees C, 0.35 M ionic strength) and often produces cross-subunit effects while hemoglobin remains in the T-state. In doubly modified hemoglobins, different changes that break the same links produce identical destabilization, changes that are structurally independent show energetic additivity, acid changes that intersect show energetic overlap. For the overall T-state to R-state transition and for some but not all modifications within the T-state, the summed loss in stabilization free energy measured at the two chain termini matches the total loss in allosteric free energy measured by global methods. These observations illustrate the importance of evaluating the detailed energetics and the modes of energy transfer that define the allosteric machinery. (C) 1998 Academic Press.

[发布日期] 1998-12-18

[关键词] allostery;hemoglobin;hydrogen exchange;structure change;free energy