The Effect of Water on the Rate of Conformational Change in Protein Allostery

Abstract

The influence of solvation on the rate of quaternary structural change is investigated in human hemoglobin, an allosteric protein in which reduced water activity destabilizes the R state relative to T. Nanosecond absorption spectroscopy of the heme Soret band was used to monitor protein relaxation after photodissociation of aqueous HbCO complex under osmotic stress induced by the nonbinding cosolute poly(ethylene glycol) (PEG). Photolysis data were analyzed globally for six exponential time constants and amplitudes as a function of osmotic stress and viscosity. Increases in time constants associated with geminate rebinding, tertiary relaxation, and quaternary relaxation were observed in the presence of PEG, along with a decrease in the fraction of hemes rebinding CO with the slow rate constant characteristic of the T state. An analysis of these results along with those obtained by others for small cosolutes showed that both osmotic stress and solvent viscosity are important determinants of the microscopic R --> T rate constant. The size and direction of the osmotic stress effect suggests that at least nine additional water molecules are required to solvate the allosteric transition state relative to the R-state hydration, implying that the transition state has a greater solvent-exposed area than either end state.

Time-resolved Absorption and UV Resonance Raman Spectra Reveal Stepwise Formation of T Quaternary Contacts in the Allosteric Pathway of Hemoglobin

“…Our values agree well with the recent determinations by Goldbeck et al, 45 and are not far from those reported earlier by Hofrichter et al 48 The last two time constants represent CO recombination from solution, and can be associated with molecules in the R and T quaternary states. Matthews & Olson 50 cite second-order rate constants of 2.9 £ 10 6 M 21 s 21 and 7.1 £ 10 6 M 21 s 21 for CO binding to the a and b subunits within the R state and 0.12 £ 10 6 M 21 s 21 and 0.18 £ 10 6 M 21 s 21 in the T state.…”

“…Correcting for this effect, they found an additional fast optical relaxation (t 1 0 in Table 1) at 20 ns. Goldbeck et al 44,45 corrected for photoselection by setting the probe polarization to the magic angle (54.78), and likewise found a 22 ns relaxation. We have not collected data at these early times.…”

Differential influence of dynamic processes on forward and reverse electron transfer across a protein-protein interface

“…1 has disclosed a fruitful area for examination, but falls far short of a complete picture of the influence of intracomplex dynamics on interprotein ET reactions. Among the issues to be addressed are the degree to which the addition of glycerol may be (i) changing the energy landscapes themselves, not merely the rates with which the landscapes are traversed (48), and (ii) influencing water activity (49)(50)(51).…”

Role of Heme Pocket Water in Allosteric Regulation of Ligand Reactivity in Human Hemoglobin

“…The tetramer loses ~60 surface-bound water molecules overall in the R → T transition, after first picking up ~10 additional water molecules in passing through the allosteric transition state. 25,26 However, the coupling of quaternary structure to hydration changes internal to the protein remains an open question. 27 This issue is addressed in the study presented here by using spectrokinetic techniques that monitor the entry of water into the distal pockets of the hemoglobin tetramer after CO photodissociation triggers protein structural relaxation from the R to the T quaternary state.…”