The Monod-Wyman-Changeux allosteric model accounts for the quaternary transition dynamics in wild type and a recombinant mutant human hemoglobin

Abstract

The acknowledged success of the Monod-Wyman-Changeux (MWC) allosteric model stems from its efficacy in accounting for the functional behavior of many complex proteins starting with hemoglobin (the paradigmatic case) and extending to channels and receptors. The kinetic aspects of the allosteric model, however, have been often neglected, with the exception of hemoglobin and a few other proteins where conformational relaxations can be triggered by a short and intense laser pulse, and monitored by time-resolved optical spectroscopy. Only recently the application of time-resolved wide-angle X-ray scattering (TR-WAXS), a direct structurally sensitive technique, unveiled the time scale of hemoglobin quaternary structural transition. In order to test the generality of the MWC kinetic model, we carried out a TR-WAXS investigation in parallel on adult human hemoglobin and on a recombinant protein (HbYQ) carrying two mutations at the active site [Leu(B10)Tyr and His(E7) Gln]. HbYQ seemed an ideal test because, although exhibiting allosteric properties, its kinetic and structural properties are different from adult human hemoglobin. The structural dynamics of HbYQ unveiled by TR-WAXS can be quantitatively accounted for by the MWC kinetic model. Interestingly, the main structural change associated with the R-T allosteric transition (i.e., the relative rotation and translation of the dimers) is approximately 10-fold slower in HbYQ, and the drop in the allosteric transition rate with ligand saturation is steeper. Our results extend the general validity of the MWC kinetic model and reveal peculiar thermodynamic properties of HbYQ. A possible structural interpretation of the characteristic kinetic behavior of HbYQ is also discussed.time-resolved X-ray scattering | protein conformational changes | cooperativity | flash photolysis E ver since the publication of the Monod-Wyman-Changeux paper on allostery (1), hemoglobin (Hb) has been considered the prototype of an allosteric protein; the molecular basis of positive cooperativity in O 2 binding involving a ligand-linked shift between two different quaternary states. The dynamics of ligand rebinding and of the tertiary and quaternary allosteric changes of tetrameric human Hb have been investigated, by-and-large, using transient spectroscopy in the picosecond to millisecond time range, following laser-induced photolysis of the ligand-heme iron bond. Starting with the carbon monoxide adduct HbCO in the allosteric quaternary state called R 4 , complete photolysis yields the unliganded R 0 state; the destiny of this photoproduct is a complex time-dependent process involving competing events such as ligand rebinding and (tertiary and quaternary) conformational decays. Changes in the optical and resonance Raman spectra of the different states have provided, over the last four decades, a quantitative estimate of the rates of the competing events (2-5). For a review on time-resolved optical absorption (TR-OA) data describing conformational decays as well as rebinding in the dark of a ligand...

Fine Sampling of the R→T Quaternary‐Structure Transition of a Tetrameric Hemoglobin

“…After decades of investigations, tetrameric Hbs are still one of the favorite systems for the analysis of structure–function relationships in proteins, including cooperativity and allostery . The sequential and detailed snapshots of the Hb R–T transition provided by the present study are the best atomic‐level representation hitherto available for this widely studied structural transition.…”

Statistical Mechanics of Monod–Wyman–Changeux (MWC) Models

“…Though the original formulation of the MWC model was primarily an equilibrium concept, generalization to the dynamical situation is relatively straightforward and has been undertaken by many workers in the meantime, 8,75–81 sometimes under the heading of the “kinetic allosteric model”. 82–84 The chemical reactions underlying the dynamical MWC model were outlined in one of the original papers, 4 and an application of transition state theory 85–87 to those chemical reactions yields the kinetic allosteric model.…”

Immobilization of Proteins in Silica Gel: Biochemical and Biophysical Properties

“…Hb is a tetrameric protein that has long served as a prototypical model system for our understanding of allosteric mechanisms [123]. In solution, Hb undergoes a quaternary transition following ligand release with a timescale of ~1 Bs [124,125]. This transition can be significantly slowed down by encapsulating Hb in silica gels [82].…”