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.

Fig. 1.

TR-WAXS difference patterns of HbA (A) and HbYQ (B) measured at different time delays from a 3-ns-long photolysis pulse. Continuous lines are fitting curves obtained from a global analysis of the experimental patterns at time delays longer than 250 ns as described in the text. A fast local structural change is detected at 3.16 ns in both HbA and HbYQ. At 100 μs the typical pattern assigned to the αβ dimers relative rotation and translation is observed (12, 13); its amplitude is larger for HbYQ than for HbA due to the large difference in geminate rebinding. At 100 ms, while HbA has already recombined with CO, HbYQ is still mostly in the deoxy state due to the smaller bimolecular rebinding rate.

Fig. 2.

(A) Time dependence of the TR-WAXS signal at ∼0.19 Å^-1. For HbA (open squares) the maximum value of the signal is smaller than for HbYQ (closed circles); both sets of data have been obtained using a 3-ns-long photolysis pulse. (B) HbYQ signal obtained with a 3-ns-long photolysis pulse (closed circles) compared with the HbA signal obtained with a 230-ns photolysis pulse (open triangles, data from ref. 13); both signals have been normalized to their maximum. Error bars have been estimated from the noise of the difference patterns.

Fig. 3.

Results of the global analysis of TR-WAXS patterns according to the MWC allosteric kinetic model for HbA and HbYQ based on data obtained with a 3-ns laser pulse for both proteins. Tops: normalized basis patterns obtained for HbA (A) and HbYQ (B). Bottoms: relative R-like and T-like populations for HbA (C) and HbYQ (D) as obtained from the decomposition of experimental patterns in terms of a linear combination of the basis patterns reported in A and B (symbols), and from the kinetic model (lines).

Fig. 4.

Close-up view of T state HbYQ α chain (A) and β chain (B) in the proximity of the heme; notice that CO is bound to the β chains only. The contacts that could allow distal tertiary changes to be transmitted from the heme pocket to the hinge and the switch regions of the α₁β₂ interface are indicated by orange arrows.