Experimental basis for a new allosteric model for multisubunit proteins

Abstract

Monod, Wyman, and Changeux (MWC) explained allostery in multisubunit proteins with a widely applied theoretical model in which binding of small molecules, so-called allosteric effectors, affects reactivity by altering the equilibrium between more reactive (R) and less reactive (T) quaternary structures. In their model, each quaternary structure has a single reactivity. Here, we use silica gels to trap protein conformations and a new kind of laser photolysis experiment to show that hemoglobin, the paradigm of allostery, exhibits two ligand binding phases with the same fast and slow rates in both R and T quaternary structures. Allosteric effectors change the fraction of each phase but not the rates. These surprising results are readily explained by the simplest possible extension of the MWC model to include a preequilibrium between two tertiary conformations that have the same functional properties within each quaternary structure. They also have important implications for the long-standing question of a structural explanation for the difference in hemoglobin oxygen affinity of the two quaternary structures.S mall molecules can regulate protein reactivity by binding to residues distant from the active site, a phenomenon known as allostery. The classic theoretical model proposed by Monod, Wyman, and Changeux (MWC) (1, 2) for explaining this phenomenon considered only proteins with multiple subunits, and the two-state allosteric model of MWC was originally used by them to explain the functional properties of the hemoglobin tetramer, the "honorary enzyme" (1, 3). This famous model has since been applied to a wide variety of other systems in biology, including ligand-gated ion channels, G-protein-coupled receptors, nuclear receptors, and supramolecular assemblies such as chaperonins (4-7). In the case of hemoglobin, the paradigm of allostery, MWC makes two key postulates that have been extensively tested by experiments. Oxygen binding to the deoxy quaternary structure (T) is noncooperative; cooperativity arises from a shift in the population from the low-affinity T quaternary structure to the high-affinity R quaternary structure as the oxygen concentration is increased. The second postulate is that allosteric effectors regulate oxygen affinity by altering only the R ⇄ T preequilibrium. Investigations of hemoglobin focused primarily on the first postulate, which was finally confirmed by single-crystal oxygen-binding measurements that ruled out a sequential model (8) and ended a 25-y controversy (9-12). The second is of more general interest because it applies to all multisubunit proteins exhibiting allosteric behavior and has been known for many years to be inconsistent with the fact that allosteric effectors markedly affect oxygen affinity without changing the quaternary preequilibrium [see data summaries by Imai, Yonetani, and coworkers (13,14)]. The change in oxygen affinity constants, K T and K R , with conditions was interpreted as indicating that tertiary conformations must be affected or that more than two quat...

Experiments on Hemoglobin in Single Crystals and Silica Gels Distinguish among Allosteric Models

“…Finally, photolysis experiments on gel-encapsulated hemoglobin revealed that in the absence of allosteric effectors, subunits of the T quaternary structure can bind carbon monoxide (CO) at the same fast rate as subunits in the R quaternary structure, and that subunits of the R quaternary structure can bind CO with the same slow rate as subunits in the T quaternary structure. More specifically, nanosecond pulsed-laser photolysis measurements for the CO complex of hemoglobin trapped by the gel in the T quaternary structure in the absence of allosteric effectors showed that a large fraction of the photolyzed subunits bind CO at rates identical to those found for the R quaternary structure (30), whereas CO rebinding after continuous-wave (cw) photolysis of the R quaternary structure encapsulated in the gel showed the stretched exponential appearance of a fraction of subunits that bind CO at the Hill plots comparing oxygen binding curves of human hemoglobin in solution, in single crystals, in the sickle hemoglobin fiber, and encapsulated in gels as either the T or R quaternary structure (from Viappiani et al (37)). The superscripts indicate whether (þ) or not (À) saturating concentrations of allosteric effectors inositol hexaphosphate and bezafibrate are present.…”

Immobilization of Proteins in Silica Gel: Biochemical and Biophysical Properties

“…The low affinity T-state and the high affinity Rstate of Hb can be trapped by encapsulating Hb either as fully deoxygenated or fully oxygenated molecules, respectively [57,82,83,100]. Indeed, non-cooperative oxygen binding is observed both when Hb is encapsulated in the high-affinity R state or in the lowaffinity T state [57,58,82,83,85,100] (Fig. 3).…”

“…The possibility provided by silica gel encapsulation to reduce the rate of conformational changes by orders of magnitude, increasing the kinetic separation of tertiary and quaternary relaxations [57,58,[80][81][82][83][84][85][86][87][88][89][90][91][92], has been extensively exploited by several groups to make accessible to spectroscopic investigation species that are normally poorly populated, or metastable on the time scale of solution experiments. Not surprisingly, many of these studies concern hemoglobin (Hb), myoglobin and other heme binding proteins, since the availability of a sensitive, intrinsic chromophoric signal allowed to achieve an unprecedented level of detail in the comprehension of structure-dynamic-function relationships.…”

“…By encapsulating the enzyme either in the T or in the R state, it was possible to investigate the functional relevance of ligand-associated tertiary structural changes [121]. Results were interpreted on the basis of the known crystallographic structures and in the frame of the tertiary two-state allosteric model of Eaton and coworkers [58,85,122], assuming an equilibrium of t and r tertiary conformations, within each quaternary state, regulated by allosteric effectors.…”

“…Using a cw laser photolysis scheme, Eaton and coworkers recently demonstrated that a tertiary t conformation can be identified in deoxy R, with functional properties comparable to those of deoxy T subunits [85]. The deoxy R species is obtained by photodissociating HbCO gels with a cw laser for a long enough time span to allow tertiary, but not quaternary relaxation.…”