Speed of intersubunit communication in proteins

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...

Section: Resultssupporting

confidence: 90%

Section: Resultsmentioning

confidence: 99%

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

Levantino

Spilotros

Cammarata

et al. 2012

“…However, there is little known about speed limits for conformational change in native proteins. Eaton et al (39) estimated that the time required for communication between subunits in hemoglobin during the R-to-T allosteric transition is Ϸ1 s. With regard to ion channels, a fast component of the Shaker gating current has a time constant of Ϸ5 s and has been interpreted as arising from the diffusion of charges in a potential well (40)(41)(42). In gramicidin, the open-channel noise spectrum has an Ϸ1-s component that has been attributed to structural fluctuations of pore residues (43).…”

Section: [4]mentioning

confidence: 99%

A speed limit for conformational change of an allosteric membrane protein

Chakrapani

Auerbach

2004

Neuromuscular acetylcholine receptors are synaptic ion channels that open and close with rate constants of Ϸ48,000 s ؊1 and Ϸ1,700 s ؊1 , respectively (in adult mouse, at 24°C, ؊100 mV membrane potential). Perturbations of many different sites in the protein can change these rate constants, with those in the extracellular domain mainly affecting channel-opening and many of those in the membrane and intracellular domains mainly affecting channel-closing. We used single-channel recordings to measure the total open time per activation ( b) elicited by a low concentration of the natural transmitter, acetylcholine. b increased in constructs with mutations that increased the gating equilibrium constant by either increasing the opening or decreasing the closing rate constant. However, b did not approach the same asymptote in fast-opening and slow-closing constructs. The maximum value for the slow closers was about twice that for the fast openers. One interpretation of this difference is that there is an upper limit to the channel-opening rate constant, which we estimate to be Ϸ0.86 s ؊1 . One possibility is that this limit is the rate of conformational change in the absence of an overall activation barrier and thus reflects the kinetic prefactor for the acetylcholine receptor opening isomerization.channel gating ͉ nicotinic acetylcholine receptor ͉ energy landscape ͉ transition state A t cholinergic synapses, transmitter molecules trigger acetylcholine (ACh) receptor channels (AChRs) to switch from a stable conformation in which ion permeation is forbidden (''closed'') to one in which cations can permeate rapidly (''open''). This reversible change in structure, known as gating, involves the organized movements of many of the Ͼ2,300 residues within the five subunits that comprise this allosteric membrane protein (1-3). The driving energy for the reaction is a change in binding energy for the ligand; thus, gating must, at least, entail movements of residues at the two transmitter binding sites and in the ion conduction pathway.When activated by the natural transmitter ACh, mouse neuromuscular AChRs open rapidly (Ϸ48,000 s Ϫ1 ) and close relatively slowly (Ϸ1,700 s Ϫ1 ) (at 24°C, Ϫ100-mV membrane potential) (4). In single-molecule patch-clamp recordings with a time resolution of Ϸ25 s, the closed^open isomerization appears to be a two-state reaction with no directly detectable intermediate states. However, these states can be probed indirectly by using rate-equilibrium free energy (REFER) analyses (5-7). For a series of point mutations, the slope (⌽) of a log-log plot of the opening rate constant vs. the equilibrium constant is an index of the extent of progress of the perturbed site at the transition state of the reaction. In fully liganded AChRs, there is, to a first approximation, a longitudinal gradient in ⌽-values, with residues near the transmitter binding sites being open-like (⌽ Ϸ 1) and some of those in the transmembrane region being closed-like (⌽ Ϸ 0) at the transition state (8, 9). These results have been inte...

“…6). This is the classic test for distinguishing tertiary and quaternary kinetics, because the amplitude of the R-to-T quaternary transition is highly dependent on the fraction of subunits with ligand bound (41).…”

Section: Resultsmentioning

confidence: 99%

Experimental basis for a new allosteric model for multisubunit proteins

Viappiani

Abbruzzetti

Ronda³

et al. 2014

Self Cite

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...