Can a Two-State MWC Allosteric Model Explain Hemoglobin Kinetics?

Henry, Eric R.; Jones, Colleen M.; Hofrichter, James; Eaton, William A.

doi:10.1021/bi9619177

Cited by 107 publications

(131 citation statements)

References 67 publications

(135 reference statements)

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“…With time, r converts to t within R with a highly stretched exponential time course as observed in solution, and the rebinding rate to t in R asymptotically approaches the same rate as t in T. The fractional population of 20% for the t subunits, moreover, is very close to the 30-40% predicted from fitting the kinetics in solution with the TTS model under conditions that would be expected to favor more t (27). A result that strongly supports our interpretation of these experiments is that the stretching exponent of ∼0.2 is nearly identical to the exponent of ∼0.3 observed in solution for the unliganded heme spectral changes that were interpreted as corresponding to the r → t relaxation (27,42), but with a half-time that is ∼10 6 -fold larger due to slowing by the gel. The highly stretched exponential time course for the r → t tertiary conformational relaxation was previously explained by a theoretical model (43) that takes into account the dynamics of interconversion within the ensemble of conformational substates (21,44).…”

Section: Resultssupporting

confidence: 83%

Experimental basis for a new allosteric model for multisubunit proteins

Viappiani

Abbruzzetti

Ronda³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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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...

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Section: Resultssupporting

confidence: 83%

Experimental basis for a new allosteric model for multisubunit proteins

Viappiani

Abbruzzetti

Ronda³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…3. Measurements made by linearly polarized absorption spectroscopy of T state crystals showed that the Hill coefcient of 1.0 in the crystals resulted from the ®¯inequiv-alence (which would produce anti-cooperativity) being compensated by an approximately equal amount of positive cooperativity within the ®¯dimer in the T state (37 ). These departures from a pure MWC model are small; the ®¯inequivalence being 5 or less, and the dimer inequivalence being »2% at the quaternary-linked binding strengths and cooperativity.…”

Section: Appendixmentioning

confidence: 97%

Spectroscopic Contributions to the Understanding of Hemoglobin Function: Implications for Structural Biology

Shulman¹

2001

IUBMB Life

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“…This approach has been very useful when starting with ligand-saturated hemeproteins. Nanosecond and faster photodissociation of ligand-bound hemoglobins has shed considerable light on the sequence of molecular steps associated with the tertiary and quaternary structure changes in the globin that follow ligand dissociation (2)(3)(4)(5)(6)(7)(8)(9)(10).…”

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confidence: 99%

UV Resonance Raman Spectra of Ligand Binding Intermediates of Sol-Gel Encapsulated Hemoglobin

Juszczak

Friedman

1999

Journal of Biological Chemistry

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We report for the first time specific conformational changes for a homogeneous population of ligand-bound adult deoxy human hemoglobin A (HbA) generated by introducing CO into a sample of deoxy-HbA with the effector, inositol hexaphosphate, encapsulated in a porous sol-gel. The preparation of ligand-bound deoxyHbA results from the speed of ligand diffusion relative to globin conformational dynamics within the sol-gel (1). The ultraviolet resonance Raman (UVRR) difference spectra obtained reveal that E helix motion is initiated upon ligand binding, as signaled by the appearance of an ␣14␤15 Trp W3 band difference at 1559 cm ؊1 . The subsequent appearance of Tyr (Y8a and Y9a) and W3 (1549 cm ؊1 ) UVRR difference bands suggest conformational shifts for the penultimate Tyr␣140 on the F helix, the "switch" region Tyr␣42, and the "hinge" region Trp␤37. The UVRR results expose a sequence of conformational steps leading up to the ligation-induced T to R quaternary structure transition as opposed to a single, concerted switch. More generally, this report demonstrates that sol-gel encapsulation of proteins can be used to study a sequence of specific conformational events triggered by substrate binding because the traditional limitation of substrate diffusion times is overcome.A molecular level understanding of how proteins function requires not only a three-dimensional picture of equilibrium structures but also a time-ordered sequence of functionally related molecular events. The second requirement is difficult because of the challenge of either trapping detectable populations of nonequilibrium structures or creating a synchronized nonequilibrium population whose dynamics can be followed.Because protein dynamics can span many decades in time starting on the picosecond time scale, it is often necessary to prepare the evolving population using a short, laser-derived excitation pulse. This approach has been very useful when starting with ligand-saturated hemeproteins. Nanosecond and faster photodissociation of ligand-bound hemoglobins has shed considerable light on the sequence of molecular steps associated with the tertiary and quaternary structure changes in the globin that follow ligand dissociation (2-10).The sequence of conformational events following solventderived (non-geminate) ligand binding to deoxy hemoglobin is much more of a challenge because of the temporal constraints imposed by ligand diffusion. The initial conformational responses to ligand or substrate binding to a given protein will typically be faster than the diffusion time that is inherent to any rapid mixing experiments. In the present work, an approach to overcoming this limitation, based on sol-gel encapsulation, is presented in which the conformational response time is extended well beyond the ligand diffusion time. Thus we have been able to use UVRR 1 to probe the initial sequence of conformation changes in hemoglobin when CO binds to the equilibrium form of deoxy-HbA.The encapsulation of proteins in porous TMOS-derived solgels has been shown to oc...

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Can a Two-State MWC Allosteric Model Explain Hemoglobin Kinetics?

Cited by 107 publications

References 67 publications

Experimental basis for a new allosteric model for multisubunit proteins

Experimental basis for a new allosteric model for multisubunit proteins

Spectroscopic Contributions to the Understanding of Hemoglobin Function: Implications for Structural Biology

UV Resonance Raman Spectra of Ligand Binding Intermediates of Sol-Gel Encapsulated Hemoglobin

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