Effects of surface hydrophobicity on the removal of F-specific RNA phages from reclaimed water by coagulation and ceramic membrane microfiltration.

Microfiltration (MF) has been widely adopted as an advanced treatment process to reduce suspended solids and turbidity in treated wastewater effluents designated for potable reuse. Although microfilter pores are much larger than viruses, the addition of a coagulant upstream of a microfilter system can achieve stable virus removal. Ceramic membranes have a narrow pore size distribution to achieve the high removal of contaminants.

Usefulness of myoglobin containing cobalt heme cofactor in designing a myoglobin-based artificial oxygen carrier.

The structure and reactivity of cobalt-replaced myoglobin (Mb) were investigated to explore its possible application as an artificial oxygen carrier. Ligand binding analysis with relaxation kinetics revealed that various ligands bind to Co(III) Mb, contrary to the earlier thoughts. The equilibration process, however, was so slow that it proceeded over 90 min.

How does hemoglobin generate such diverse functionality of physiological relevance?

The absolute values of the O2-affinities (P50, Klow, and Khigh) of hemoglobin (Hb) are regulated neither by changes in the static T-/R-quaternary and associated tertiary structures nor the ligation states. They are pre-determined and regulated by the extrinsic environmental factors such as pH, buffers, and heterotropic effectors. The effect and role of O2 on Hb are reversibly to drive the structural allosteric equilibrium between the T(deoxy)- and R(oxy)-Hb toward R(oxy)-Hb (the structural allostery).

T-quaternary structure of oxy human adult hemoglobin in the presence of two allosteric effectors, L35 and IHP.

The cooperative O(2)-binding of hemoglobin (Hb) have been assumed to correlate to change in the quaternary structures of Hb: T(deoxy)- and R(oxy)-quaternary structures, having low and high O(2)-affinities, respectively. Heterotropic allosteric effectors have been shown to interact not only with deoxy- but also oxy-Hbs causing significant reduction in their O(2)-affinities and the modulation of cooperativity. In the presence of two potent effectors, L35 and inositol hexaphosphate (IHP) at pH 6.

Millisecond time-scale protein dynamics exists prior to the activation of the bulk solvent matrix.

Conformational dynamics of proteins is of fundamental importance in their physiological functions. The exact mechanisms and determinants of protein motions, including the regulatory interplay between protein and solvent motions, are not yet fully understood. In the present work, the thermal activation of phosphorescence quenching was measured in oxygen-saturated aqueous protein solutions to explore protein dynamics in the millisecond range.

Protein dynamics explain the allosteric behaviors of hemoglobin.

Bohr, Hasselbalch, and Krogh discovered homotropic and heterotropic allosteric behaviors of hemoglobin (Hb) in 1903/1904. A chronological description since then of selected principal models of the allosteric mechanism of Hb, such as the Adair scheme, the MWC two-state concerted model, the KNF induced-fit sequential model, the Perutz stereochemical model, the tertiary two-state model, and the global allostery model (an expanded MWC models), is concisely presented, followed by analysis and discussion of their limitations and deficiencies. The determination of X-ray crystallographic structures of deoxy- and ligated-Hb and the structure-based stereochemical model by Perutz are an epoch-making event in this history.

Molecular dynamics simulations of hemoglobin A in different states and bound to DPG: effector-linked perturbation of tertiary conformations and HbA concerted dynamics.

Recent functional studies reported on human adult hemoglobin (HbA) show that heterotropic effector-linked tertiary structural changes are primarily responsible for modulating the oxygen affinity of hemoglobin. We present the results of 6-ns molecular dynamics simulations performed to gain insights into the dynamical and structural details of these effector-linked tertiary changes. All-atom simulations were carried out on a series of models generated for T- and R-state HbA, and for 2,3-diphosphoglycerate-bound models.

Common dynamics of globin family proteins.

The recently discovered new members of the globin family, neurogobin and cytoglobin, are the object of sustained structural and functional studies aimed at understanding their physiological role and elucidating the impact of their bis-his heme hexacoordination. However, no studies have yet considered the dynamics of this protein family, an essential link between structure and function. In this communication, we present normal mode analysis results for neuroglobin, cytoglobin, hemoglobin and myoglobin to provide exploratory insights into globin characteristic motions.

Method for determination of association and dissociation rate constants of reversible bimolecular reactions by isothermal titration calorimeters.

The rate law equation for reversible bimolecular reactions, which are describable by association and dissociation rate constants (k1 and k-1), is not solvable to a plain formula under stoichiometric reaction conditions. Therefore, it is a general technique to observe such reactions under pseudo first-order conditions, which make the reactions a single-exponential process, and enable us to determine k1 and k-1 without any complicated iterative computations needed to analyze the same reactions under stoichiometric reaction conditions. However, the accelerated reaction rates under pseudo first-order conditions are not always favorable to the physicochemical tools employing a slow or medium response time, such as thermal analysis instruments.

Erythrocytes with T-state-stabilized hemoglobin as a therapeutic tool for postischemic liver dysfunction.

This study aimed to examine if T-state stabilization of hemoglobin in erythrocytes could protect against postischemic organ injury. Human erythrocytes containing three different states of Hb allostery were prepared: control Hb (hRBC), CO-Hb that is stabilized under R-state with the 6-coordinated prosthetic heme (CO-hRBC), and alpha-NO-deoxyHb stabilized under T-state (alpha-NO-hRBC). To prepare alpha-NO-RBC, deoxygenated RBC was treated with FK409, a thiol-free NO donor, at its half molar concentration to that of Hb; this procedure resulted in the 5-coordinated NO binding on the alpha-subunit heme, as judged by electron spin resonance spectrometry.

Allosteric effectors influence the tetramer stability of both R- and T-states of hemoglobin A.

The contribution of heterotropic effectors to hemoglobin allostery is still not completely understood. With the recently proposed global allostery model, this question acquires crucial significance, because it relates tertiary conformational changes to effector binding in both the R- and T-states. In this context, an important question is how far the induced conformational changes propagate from the binding site(s) of the allosteric effectors.

Normal mode analysis of the horseradish peroxidase collective motions: correlation with spectroscopically observed heme distortions.

Horseradish peroxidase C is a class III peroxidase whose structure is stabilized by the presence of two endogenous calcium atoms. Calcium removal has been shown to decrease the enzymatic activity of the enzyme and significantly affect the spectroscopically detectable properties of the heme, such as the spin state of the iron, heme normal modes, and distortions from planarity. In this work, we report on normal mode analysis (NMA) performed on models subjected to 2 ns of molecular dynamics simulations to describe the effect of calcium removal on protein collective motions and to investigate the correlation between active site (heme) and protein matrix fluctuations.

R-state haemoglobin with low oxygen affinity: crystal structures of deoxy human and carbonmonoxy horse haemoglobin bound to the effector molecule L35.

Although detailed crystal structures of haemoglobin (Hb) provide a clear understanding of the basic allosteric mechanism of the protein, and how this in turn controls oxygen affinity, recent experiments with artificial effector molecules have shown a far greater control of oxygen binding than with natural heterotropic effectors. Contrary to the established text-book view, these non-physiological compounds are able to reduce oxygen affinity very strongly without switching the protein to the T (tense) state. In an earlier paper we showed that bezafibrate (BZF) binds to a surface pocket on the alpha subunits of R state Hb, strongly reducing the oxygen affinity of this protein conformation.

Quaternary structures of intermediately ligated human hemoglobin a and influences from strong allosteric effectors: resonance Raman investigation.

The Fe-histidine stretching (nu(Fe-His)) frequency was determined for deoxy subunits of intermediately ligated human hemoglobin A in equilibrium and CO-photodissociated picosecond transient species in the presence and absence of strong allosteric effectors like inositol(hexakis)phosphate, bezafibrate, and 2,3-bisphosphoglycerate. The nu(Fe-His) frequency of deoxyHb A was unaltered by the effectors. The T-to-R transition occurred around m = 2-3 in the absence of effectors but m > 3.

R-state hemoglobin bound to heterotropic effectors: models of the DPG, IHP and RSR13 binding sites.

We performed a docking study followed by a 500-ps molecular dynamics simulation of R-state human adult hemoglobin (HbA) complexed to different heterotropic effectors [2,3-diphosphoglycerate (DPG), inositol hexaphosphate (IHP), and 2-[4-[(3,5-dichlorophenylcarbamoyl)-]methyl]-phenoxy]-2-methylpropionic acid (RSR13)) to propose a molecular basis for recently reported interactions of effectors with oxygenated hemoglobin. The simulations were carried out with counterions and explicit solvation. As reported for T-state HbA, the effector binding sites are also located in the central cavity of the R-state and differ depending on effector anionic character.

Semihemoglobins, high oxygen affinity dimeric forms of human hemoglobin respond efficiently to allosteric effectors without forming tetramers.

Significant reduction in oxygen affinity resulting from interactions between heterotropic allosteric effectors and hemoglobin in not only the unligated derivative but also the fully ligated form has been reported (Tsuneshige, A., Park, S. I.

Normal coordinate structural decomposition of the heme distortions of hemoglobin in various quaternary states and bound to allosteric effectors.

The distortions of the alpha1, alpha2, beta1, and beta2 hemes of human hemoglobin (HbA) in various quaternary states and as affected by the presence of allosteric effectors was investigated by subjecting CHARMM energy-minimized models to normal coordinate structural decomposition (NSD) analysis. NSD was applied to the individual hemes extracted from the R, T, and R2-state models of HbA and to HbA bound to DPG and to IHP. Overall, NSD results are indicative of characteristic distortions, not only for the hemes of the different HbA quaternary states, but also for the hemes of the HbA models bound to allosteric effectors.

The global allostery model of hemoglobin: an allosteric mechanism involving homotropic and heterotropic interactions.

Studies of the allosteric mechanism of hemoglobin (Hb) have evolved from phenomenological descriptions to structure-based molecular mechanisms, as the molecular structures of Hb in deoxy and ligated states have been elucidated. The MWC two-state concerted model has been the widely accepted as the most plausible of the allosteric mechanisms of Hb. It assumes that the O2-affinity of Hb is regulated/controlled primarily by the T/R quaternary structural transition and that heterotropic effectors bind preferentially to T (deoxy) Hb to shift the T/R allosteric equilibrium toward the T state.

Description of hemoglobin oxygenation under universal solution conditions by a global allostery model with a single adjustable parameter.

The Monod-Wyman-Changeux allosteric model parameters evaluated from accurate oxygen equilibrium curves (OECs) of hemoglobin that were measured in an extremely wide range of structural constraints, imposed by allosteric effectors, yielded a closed circle when log K(T) and log K(R) were plotted against log L(0) and log L(4), respectively, showing novel phenomena that L(0) and L(4) have a maximal value and a minimal value, respectively, and K(T) and K(R) vary by more than three orders of magnitude. These phenomena were successfully described by a global allostery model, which mathematically keeps the frame work of the MWC model, but allows that K(T) under a set of solution conditions becomes larger than K(R) under another set of solution conditions and postulates that a representative allosteric effector binds to both the T and R states with a lower affinity but with a larger stoichiometry for the R state than for the T state. Thus, this global model can describe any given OEC measured under universal solution conditions with the single adjustable parameter, the concentration of the representative effector.

Heterotropic effectors control the hemoglobin function by interacting with its T and R states--a new view on the principle of allostery.

Careful analyses of precise oxygenation curves of hemoglobin (Hb) clearly indicate that, contrary to the common belief, allosteric effectors exert a dramatic control of the oxygenation characteristics of the protein by binding not only to the T (unligated), but also to the R (ligated) state, in a process that is proton-driven and involves proton uptake. The most striking functional changes were obtained when the allosteric effectors were bound to the fully ligated Hb: the oxygen affinity decreased dramatically, Bohr effect was enhanced, and cooperativity of oxygen ligation was almost absent, emulating a Root effect-like behavior. However, structural analysis, such as Cys beta 93 sulfhydryl reactivity and ultraviolet circular dichroism, confirmed that the ligated Hb was in fact in the R state, despite its extremely low affinity state features.

Crystal structure of horse carbonmonoxyhemoglobin-bezafibrate complex at 1.55-A resolution. A novel allosteric binding site in R-state hemoglobin.

Bezafibrate, an antilipidemic drug, is known as a potent allosteric effector of hemoglobin. The previously proposed mechanism for the allosteric potency of this drug was that it stabilizes and constrains the T-state of hemoglobin by specifically binding to the large central cavity of the T-state. Here we report a new allosteric binding site of fully liganded R-state hemoglobin for this drug.

Global allostery model of hemoglobin. Modulation of O(2) affinity, cooperativity, and Bohr effect by heterotropic allosteric effectors.

The O(2) equilibria of human adult hemoglobin have been measured in a wide range of solution conditions in the presence and absence of various allosteric effectors in order to determine how far hemoglobin can modulate its O(2) affinity. The O(2) affinity, cooperative behavior, and the Bohr effect of hemoglobin are modulated principally by tertiary structural changes, which are induced by its interactions with heterotropic allosteric effectors. In their absence, hemoglobin is a high affinity, moderately cooperative O(2) carrier of limited functional flexibility, the behaviors of which are regulated by the homotropic, O(2)-linked T/R quaternary structural transition of the Monod-Wyman-Changeux/Perutz model.