Mb, Hb, Allostery, and Motors - Biology

Mb, Hb, Allostery, and Motors - Biology

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Advantages of complex (quaternary) protein structures

• Stability: decreased surface-to-volume -> more hydrophobic interactions

• New sites: e.g., immunoglobulin binding sites

• Coupled reactions:

trptophan synthetase:

Indoleglycerol-P --> G3P + indole

Indole + ser --> trp

purine (A, G) synthesis: 10 reactions, 6

enzymes, 1 complex (in purine depleted medium)

• Cooperativity: e.g., allostery

Example of Cooperativity: Mb/Hb (Myoglobin and Hemoglobin)

  • shows advantage of quaternary structure
  • shows examples of flexibility: low ΔG of shape change


• MW ca 17,000 daltons

• 75% -helix

• Heme prosthetic group: protoporphyrin ring plus Fe2+

• Heme binds O2 as porphyrin-Fe2+-O2, color change from brown --> red

• Serves as an O2 buffer

• Hyperbolic saturation curve shows that there is no coordinate activity

Why is this an O2 buffer? High slope below the P50 means that considerable Mb is charged (or uncharged) for a small change in pO2 (as pO2 drops, MbO2 replenishes O2)


• Tetramer of myoglobin-like subunits, each with...

• Heme prosthetic groups: protoporphyrin ring plus Fe2+

• MW ca 4 x 17,000 daltons

• 75% -helix

• Complexed with O2, porphyrin-Fe2+-O2, brown --> red

• Better O2 buffer (at a higher [O2])

• Sigmoid saturation curve shows that there is coordinate activity: “positive, homotropic, allosteric effector"

Bohr effect: H+, CO2 promote dissociation of O2 from Hb-O2: "negative, heterotropic, allosteric effector." The Bohr effect in hemoglobin can also be depicted as an oxygen-binding curve. There is a proportional relationship between the affinity of pxygen and pH level. As the pH level decreases, the affinity of oxygen in hemoglobin also decreases. As hemoglobin approaches low pH, more oxygen is released.

2,3-bisphosphoglycerate also promotes dissociation of O2. Purified hemoglobin binds much more tightly to the oxygen, making it less useful in oxygen transport. The difference in characteristics is due to the presence of 2,3-Bisphosphoglycerate(2,3-BPG) in human blood, which acts as an allosteric effector. An allosteric effector binds in one site and affects binding in another. 2,3-BPG binds to a pocket in the T-state (taut) of hemoglobin and is released as it forms the R-state (relaxed). The presence of 2,3-BPG means that more oxygen must be bound to the hemoglobin before the transition to the R-form is possible.

Lung conditions (Low H+, CO2) promotes O2 saturation; tissue conditions (high H+, CO2) promote O2 release; 2,4-BPG magnifies the allosteric effects. Allosteric effects match the saturation curve to the conditions in lung and tissue.

Motor Proteins

Types (substrate-motor)


• M icrotubules (tubulin) – dynein (+ to -), kinesin (- to +, with exception—Science 1April 2011)

• DNA – helicases

• Microfilaments (actin) – myosin


• Bacterial flagella

• F0F1 ATP synthases

Motion depends on

• Flexible 3o structure

• Reversible binding

• ATP hydrolysis affecting binding


Microtubule: right handed hallow helix of tubulin α/β dimers

kinesin: left-handed helix with two globular heads

Each step depends on flexibility (“rotation”)

Each step hydrolyzes one ATP (--> ADP + Pi)

Each step involves an exchange reaction

(There is another motor protein, dynein, which moves along microtubules.

Its 4-A crystal structure was recently reported (Science 331:1159, 3/4/11),

but its mechanism of action is still unknown.)


Microfilaments: right-handed double helix of actin monomers

myosin: left-handed coil- (alpha-helix) coil

Myosin is an ATPase. Would you expect the addition of actin to increase or decrease ATP hydrolysis activity?

(Reaction rate: 0.05 s-1 --> 10 s-1)

Actomyosin in Muscles

Contraction: sliding in the A band from myosin-actin connections

Why rigor mortis? When there is a loss of ATP, the muscles cannot relax because it cannot be broken down into ADP


Flexibility in protein structures allows more complex functions

  • Reversible O2 and CO2 binding
  • Reversible protein-protein (kinesin-MT) binding

Shows the importance of low ΔG in protein shape changes

Allosteric Regulation


Allosteric regulation refers to the process for modulating the activity of a protein by the binding of a ligand, called an effector, to a site topographically distinct from the site of the protein, called the active site, in which the activity characterizing the protein is carried out, whether catalytic (in the case of enzymes) or binding (in the case of receptors) in nature. The word allosteric, Greek for other site, was coined to emphasize this distinctness. The modulation of protein activity is accomplished by the reversible alteration of the protein conformation that accompanies effector binding. Effectors that increase activity are called activators, while those that decrease activity are called inhibitors. For the purposes of this article, we will use the term substrate to indicate a ligand bound to the active site of either an enzyme or a receptor that undergoes the characteristic activity of the protein.

Hemoglobin contains four heme units each embedded in a globular protein sub-unit. There are two types of protein sub-units i.e., &alpha and &beta.

Myoglobin contains only one heme unit surrounded by a globular protein, containing seven &alpha-helical and six non helical segments, made up of 153 amino acids .

Note: Heme moieties are shown in green color in above diagram. Fe(II) ion is shown in red color.

Heme is a porphyrin ring system made up of four pyrrole rings with an Fe(II) ion coordinated to nitrogens of pyrrole rings.

Globin part prevents irreversible oxidation of Fe(II) ion by providing hydrophobic environment. It enhances the selectivity for O2 binding. In hemoglobin, the tetramer arrangement allows for co-operativity by making it more efficient in binding to dioxygen.

Hemoglobin and Myoglobin exist in two forms i.e.,

1) deoxy form: No oxygen is bound to iron.

2) oxy form: dioxygen is bound to iron.

In deoxy-hemoglobin, four of the coordinated sites of iron are occupied by nitrogens of porphyrin ring. The fifth site is occupied by Histidine residue (called proximal histidine) of globin. The sixth position is occupied by weakly bonded water molecule. Hence some authors tend to report Fe(II) ion in deoxy form as pentacoordinated. Deoxy-hemoglobin is said to be in T-state (tense).

On the opposite side of the proximal histidine, there is one more histidine group (called distal histidine) placed near the iron ion. It forces the binding of dioxygen in "end on bent" confirmation.

Note: The bent confirmation discourages the binding of CO to heme iron. Otherwise, CO may have even more affinity with the iron ion. It is observed that CO binds to hemoglobin 200X stronger than dioxygen but binds 20,000X stronger with unprotected heme.

Hemoglobin coordinated to dioxygen is called oxy-hemoglobin. It is also referred to as R-state (relaxed). In oxy-hemoglobin the sixth coordinated position of iron is occupied by dioxygen in "end on bent" geometry.


In deoxy-Hemoglobin, the porphyring ring is dome shaped. The Fe(II) is in high spin state and is paramagnetic. Its size is 0.78 A o and is positioned above the plane of the porphyrin ring.

However, in oxy-Hemoglobin, the size of iron ion is reduced to 0.61 A o and can fit into the cavity of planar porphyrin ring and hence moves into the cavity of porphyrin ring with concomitant dragging of proximal histidine that triggers the conformational changes in other globin subunits and thus by opening other heme sites. As a result, the binding capacity of other heme irons with dioxygen is enhanced. This is best example for co-operativity through allostery.

The nature of Fe in oxy-Hemoglobin or in oxy-Myoglobin is controversial.

According to old Pauling model, there is a low spin Fe(II) ion that is bound to singlet O2 in oxy-Hb. Both are diamagnetic.

However, according to Weiss model, there is Fe(III) ion bound to superoxide radical anion (O 2- ). Though both are paramagnetic, a strong paramagnetic coupling between them ensues diamagnetic behavior . This model is supported by the O-O stretching frequency at 1105 cm -1 in resonance raman spectrum that is consistent with the fact that O2 is in superoxide form. This deems to be more accurate and modern explanation.

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University La Sapienza, Rome, Italy

University La Sapienza, Rome, Italy

University La Sapienza, Rome, Italy

University La Sapienza, Rome, Italy


Hemoglobin (Hb) is the generic name for a vital protein, basic to oxygen (O2) metabolism of all vertebrates, some invertebrates, and plants that perform nitrogen fixation. The only vertebrates that survive without Hb in their blood are antarctic fishes (such as Chaenocephalus aceratus Lonnberg ) that bear the Hb genes but do not express the proteins. Structural and functional properties of Hb vary widely in the different species, according to physiological requirements. The multifaceted behavior of Hb has challenged the interest of many scientists with different backgrounds (biochemists, physiologists, geneticists, and biophysicists), who have investigated this protein from extremely diverse points of view (from the quantum chemistry of the heme iron to the unloading of O2 into the swim bladder of fish, thus regulating the creature's buoyancy). Increasing concern over viral contamination of blood is spurring the development of a blood substitute solutions of chemically modified hemoglobin represent one option.


Our ensemble MD simulation revealed that, even without the heme deformation by the O2 binding to the heme, the solution environment of high O2 partial pressure itself enhances the quaternary structural change from T to R. This means that the traditional site-specific allosteric regulation by O2 binding to the heme is not necessarily the only one unique mechanism of O2 allostery and the additional non-site-specific regulation does exist. From a molecular point of view, we propose following two hypothetical mechanisms of the non-site-specific allostery of O2.

First is the internal effect: O2 molecules enter the hydrophobic cavities inside HbA subunits 20 and trigger the tertiary structural changes to enhance the T to R quaternary change. In MD O2 simulations, hydrophobic O2 molecules tended to escape from water and prefer the hydrophobic environment on the surface or inside HbA subunits 14 . The O2 migrations between inside cavities would bring about the tertiary structural changes of HbA subunits since it was observed that, in Mb, structural changes occur in response to the ligand migrations between cavities 21,22,23 . In particular, these reported structural changes in response to the CO transitions between the heme pocket and Xe4 cavity included displacements of the F helix, which forms the switch region of HbA β subunit 22 and hence would affect the stability of the T state structure.

Second is the surface effect: the existence of O2 around the subunit interfaces weakens the contacts which stabilize the T state structure 3 by affecting the surface residues directly and/or indirectly through perturbing the behavior of water molecules. In fact, there are obviously high O2 density locations in the vicinity of the switch region (Fig. 4). Thus it should be possible that the existance of O2 directly affects the switch region residues through steric hinderance to weaken the contacts, while O2 also influences surface water molecules to indirectly affect the switch region residues. In general, the characteristic constants of water molecules near the protein surfaces, such as diffusion constant 24 , can be different from that in bulk solvent. Moreover, it has been reported that the number of interfacial water molecules changes during quaternary changes in hemoglobins 25,26,27,28 , suggesting the important roles of the number of interfacial water molecules in the quaternary regulation. Thus, we can conjecture that the existence of O2 would perturb the behavior of water molecules, e.g., as the deviation of diffusion constants and the number of interfacial water molecules around the surfaces, and, as a result, indirectly weaken the intersubunit contacts to enhance the T to R quaternary change.

Distribution of O2 oxygen atoms around switch region.

Distribution of O2 oxygen atoms calculated from 128 MD trajectories during 7 to 8 ns in the vicinity of the switch region at the α1β2-interface switch region. Locations with density >0.05 Å −3 and >0.1 Å −3 are drawn with red wire frame and solid surface, respectively. Since the average density in the bulk solvent region (distance to the nearest protein atom >4.0 Å) is about 0.005 Å −3 , the wire frame and solid surface regions are 10 and 20 times denser than that in the bulk solvent, respectively. Four high density locations around the switch region are numbered in descending order of the peak density.

The O2 partial pressure applied in this work (0.55 mol/L) is about 500 times higher than that in the O2 saturation concentration at ambient conditions (

1 mmol/L). We anticipate that the non-site-specific effects should be also observed under the ambient conditions for the following three reasons. First, our previous computational analysis with the same concentration 0.55 mol/L reproduced the rate constants of O2 entry into the binding sites of HbA subunits 14 . Assuming a kinetic model of O2 entry

where x denotes subunit (x = α or β) and is the situation of O2 in the binding site, the rate of O2 entry is and the rate constants and are calculated to be 45 and 99 (μmol/L) −1 s −1 , respectively. These values are consistent with experimentally observed values after temperature correction, 48–69 and 81–131 (μmol/L) −1 s −1 for α and β subunits, respectively. This consistency indicates that the concentration 0.55 mol/L is within the linearly extrapolatable range by the O2 concentration, as the rate constants can be estimated by the formula . Second, the “effective” O2 concentration around HbA is higher than the bulk concentration. As discussed above, hydrophobic O2 molecules prefer the hydrophobic environment near the HbA surface and there are several high O2 density regions, whose density is an order of magnitude higher than that in the bulk solvent region (Fig. 4). Therefore, compared to the bulk concentration, the “effective” O2 concentration around HbA is higher. This should partially narrow the concentration gap between the current simulation conditions and ambient conditions. Third, a preliminary ensemble MD simulation at one-tenth concentration, 0.055 mol/L (MD lowO2 with 12 O2 and

12000 H2O molecules) also revealed the non-site-specific effect. We executed 40 MD simulations for 8 ns at the 0.055 mol/L concentration and calculated the χ distribution during 7–8 ns (Supplementary Fig. S6). The MD lowO2 distribution is apparently shifted toward the R state as in MD O2 .

From an experimental point of view, it is necessary to analyze the number of O2 molecules around or inside HbA subunits because the number of O2-bound heme in tetrameric HbA, which is the traditional index of O2 allostery and is easily observed by spectroscopy, cannot capture the non-site-specific O2 allosteric contributions. In particular, as Tomita et al. discussed in Mb case 29 , accurate measurement of the heme:O2 stoichiometry with modern instrumentation is desired to verify the traditionally employed stoichiometry 1:1, which assumes that only one O2 binds to the heme and there is effectively no O2 in the hydrophobic cavities or near the surfaces of HbA subunits. The stoichiometry greater than 1:1 is plausible by hydrophobic interactions between O2 and HbA and means that HbA subunits can carry excess O2 in their cavities and/or surfaces, or the “effective” O2 concentration around HbA is higher than bulk concentarion and support the existence of the non-site-specific effect.

The current non-site-specific effect should provide a complementary mechanism to account for the cleavages of intersubunit contacts during the T to R quaternary change of HbA in high O2 concentration environment from the molecular point of view. With respect to the displacements in the heme average strucutre, the O2-heme binding itself brings about very small structural rearrangements: a comparison of high resolution crystal structures of Mb revealed that the heme iron atom is 0.290 Å displaced by ligand binding 10 . Since the immediate displacements in the residues distant from the heme within 20 ps after ligand phosolysis are smaller than those neighboring to the heme because of the elastic behavior of Mb 11 , the immediate displacements propagated to the residues composing the intersubunit contacts must be smaller than the iron displacement. It is not obvious how such a small immediate displacements could cleavage the contacts, although it was in fact experimentally obserbed that the T to R quaternary change occurs after microseconds of ligand dissociation 30 . Meanwhile, by the dynamic non-equilibrium MD approach in Mb, it was shown that a large structural fluctuation in the FG-corner (that of β subunit forms the switch region in HbA) was brought about by the ligand dissociation and recombination to the heme 31 . Together with this structural fluctuation effect, O2 molecules located near the intersubunit contacts as in Fig. 4 can interact with the contacts, playing a complementary role in cleavage of the contacts.

The non-site-specific effect can also be an important factor in allosteric structural regulation in other proteins. For example, in muscarinic receptors, orthosteric ligands were observed to function as an allosteric modulator 32 or weakly bind to the allosteric sites 33 , indicating that the ligands can interact with multiple sites not only the orthosteric site but also allosteric sites. In particular, for allosteric proteins in which the distance between orthosteric and allosteric sites is so far-away that direct structural perturbation between the sites seems to be unlikely, the multiple interaction sites between proteins and ligands, most of which are invisible by the X-ray crystallography because of their small occupancy, can help the structural rearrangements at the orthosteric sites. The concept of non-site-specific allostery should facilitate further understanding of allosteric regulation process depending on the concentration of effectors from the atomistic point of view.


Carbon monoxide (CO) poisoning causes between 5,000−6,000 deaths per year in the US alone. The development of small molecule allosteric effectors of CO binding to hemoglobin (Hb) represents an important step toward making effective therapies for CO poisoning. To that end, we have found that the synthetic peptide IRL 2500 enhances CO release from COHb in air, but with concomitant hemolytic activity. We describe herein the design, synthesis, and biological evaluation of analogs of IRL 2500 that enhance the release of CO from COHb without hemolysis. These novel structures show improved aqueous solubility and reduced hemolytic activity and could lead the way to the development of small molecule therapeutics for the treatment of CO poisoning.


Protein uses allostery to execute biological function. The physical mechanism underlying the allostery has long been studied, with the focus on the mechanical response by ligand binding. Here, we highlight the electrostatic response, presenting an idea of “dielectric allostery”. We conducted molecular dynamics simulations of myosin, a motor protein with allostery, and analyzed the response to ATP binding which is a crucial step in force-generating function, forcing myosin to unbind from the actin filament. We found that the net negative charge of ATP causes a large-scale, anisotropic dielectric response in myosin, altering the electrostatic potential in the distant actin-binding region and accordingly retracting a positively charged actin-binding loop. A large-scale rearrangement of electrostatic bond network was found to occur upon ATP binding. Since proteins are dielectric and ligands are charged/polar in general, the dielectric allostery might underlie a wide spectrum of functions by proteins.

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In the early 1900s, Christian Bohr was a professor at the University of Copenhagen in Denmark, already well known for his work in the field of respiratory physiology. [3] He had spent the last two decades studying the solubility of oxygen, carbon dioxide, and other gases in various liquids, [4] and had conducted extensive research on haemoglobin and its affinity for oxygen. [3] In 1903, he began working closely with Karl Hasselbalch and August Krogh, two of his associates at the university, in an attempt to experimentally replicate the work of Gustav von Hüfner, using whole blood instead of haemoglobin solution. [1] Hüfner had suggested that the oxygen-haemoglobin binding curve was hyperbolic in shape, [5] but after extensive experimentation, the Copenhagen group determined that the curve was in fact sigmoidal. Furthermore, in the process of plotting out numerous dissociation curves, it soon became apparent that high partial pressures of carbon dioxide caused the curves to shift to the right. [4] Further experimentation while varying the CO2 concentration quickly provided conclusive evidence, confirming the existence of what would soon become known as the Bohr effect. [1]

Controversy Edit

There is some more debate over whether Bohr was actually the first to discover the relationship between CO2 and oxygen affinity, or whether the Russian physiologist Bronislav Verigo [ru] beat him to it, allegedly discovering the effect in 1898, six years before Bohr. [6] While this has never been proven, Verigo did in fact publish a paper on the haemoglobin-CO2 relationship in 1892. [7] His proposed model was flawed, and Bohr harshly criticized it in his own publications. [1]

Another challenge to Bohr's discovery comes from within his lab. Though Bohr was quick to take full credit, his associate Krogh, who invented the apparatus used to measure gas concentrations in the experiments, [8] maintained throughout his life that he himself had actually been the first to demonstrate the effect. Though there is some evidence to support this, retroactively changing the name of a well-known phenomenon would be extremely impractical, so it remains known as the Bohr effect. [4]

The Bohr effect increases the efficiency of oxygen transportation through the blood. After hemoglobin binds to oxygen in the lungs due to the high oxygen concentrations, the Bohr effect facilitates its release in the tissues, particularly those tissues in most need of oxygen. When a tissue's metabolic rate increases, so does its carbon dioxide waste production. When released into the bloodstream, carbon dioxide forms bicarbonate and protons through the following reaction:

Although this reaction usually proceeds very slowly, the enzyme carbonic anhydrase (which is present in red blood cells) drastically speeds up the conversion to bicarbonate and protons. [2] This causes the pH of the blood to decrease, which promotes the dissociation of oxygen from haemoglobin, and allows the surrounding tissues to obtain enough oxygen to meet their demands. In areas where oxygen concentration is high, such as the lungs, binding of oxygen causes haemoglobin to release protons, which recombine with bicarbonate to eliminate carbon dioxide during exhalation. These opposing protonation and deprotonation reactions occur in equilibrium resulting in little overall change in blood pH.

The Bohr effect enables the body to adapt to changing conditions and makes it possible to supply extra oxygen to tissues that need it the most. For example, when muscles are undergoing strenuous activity, they require large amounts of oxygen to conduct cellular respiration, which generates CO2 (and therefore HCO3 − and H + ) as byproducts. These waste products lower the pH of the blood, which increases oxygen delivery to the active muscles. Carbon dioxide is not the only molecule that can trigger the Bohr effect. If muscle cells aren't receiving enough oxygen for cellular respiration, they resort to lactic acid fermentation, which releases lactic acid as a byproduct. This increases the acidity of the blood far more than CO2 alone, which reflects the cells' even greater need for oxygen. In fact, under anaerobic conditions, muscles generate lactic acid so quickly that pH of the blood passing through the muscles will drop to around 7.2, which causes haemoglobin to begin releasing roughly 10% more oxygen. [2]


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Keywords: Mac-1, GPIbα, molecular dynamics simulation, structure𠄿unction relation, leukocyte–platelet interaction

Citation: Jiang X, Sun X, Lin J, Ling Y, Fang Y and Wu J (2021) MD Simulations on a Well-Built Docking Model Reveal Fine Mechanical Stability and Force-Dependent Dissociation of
Mac-1/GPIbα Complex. Front. Mol. Biosci. 8:638396. doi: 10.3389/fmolb.2021.638396

Received: 06 December 2020 Accepted: 12 February 2021
Published: 22 April 2021.

Agnel Praveen Joseph, Science and Technology Facilities Council, United Kingdom

Matteo Degiacomi, Durham University, United Kingdom
Jinan Wang, University of Kansas, United States

Copyright © 2021 Jiang, Sun, Lin, Ling, Fang and Wu. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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