Student Projects for UMass Chemistry 423 Spring 2012-8

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Hemoglobin 1qxd


Polymerization of hemoglobin occurs when a mutant HbS molecule, in which the Glu6 residues have been replaced by a Val6 residue, binds to the another hemoglobin molecule at the region defined by Phe85 - Leu88. In other words, a hydrophobic interaction is formed between the Glu6 residue of one hemoglobin molecule, and the Phe85 - Leu88 region of another hemoglobin molecule

Individuals with Sickle Cell Anemia, or Sickle Cell Disease, contain a mutated form of hemoglobin, the oxygen binding protein found in red blood cells. Mutated hemoglobin causes normal disk-shaped red blood cells to become sickle-shaped. These sickle cells are fragile, deliver less oxygen to the body's tissues, and clog small blood vessels and capillaries, which results in a variety of adverse symptoms and detrimental complications. Some of these symptoms include abdominal and bone pain, breathlessness, fatigue, and rapid heart rate. Over time, irreversible tissue damage leads to the failure of many organ systems.[1]

Sickle Cell Disease results from a single point mutation in the Hemoglobin amino acid sequence. Normal Hb contains hydrophilic Glu6 residues in the 2 beta strands, shown , whereas in HbS, these residues have been changed to hydrophobic Val6. The mutation region of one HbS molecule will then bind to a region defined by β Phe85, βAla86, βThr87, β Leu88 in the Heme pocket of another HbS molecule via noncovalent hydrophobic interactions.. The subsequent polymerization of HbS molecules leads to the sickling of red blood cells. [2]

The cooperative binding of oxygen leads to a conformation change in hemoglobin from the tense, or T state, to the R, or relaxed state. Recently, studies have shown that multiple relaxed Hb conformers exist, such as the R2, RR2, and R3 states.[3] It has been proven that sickling only occurs with the deoxygenated T-state Hb, and it is therefore desirable to explore ways in which allosteric equilibrium can be shifted toward the oxygenated R-state conformations. Compounds that achieve such an equilibrium shift are being sought. Vanillin, a food flavoring compound, as well as the furanic aldehyde compounds 5-hydroxymethyl-2-furfural (5HMF), 5-methyl-2-furfural (5MF), 5-ethyl-furfural (5EF), and furfural (FUF) all exhibit such antisickling properties and are nontoxic to humans. These compounds are therefore promising candidates for potential SCD drug treatments. Of the compounds studied, 5HMF was the most potent, shifting the oxygen equilibrium curve to the left by over 25 mmHg. Additionally, an equilibrium shift of approximately 16 mmHG was observed in FUF. It will later be seen that these compounds bind and stabilize the R2 conformation.[4].

Overall Structure

Hemoglobin is a tetramer of two types of globular subunits: Alpha Chains, shown initially in Blue and Pink, and Beta Chains, shown in Yellow and Green

Ordinary human hemoglobin is a tetramer of globular protein subunits: two α chains and two β chains. Both the α and β subunits are identical, and form two identical αβ dimers, which in turn form a dimer to create the complete structure of hemoglobin. Each of the subunits consists largely of alpha helices, with 8 in both the α and β chains and short, non-helical residue sequences binding them. In total, each α chain contains 141 residues, and each β chain contains 146.

Hemoglobin can exist in two possible conformations of its quaternary structure, depending on whether it is bound to oxygen. The state shown in our green scenes is the T-state (tense state), so named because it's structure is constrained by interactions between subunits. The fully oxygenated state is known as the R-state (relaxed state), as the binding of oxygen results in a 15 degree rotation between the two αβ dimers, thus disrupting many subunit interactions.

The structure of sickle hemoglobin is identical to that of healthy hemoglobin, save for the substitution of a single residue within the β chains. The sixth residue in each chain, a glutamic acid, has been changed to a valine, resulting from a single point mutation. This mutation doesn't actually lead to any interference with the bonding interactions that lend hemoglobin its quaternary structure, and doesn't lead to structural changes within the hemoglobin or the β chain. It does, however, allow for hydrophobic interactions between separate hemoglobin proteins, leading to the polymerization of Hb molecules that causes sickling.

Each of the four globular domains within hemoglobin contains a heme group, the non-protein components that allow hemoglobin to bind to oxygen. Each heme group consists of an iron ion bound within four cyclically bonded pyrrole molecules, referred to as a whole as a porphyrin ring. Each pyrrole molecule consists of a heterocyclic ring of four carbons and one nitrogen, with the nitrogen from each ring bound to the Fe ion at the heme group's center. Each heme group is anchored in place in its respective subunit primarily by a histidine sidechain; a nitrogen atom in the imidazole ring on the sidechain anchors to the iron ion in the heme group, and the propanoate groups attached to the porphyrin ring are held in place by hydrophobic interaction with the hydrophobic residues within the subunit.

Binding Interactions

Binding of 5HMF involves 6 water-mediated hydrogen bonds that link the 2 alpha chains together, stabilizing the R2 Hb conformation

The binding interactions of two of the four aforementioned heterocyclic aldehydes, (FUF) and (5HMF), were investigated. Both bind to the N-terminal αVal1 residues in the α cleft (two binding sites, since hemoglobin is a tetramer with two α chains).

A covalent bond is formed between the ligand aldehyde and the Val1 nitrogen. . Furfural can assume two different conformations when compelxed with Hemoglobin. In one conformer, the ring oxygen faces the α2Ser138 residue and forms a weak intersubunit hydrogen bond with this moiety. In the second conformer, the ring oxygen faces the water cavity, and forms a weak hydrogen bond with α1Ser131. , in which the the ring oxygen is facing the water cavity. Notice that the furfural molecule bound to the α2 chain is interacting with the α1Ser131 residue via water-mediated hydrogen bonds, thus linking the α1 and α2 chains together. Weak hydrophobic interactions are also formed between the furan ring and Lys127 and Ala130 residues on the same chain. For simplicity, interactions involving only one of the furfural molecules is depicted, as those involving the other furfural are identical.

Unlike FUF, cannot rotate within the α cleft and thus assumes only a single conformation, in which the ring oxygen faces the water cavity. Also, 5HMF cannot form a hydrogen bond with α2Ser138, as does FUF in one of its conformations. 5HMF does form an intrasubunit hydrogen bond with α1Ser131 that is stronger than that formed by FUF. In addition, the 5-hydroxymethyl group of 5HMF forms a strong intrasubunit hydrogen bond with the α1Thr134 residue. As mentioned before, FUF linked together the two α chains via hydrogen bonds with Ser131. When 5HMF is bound, this feat is accomplished by a strong network of 6 water-mediated hydrogen bonds via the ring oxygens and hydroxyl groups between the two 5HMF molecules. to the α1 chain forming these interactions.

Additional Features

Hemoglobin polymerization

A hemoglobin polymer looks like this: The polymer shows how sickle cell hemoglobin looks when polymerized and the picture also shows where the mutation of replacing a Glu6 residue with Val6 occurs in the structure. [5]

As discussed earlier in the introduction section, normal Hb contains a hydrophilic Glu6 residues in the 2 beta strands, whereas in HbS, these residues have been changed to hydrophobic Val6. In normal Hb, the hydrophillic negatively charged Glu6 residues do not interact with the hydrophobic Ala86, Phe85, Thr87, and Leu88 residues. This ensures that hemoglobin does not polymerize. The contrast happens in sickle-cell hemoglobin. Since in sickle-cell hemoglobin Glu6 is mutated and is replaced with Val6, hydrophobic interactions occurs with val6 and Ala86, Phe85, Thr87, and Leu88 residues. The reason for this is because valine, leucine, phenylalanine, threonine, and alanine are all hydrophobic due to their non-polar attributes. Val6 from one hemoglobin interacts with the hydrophobic patch formed by Ala86, Phe85, Thr87, and Leu88 residues of another deoxygenated form of hemoglobin leads to polymerization of hemoglobin under low oxygen conditions.[6]


Introduction - Ryan Colombo

Overall Structure - Will Yarr

Drug Binding Site - Jacqueline Pasek-Allen

Additional Features - Joey Nguyen


  1. Geller AK, O'Connor MK. The sickle cell crisis: a dilemma in pain relief. Mayo Clin Proc. 2008;83:320-323.
  2. Safo MK, Abdulmalik O, Danso-Danquah R, Burnett JC, Nokuri S, Joshi GS, Musayev FN, Asakura T, Abraham DJ. Structural basis for the potent antisickling effect of a novel class of five-membered heterocyclic aldehydic compounds. J Med Chem. 2004 Sep 9;47(19):4665-76. PMID:15341482 doi:10.1021/jm0498001
  3. Shibayama N, Sugiyama K, Park SY. Structures and oxygen affinities of crystalline human hemoglobin C (β6 Glu->Lys) in the R and R2 quaternary structures.J Biol Chem. 2011 Sep 23;286(38):33661-8.
  4. Safo MK, Abdulmalik O, Danso-Danquah R, Burnett JC, Nokuri S, Joshi GS, Musayev FN, Asakura T, Abraham DJ. Structural basis for the potent antisickling effect of a novel class of five-membered heterocyclic aldehydic compounds. J Med Chem. 2004 Sep 9;47(19):4665-76. PMID:15341482 doi:10.1021/jm0498001
  5. Dutta, Shuchismita; Goodsell, David, May 2003, Hemoglobin. RCSB Protein Data Bank, 2012.
  6. Harrington DJ, Adachi K, Royer WE Jr. The high resolution crystal structure of deoxyhemoglobin S. J Mol Biol. 1997 Sep 26;272(3):398-407. PMID:9325099 doi:10.1006/jmbi.1997.1253

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