Chapter 5 - Protein Function

What is a "salt bridge"?

A salt bridge is a pair of opposite charges held together by electrostatic attraction. Oppositely charged amino acid sidechains are generally salt-bridged when their charged atoms are less than 4 Å apart.

How does a slightly acidic pH affect charge?

A slightly acidic pH, around 6.5, increases the partial charges on the sidechains of histidines. This is because their sidechain [pKa's are about 6.5. In contrast, lysine and arginine have full positive charges at any physiological pH because their sidechain pKa's are 11-12, while aspartic acid and glutamic acid have full negative charges at any physiological pH because their sidechain pKa's are about 4.

What protein secondary structures make up each chain of hemoglobin?

Alpha helices and loops (including turns) make up the alpha and beta chains of hemoglobin. They contain no beta strands or sheets.

What does "amphipathic" mean?

Amphipathic means having one part that is hydrophobic, and another part that is hydrophilic.

Red blood cells contain one molecule of diphosphoglycerate (DPG) for each molecule of hemoglobin. In what feature of the hemoglobin molecule does DPG bind, favoring the release of oxygen?

DPG binds in a crevice between the beta chains. That crevice is open only in the deoxy conformation. It closes up in the oxy conformation. DPG act like a chock holding the beta chains apart in the low-affinity conformation, facilitating the release of oxygen in tissues.

How many heme-iron complexes are in each hemoglobin molecule?

Each chain in the hemoglobin molecule has a pocket to hold one heme-iron complex. Thus, there are four heme-iron complexes in each hemoglobin molecule.

How many protein chains are in each hemoglobin molecule?

Each hemoglobin molecule is made up of four protein chains.

What holds each iron atom in place?

Each iron atom is held by five nitrogen atoms: four nitrogens in heme, plus one nitrogen in the sidechain of a histidine amino acid.

When saturated with oxygen, how many oxygen atoms are bound to each hemoglobin molecule?

Each of the heme-iron complexes can bind one O2 molecule. Thus, each hemoglobin molecule can bind up to eight atoms of oxygen.

Certain salt bridges stabilize the low oxygen affinity conformation. Which amino acid is crucial in breaking those salt bridges at the highter pH in the lungs?

Histidine is crucial in breaking the salt bridges that favor the low-affinity conformation. The higher pH in the lungs reduces the partial positive charge on histidine, weakening the salt bridge.

Are those chemical bonds covalent or non-covalent in the alpha helix?

Hydrogen bonds are non-covalent bonds.

What kind of chemical bonds stabilize the conformation of an alpha helix?

Hydrogen bonds stabilize the conformation of an alpha helix.

What does "hydrophobic" mean?

Hydrophobic (literally "water fearing") means structures that avoid contact with water because such contact is energetically unfavorable. Hydrophobic structures are apolar. In contrast, hydrophilic structures favor contact with water molecules, usually because they can form hydrogen bonds with water. Hydrophilic structures include polar and charged structures.

When the mutant sickle hemoglobin is the only form present, what happens to it in deoxygenated red blood cells? How does that cause disease?

In sickle homozygotes, the mutant hemoglobin, when deoxygenated, forms chains and fibers stuck together with the mutant hydrophobic amino acid. The fibers distort red blood cells, blocking blood flow (vaso-occlusion) and greatly shortening the lifetime of red blood cells. Vaso-occlusion causes pain and can damage bone, spleen, cause strokes, and impair respiration by damaging lungs. The breaking of red blood cells causes anemia and the excess hemoglobin released free in the blood, which is toxic. The body responds by producing red blood cells faster, which causes problems in bone and liver. For a more complete overview, please see this excellent 8-minute video.

What does oxygen bind to in hemoglobin? (Remember, two sides of the oxygen molecule each bind to different things.)

Molecular oxygen binds between iron (Fe++) and a nitrogen in the sidechain of histidine. The oxygen-binding histidine is on the opposide side of the iron from the iron-binding histidine.

Most people who have sickle hemoglobin have little or no sickle cell disease. Why?

Most people who have sickle hemoglobin are heterozygotes, with one normal and one mutant gene. Half of their hemoglobin is normal, which largely prevents fiber formation and disease. Homozygotes have only sickle hemoglobin, with no normal hemoglobin to impede fiber formation and disease.

Which chemical element in the sidechains of histidines is crucial to those functions?

Nitrogen. The lone-electron pairs of nitrogen are attracted to electron-seeking Fe++ and O2.

What are the functions of the two histidines closest to heme?

One histidine (sequence number 87 in the beta chain) binds to iron (Fe++), helping to hold the iron and heme in place. The other histidine (sequence number 58 in the beta chain) helps to bind molecular oxygen, and probably also serves as a gate to admit and release oxygen (see text under View 11 in Chapter 2).

How do the core and the surface of each protein chain differ?

Protein chains or domains that are soluble in water, such as the alpha and beta chains of hemoglobin, have hydrophobic cores. Amino acids with hydrophilic sidechains are mostly on the surfaces of these chains.

Sickle carriers with little or no disease have an advantage in some parts of the world. What is it?

Sickle hemoglobin reduces the severity of malaria, although it does not prevent infection. Sickle heterozygotes have an advantage where malaria is endemic. (Malaria actually worsens sickle disease in homozygotes, so they have no advantage.)

What holds the polypeptide chains together in one hemoglobin molecule?

The four chains that make up one hemoglobin molecule are held together by non-covalent interactions (non-covalent bonds).

What drives that difference in in how the core and the surface of each protein chain differ.

The hydrophobic cores of proteins are devoid of water. It is the avoidance of water that drives amino acids with hydrophobic sidechains to pack together in the core, while the attraction of hydrophilic amino acids to water favors their locations on the surface of the folded protein chain.

What is the main function of hemoglobin?

The main function of hemoglobin is to transport oxygen from the lungs to tissues throughout the body.

What types of heme-protein contacts predominate in the pocket that holds heme?

The protein pocket that holds heme is predominantly hydrophobic, as is heme itself. Thus, the avoidance of contact with water is partly what holds the heme in the pocket. The histidine that contacts the iron in heme is an exception, since its sidechain is hydrophilic.

The beta chain of hemoglobin contains 146 amino acids. How many amino acids are changed in sickle hemoglobin?

The sickle mutation changes only one amino acid among the 146 in the beta chain. Since there are two beta chains per hemoglobin molecule, two amino acids are changed in each molecule.

What does the mutation do to the chemical properties of the protein?

The sickle mutation puts a hydrophobic valine where a hydrophilic (charged) glutamic acid is present on the surface of the normal, wild type. This makes hemoglobin more hydrophobic on its surface.

What does the slightly acidic pH of tissues do to hemoglobin's affinity for oxygen?

The slightly acidic pH of tissues, due in part to CO2 accumulation, reduces the affinity of hemoglobin for oxygen, fostering the release of the oxygen brought from the lungs. The effect of lower pH is in part because it increases the partial positive charge on His146, favoring its salt bridge with Asp94, which helps to hold hemoglobin in the low-affinity T conformation.

What happens to the iron atom when oxygen binds to it?

When oxygen binds to the Fe++ in heme, the Fe++ moves into the plane of the heme. This pulls the histidine attached to the iron, which in turn pulls the alpha helix containing that heme, which in turn favors the conformational change of the entire hemoglobin molecule from the low-oxygen-affinity deoxy T state to the higher oxygen-affinity oxy R conformation.