5: Protein-Ligand Interactions

Promote the formation of highly reactive oxygen species, such as hydroxyl radicals, which can damage DNA.

Heme is the protein-bound prosthetic group within hemoglobin, incorporating iron that tends to bind oxygen. Hemoglobin is the entire protein-complex: a globulin protein plus heme prosthetic group.

Heme is comprised of an organic ring called protoporphyrin proto-por-phy-rin. The iron is present in its Fe2+ state, with 6 coordination bonds. Four of these bonds are planar with the protoporphyrin ring, and two are perpendicular.

Reversibly, never. This is why it is important for hemoglobin to prevent irreversible oxidation of its iron.

The 4 coordinated nitrogen atoms of the polyporphyrin ring, to which the iron binds, have electron donating character.

Larger proteins such as hemoglobin shove the heme deep into the center of its structure, preventing access of molecular oxygen to the iron. Also, proximal His.

When molecular oxygen encounters an unbound heme molecule, it may bind both of its 2 free coordination bonds at the same time. This is bad!!!!!!!!!!!

The proximal His refers to the nitrogen-containing side chain of a histidine residue that is integral to maintaining a spot in one of Fe2+'s two coordination bonds. Proximal His therefore helps to prevent the oxidation of Fe2+ by acting as a placeholder.

Small molecules bind to heme with much greater affinity. Oxygen binding proteins counteract this by sequestering the heme deep into the center of the protein to prevent access to the open coordination bond.

Globin refers to the family of proteins that primarily participate in oxygen storage, transport, or sensing. They have a highly conserved tertiary structure comprised of 8 α-helical segments, connected by bends. Their folding motif is known as the globin fold.

Myoglobin: monomeric, oxygen storage. Hemoglobin: tetrameric, oxygen transport. Neuroglobin: monomeric, low-oxygen protection. Cytoglobin: monomeric, regulates [NO].

Mb = 16,700 AA = 153 Heme = 1 (monomeric)

The eight α-helices of the globin fold. (Named A-H, like anna hahn lol!!!!)

His93 (93rd residue from the amino terminus) HisF8 (8th residue within the F helix)

P + L ⇌ PL

Ka = [PL]/([P][L]) = ka/kd

It describes the affinity of a ligand to its protein. A higher Ka indicates higher affinity of ligand to protein. It also denotes the ratio of the forward (association) rate to reverse (dissociation) rate of proteins binding ligands.

For all intents and purposes, [L] is constant.

The proportion of occupied binding sites on a protein.

Y = [PL] / ([PL] + [P]) The proportion of occupied binding sites is equal to the ratio of the concentration of bound protein to the total concentration of protein.

We know that [PL] = Ka[L][P], upon substitution of [PL]: Simplifies to Y = [L] / ([L]+(1/Ka))

We know 1/Ka is equal to Kd, so those can be interchanged as well.

Half of all ligand-binding sites are occupied.

When graphed Y(y) vs. L ---> Y = 0.5 at the ligand concentration equal to its Kd, or 1/Ka.

Kd is the equilibrium constant for release of ligand. Its units are of molar concentration.

Ka is the equilibrium constant for binding of ligand. Its units are 1 / (units of molar concentration), because Ka is the reciprocal of Kd.

Distal His acts as a "revolving door" for molecules within the protein, allowing only O2 to move through.

Distal His also selectively increases a heme's affinity for O2 (as opposed to CO), by implementing stabilizing hydrogen bonds to the highly polar Fe-O2 complex.

Because Fe-CO is much less polar, distal His does not have the same stabilizing effect, thereby selectively increasing heme's affinity for O2.

His64, (64th residue from amino terminus). HisE7, (7th residue on E helix).

Hemocytoblasts are the stem cells that produce erythrocytes (red blood cells). Red blood cells carry hemoglobin.

Saturated: 96% Unsaturated: 64%

Myoglobin is not sensitive to small changes in concentration of oxygen within blood.

Hemoglobin is a tetramer, meaning it has four subunits all capable of binding oxygen. This makes it much more sensitive to small concentration changes of dissolved oxygen.

Mh = 64,500 It has 4 side chains: 2 α-chains of 141 residues each 2 β-chains of 146 residues each

Each chain contains a heme group, so 4.

Unlike side chains, α-β, interact strongly via highly stabilizing hydrophobic effect, as well as H-bonds and a few disulfide bridges.

In low-oxygen environments, hemoglobin tends to assume the T-state. It has a low affinity for binding oxygen and tends to release it. In high-oxygen environments, hemoglobin tends to assume the R-state. It has a high affinity for binding oxygen that increases as more oxygens bind to its 4 hemes.

Oxygen binding is cooperative, meaning the binding of one actively alters a heme's conformation slightly to make it easier for more oxygens to bind.

An allosteric (other-site) protein has multiple ligand binding sites. It has a ligand as well as a modulator that affects the protein's affinity to bind, either negatively or positively.

A homotropic interaction is one where a ligand is its own modulator; for example, in hemoglobin, oxygen is the main ligand, which also affects the protein's affinity to bind more oxygen.

A heterotropic interaction is one where a ligand is different from its modulator.

**NOTE: A protein is not termed homo or hetero tropic; the INTERACTION is. Hemoglobin can bind multiple ligands, like CO and NO as well, so a protein can have both homotropic and heterotropic ligands.

Lungs: 13.3kPa, R-form (high O2 binding affinity) Tissues: 4kPa, T-form (low O2 binding affinity, tends to release oxygen)

P + nL ⇌ PLn

Ka = [PLn] / ([P][L]^n

log(Y/(1-Y) = nlog[L]-log(Kd)

n (commonly with subscript H) is the Hill coefficient, which denotes how cooperative a given protein-ligand interaction is. n > 1 means it is cooperative (pos) n = 1 means it's non-cooperative (neither pos nor neg) n < 1 means it's anti-cooperative (neg)

Carbon dioxide and H+

Hydration of CO2, forming bicarbonate. The reaction is catalyzed by carbonic anhydrase

CO2 is insoluble in blood. Bubbles in blood bad! Hydration into bicarbonate makes soluble molecule! No more bubbles in blood. Great success.

H+ ions. Decreases its acidity.

1. pH; decreasing pH decreases affinity.

2. temperature; increasing temp decreases affinity.

3. [2,3-BPG]; binds to heme, decreasing its affinity.

4. [CO2] ^ditto

5. [CO] ^ditto

Inversely. Binding of H+ and CO2 favor the T state, decreasing affinity for oxygen. Binding of O2 favors R state, decreasing affinity for H+ and CO2

The effect of pH and CO2 concentration on the binding and release of oxygen.

Heterotropic allosteric modulation. Basically it is not hemoglobin's main ligand, but by binding to a different site separate from the oxygen binding site, it affects the heme's affinity for oxygen nonetheless.

2,3-BPG allows for physiological adaptation to lower pO2 environments, like at higher altitudes. It has a low effect of binding O2 in lungs, but increases efficiency in releasing O2 into tissues.

White blood cells. They are comprised of macrophages and B-lymphocytes.

Macrophages have receptors on their surfaces that bind IgG, which induces the engulfing and destruction foreign bodies.

Lymphocytes leave the blood and "patrol" tissue that recognize and signal infection.

Cellular: Directed at infected host cells, parasites, and foreign tissues. Comprised of cytotoxic T-cells, Helper T-cells.

Humoral: Directed at bacterial and viral infections. Comprised of antibodies.

Immunoglobulins are antibodies. They bind foreign molecules and mark them for death. They make up 20% of blood protein. They are produced by B-cells.

Killer T-cells recognize infected cells via receptors on their surface, and kill them.

Helper T-cells interact with macrophages and are indirectly involved in the killing of antigens by a process called clonal selection, where Th cells stimulate the proliferation of killer T-cells and B-cells.

Antigens are any molecule that can induce an immune response. Epitopes refer to the region of an antigen to which a specific antibody recognizes/binds.

Usually molecules with Mr < 5,000 are not antigenic, unless they are haptens. Haptens are molecules that bind to larger antigenic molecules, but are then broken down into substituent parts, but are still recognized as antigens.

Immunoglobulin G has 4 side chains: 2 heavy and 2 light. The chains are linked by noncovalent interactions as well as disulfide bridges. It has a Y shape in which the ends of the branches of the Y are variable antigen binding sites. The stem of the Y is highly conserved, and is known as the Fc region. The bases of the branches are also highly conserved. The branches of the Y in conjunction are known as the Fab region.

GAMED.

Allows for the separation and detection of small samples separated from SDS gel, and their approximate molecular weight.

1. Proteins are separated by gel electrophoresis

2. Proteins are transported to different membrane and washed

3. Proteins are treated with antibodies linked to enzyme + substrate

The last step causes formation of a colored band containing the protein of interest, on the column the yaxis

80%

Mr = 520,000 6 subunits: 4 light chains, 2 heavy chains. At the carboxyl ends of the heavy chains is a coiled coil of left handed alpha-helices.

MAIZ: M-disk intersects A-line, A line is comprised of thick/thin filaments. Z-disk intersects I-line, I line is comprised of thin filaments.

The entire contractile unit: myosin + actin + other regulatory protein complexes. (tropomyosin, troponin)

Myosin's thick filaments slide across actin's thin filaments.

1. Myosin head is tightly bound to actin thin filament.

2. ATP binds to myosin head, triggering the release of bound actin.

3. ATP is hydrolyzed to ADP, releasing Pi.

4. A series of conformational changes occur that cause myosin to slide down the actin filament.

5. Myosin re-binds to the actin filament at a new site, and releases ADP.

Tropomyosin: Binds actin to prevent the binding of myosin. Troponin: Ca2+ binding protein; induces contraction via conformational change.