Iron has a strong tendency to bind oxygen. Without the sequestering by larger molecules like heme, what does free iron tend to do?
Promote the formation of highly reactive oxygen species, such as hydroxyl radicals, which can damage DNA.
What is heme? What is hemoglobin?
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.
Describe the heme molecule in terms of its molecular structure. (What is the molecule that surrounds the iron? In what ionic state does the iron exist? How many bonds does the iron coordinate, and what are their orientations?)
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.
Iron in the Fe2+ state binds oxygen ____________
Iron in the Fe3+ state binds oxygen ___________
Reversibly, never. This is why it is important for hemoglobin to prevent irreversible oxidation of its iron.
What parts of the heme molecule help to prevent oxidation of Fe2+? Why?
The 4 coordinated nitrogen atoms of the polyporphyrin ring, to which the iron binds, have electron donating character.
What parts of heme containing proteins help to prevent oxidation of Fe2+? Why?
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.
How does oxidation of Fe2+ happen?
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!!!!!!!!!!!
What is proximal His?
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.
What are the general binding affinities of small molecules like CO or NO to heme, compared to that of molecular oxygen? How do heme containing proteins prevent these small molecules from binding?
Small molecules bind to heme with much greater affinity. :thumbsdown: Oxygen binding proteins counteract this by sequestering the heme deep into the center of the protein to prevent access to the open coordination bond.
What is a globin? Describe the general structure and function of a globin. What is the name of its unique folding pattern?
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.
Name four kinds of globins found in mammals, and their general function.
What is the molecular weight of myoglobin? (sorry lol)
How many amino acids make up myoglobin?
How many molecules of heme?
Mb = 16,700
AA = 153
Heme = 1 (monomeric)
In myoglobin, the majority (78%) of amino acids are found where?
The eight α-helices of the globin fold. (Named A-H, like anna hahn lol!!!!)
In myoglobin, there's two ways to denote the proximal His. What are they?
His93 (93rd residue from the amino terminus)
HisF8 (8th residue within the F helix)
What is the simple equilibrium expression of a protein binding its ligand?
P + L ⇌ PL
In the reaction of a protein binding its ligand, what is the equation denoting its equilibrium constant, Ka?
Ka = [PL]/([P][L]) = ka/kd
What is Ka, in general terms? (A higher Ka indicates what?)
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 most ligands that bind to proteins in cells, [L] is usually much greater than [P] + [PL], so "the binding of the ligand by the protein does not appreciably change [L]." Basically, this means that:
For all intents and purposes, [L] is constant.
What does the variable Y denote, in terms of proteins and ligands?
The proportion of occupied binding sites on a protein.
What equation describes the fraction Y, of ligand-binding sites of a protein that are occupied by ligand? Y = ? (There are multiple, depending on how many substitutions you use).
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.
What does it mean when Y = 0.5?
Half of all ligand-binding sites are occupied.
When graphed Y(y) vs. [L](x) ---> Y = 0.5 at the ligand concentration equal to its Kd, or 1/Ka.
What is Kd? Kd has units of:
Kd is the equilibrium constant for release of ligand. Its units are of molar concentration.
What is Ka? Ka has units of:
Ka is the equilibrium constant for binding of ligand. Its units are 1 / (units of molar concentration), because Ka is the reciprocal of Kd.
What does distal His do? How?
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.
In myoglobin, there's two ways to denote the distal His. What are they?
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.
What are the percentages of oxygen in saturated and unsaturated blood?
Why is hemoglobin better for oxygen-transport than myoglobin?
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.
What is the molecular weight of hemoglobin?
How many side chains, and how many residues in each?
How many molecules of heme?
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.
Describe the interactions between the different polypeptide chains in hemoglobin.
Unlike side chains, α-β, interact strongly via highly stabilizing hydrophobic effect, as well as H-bonds and a few disulfide bridges.
Describe conformational changes related to hemoglobin's binding of oxygen. (In low/high-oxygen environments, what conformation?)
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.
What is cooperativity, in terms of hemoglobin?
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.
What is an allosteric protein, and how are they related to cooperativity?
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.
Define homotropic and heterotropic.
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.
What is the pO2 in lungs, and what form of hemoglobin would you find there?
What is the pO2 in tissues, and what form of hemoglobin would you find there?
For a protein with multiple ligand binding sites, what is the general equilibrium expression?
P + nL ⇌ PLn
For a protein with multiple ligand binding sites, what is the expression for equilibrium association, Ka?
Ka = [PLn] / ([P][L]^n
For a protein with multiple ligand binding sites, what is the Hill expression?
log(Y/(1-Y) = nlog[L]-log(Kd)
Describe the variable n within the Hill cooperativity equation.
What does it mean when: n>1, n<1, n=1
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)
Hemoglobin also transports what, in addition to oxygen?
Carbon dioxide and H+
CO2 is a waste product of cellular function. What reaction eliminates this from the blood and what enzyme catalyzes it?
Hydration of CO2, forming bicarbonate. The reaction is catalyzed by carbonic anhydrase
Why is conversion from CO2 to bicarbonate important in blood?
CO2 is insoluble in blood. Bubbles in blood bad! Hydration into bicarbonate makes soluble molecule! No more bubbles in blood. Great success.
Hydration of CO2 increases the concentration of what in the blood?How does this affect the acidity of the blood?
H+ ions. Decreases its acidity.
What are five factors within the cellular environment that affect the binding affinity of hemoglobin to oxygen?
The binding of H+ and CO2 are (directly/inversely) related to the binding of oxygen within hemoglobin.
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
What is the Bohr effect?
The effect of pH and CO2 concentration on the binding and release of oxygen.
What kind of interaction does 2,3-BPG have with hemoglobin?
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.
What does 2,3-BPG do/allow, in general terms of an organisms body?
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.
What is a leukocyte? What are two different types of leukocytes?
White blood cells. They are comprised of macrophages and B-lymphocytes.
What does a macrophage do?
Macrophages have receptors on their surfaces that bind IgG, which induces the engulfing and destruction foreign bodies.
What does a lymphocyte do?
Lymphocytes leave the blood and "patrol" tissue that recognize and signal infection.
The immune system is made up of two main categories. What are they? What types of molecules are they comprised of?
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.
What is an immunoglobulin?
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.
What does a cytotoxic T-cell do?
Killer T-cells recognize infected cells via receptors on their surface, and kill them.
What does a helper T-cell do? Define clonal selection.
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.
Describe an antigen, and any key terms related to their structure.
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.
Describe the structure of Immunoglobulin G.
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.
What are the different types of immunoglobulins?
What is the Western Blot Assay?
List the steps.
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
Actin and myosin comprise what percent of muscle protein mass?
Describe the molecular structure of myosin. (Molecular weight, subunits, notable secondary structures)
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.
Describe notable regions of a sarcomere.
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.
What is a sarcomere?
The entire contractile unit: myosin + actin + other regulatory protein complexes. (tropomyosin, troponin)
Describe the process of muscle contraction.
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.
What are two regulators of muscle contraction?
Tropomyosin: Binds actin to prevent the binding of myosin.
Troponin: Ca2+ binding protein; induces contraction via conformational change.