Chapter 5 Protein Function (R) Flashcards Preview

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Flashcards in Chapter 5 Protein Function (R) Deck (92):


A molecule bound reversibly by its binding site



Momentary, Brief


Induced Fit

conformational changes in a protein that makes the binding site more receptive to a ligand.


Q # 1 Why is it important that iron is incorporated into the heme group, rather than free in the cell?

Iron promotes the formation ROS' (reactive oxygen species) such as hydroxyl molecules, that damage DNA and macromolecules


Prosthetic group

a compound permanently associated with a protein that contributes to the protein's function



protein bound prosthetic group associated with iron


Q # 2 What is the shape of a heme group?

A heme group consists of a complex organic ring structure (Protoporphyrin) to which is bound a single iron atom (Fe2+). Fe2+ is buried deep within the heme molecule as well as being associated with four nitrogen molecules that donate electrons This prevents Fe2+ from going to Fe3+ which irreversibly binds O2. One of the two free groups is bound by His, the other allows binding of oxygen



widespread function of proteins whose functions include; oxygen transport and storage, sensing oxygen, nitric oxide or carbon monoxide


Q # 4 How is the heme group attached to and positioned within the myoglobin molecule?

The heme group is at the center of the myoglobin, and it is surrounded by 8 alpha helical segments



Ka is an association constant that describes the equilibrium between the complex and the unbound components of the complex ;For P + L PL; Ka = [PL]/[P][L]; Where P=protein, L = ligand

;The larger the Ka, association constant, the higher the affinity there is of the ligand for a protein; Units M^-1

;also equal to ka/kd


Ka[L] = [PL]/[P]

Shows that the proportion of bound protein to free ligand is directly proportional


Theta (ø) of Ka

Where theta equals the fraction of binding sites occupied divided by total binding sites
ø = [L]/([L] + 1/Ka)


ø = 0.5?

The [L] at which half of the available ligand-binding sites are occupied corresponds to 1/Ka


Dissociation constant (Kd) is simply equal to:




the equilibrium constant for the release of a ligand ;For PL -> P + L;
Kd = [P][L]/[PL]= kd/ka


Theta (ø) of Kd

1) Where theta equals the fraction of binding sites occupied divided by total binding sites 2) When [L] equals Kd, half of the ligand binding sites are occupied. 3) A lower value of Kd results in a higher affinity of ligand for a protein 4) Units (M)

ø = [L]/([L] + Kd


What exactly is Theta ø?

Theta ø is the amount, specifically the percentage, of protein bound to its ligand.


What exactly is Kd?

Kd, the equilibrium dissociation constant is the concentration of a ligand [Lx] at which 50% of the ligand binding sites are bound


Q # 6 In an oxygen -binding curve for myoglobin, why is the term pO2 used rather than [O2]?

pO2 is used because measuring the amount of oxygen in fluid, specifically the circulatory system is tough. In laboratory settings, the partial pressure of O2 is easier to measure than the concentration of oxygen dissolved in solution. Recall, that O2 is not readily soluble in blood


Q # 7 What does the term [PL] mean in terms of specific molecules?

[PL] is the amount of ligand bound to a protein


Q#9 What is the relationship between Kd and the ability of a protein such as myoglobin to bind to its ligand?

Kd is the point at which 50% of the protein is bound by its ligand. Specifically in this problem, Kd means that 50% of the ligand, oxygen, is bound to the protein, Myoglobin


Q # 8 Why are the total number of oxygen binding sites in a solution of myoglobin equal to [PL] + [P]?

The total number is such, because [PL] is the amount of protein already bound, and [P] are the available sites that have not been bound, but have the potential to be bound..


Q # 10 Given that free heme binds CO 20,000 times better than it binds O2, why don't we all succumb to carbon monoxide poisoning?

Because carbon monoxide is not a prevalent gas in the atmosphere. Also, His E7 sterically hinders the binding of CO to myoglobin



precursor stem cell that generates hemoglobin


Does hemoglobin reproduce? Why or why not?

It does not. As hemoglobin cell undergo differentiation, they lose organelles and nucleus. They only survive for about 120 days


Q # 11 Why is it important that erythrocytes are so small?

It is important that erythrocytes are so small, because the body needs a substantial amount of transporters of oxygen throughout. Small cells, more oxygen transport.


Q # 12 Could myoglobin substitute for hemoglobin in Red Blood Cells?

No. hemoglobin functions better as a storage molecule, because it does not respond to changes in the concentration of dissolved oxygen.


What are some of the physical features associated with hemoglobin?

1) it is spherical 2) tetramer (two alpha chains and two beta chains) 3) contains four heme groups


Q #14 Why do you think the histidine residues E7 and F8 are invariant between myoglobin and the alpha and beta chains of hemoglobin?

They may be invariant, because they both have the same function, to bind oxygen. Subtle differences in overall structure give difference in affinity, but they still have the same goal. Recall, that the E7 residue is also important to sterically hinder the ability of CO to bind to hemoglobin


Q # 15 What weak bonds or interactions, other than ion pairs are important to hemoglobin's quaternary contacts?

1) Hydrophobic interactions predominate at all interfaces. 2) hydrogen bonding is also important.


What is the R state, and how does it relate to the affinity in binding of hemoglobin and oxygen?

The R state, oxyhemoglobin, is the conformation of hemoglobin that has a higher affinity for oxygen. The R state is brought about when the T state binds an oxygen molecule, which breaks some of the ion pairs which stabilize the T structure. Amino acid changes involved are the proximal His F8. Heme becomes more planar.


What is the T state, and how does it relate to the affinity in the binding of hemoglobin and oxygen?

deoxyhemoglobin. more stable when oxygen is absent. stabilized by ion pairs that lie at the a1b2 and a2b1 interface.


What happens as subsequent oxygen molecules bind to hemoglobin?

As hemoglobin adds more oxygen from the T state to the R state, it increases affinity for the addition for another oxygen molecule


Allosteric protein

The binding of a ligand to one site affects the binding properties of another site on the same protein



ligands that change the conformation of a protein


homotrophic interaction

Occurs when the modulator and the ligand are the same.


heterotrophic interaction

Occurs when the modulator is a molecule other than the normal ligand


Q # 17 How does a sigmoid binding curve illustrate the presence of a low-affinity state and a high affinity state?

The sigmoid binding curve gives us information about the affinity of a protein for its ligand. For high-affinity molecules, the ligand binds almost immediately even at low concentrations. This is bad in the case of hemoglobin (does not release to tissues) Low affinity molecules show no significant increase in the amount of bound ligand even when the ligand is in high amounts. ( this is bad because it would not be able to bind oxygen well from the lungs, and would readily drop oxygen off early on at tissues. Hemoglobin has the characteristic S sigmoid curve. As concentrations of ligand increases, as it binds to subunits, the molecules affinity for oxygen increases.


Q # 18 Why does hemoglobin have a lower affinity for the first oxygen molecule it binds than it does for the binding additional molecules?

This has been answered. T-R more affinity due to the disruption of stabilizing ionic bonds. The affinity increases with increasing oxygen binding.


Q # 19 Oxygen acts as what type of modulator for hemoglobin?

it acts as a homotrophic modulator.


Hill Equation

developed by Archibald Hill, is a way to quantitatively describe ligand binding.


Derivation of the Hill equation

1) P + nL PLn; 2) Ka = [PLn]/([P][L]^2); ø = [L]^n/([L]^n + Kd); rearranging and then taking the log of both sides, yields: *log (ø/1-ø) = n log [L] - log Kd*


Hill Coefficient

a measure of the degree of cooperativity. If nH= 1, the binding is not cooperative. Where nH is the slope. An nH of greater than 1 indicates positive cooperativity. An nH of less than 1 indicates negative cooperativity.


Q # 21 When the partial pressure of oxygen in the tissues such as a muscle is less than 1 kPa how much oxygen remains bound to hemoglobin compared to myoglobin?

Hemoglobin has a lower affinity for oxygen compared to myoglobin. Myoglobin therefore has a higher concentration of oxygen relative to myoglobin.


MWC model

a.k.a Concerted model; developed by Jeffries Wyman, Jacques Monod, and Jean-Pierre Changeux; assumptions of the model; 1) all subunits are functionally identical, 2) each subunit exists in two conformations, 3) all subunits undergo the transition from one conformation to the other simultaneously. 4) No protein has individual subunits in different conformations. 5) The two subunits are in equilibrium.


Sequential Model

Developed by Daniel Koshlan; states that ligand binding can induce a change of conformation in an individual subunit. If one unit changes shape, it causes another subunit to change shape.


What are the two models of cooperative binding?

MWC/Concerted Model and the Sequential model


Q # 23 Is the concerted model or the sequential model more consistent with the knowledge we have about T-R?

Sequential model. O2 binds to one molecule cause changes in the ion interactions that previously stabilized the protein. This causes changes in subsequent subunits.


Q # 24 The equation HHb+ + 02 HBO2 + H+ indicates that both O2 and H+ can bind to hemoglobin. Do these two ligands bind at the same site? Do they both cause the same changes in conformation?

H+ and O2 do not bind at the same site. H+ binds to amino acid residues in the protein (I.e His 146) of ß subunits. This ionization helps it bind to Asp 94 ion pairs thus stabilizing deoxyhemoglobin in the T state. O2 of course binds to heme groups. As H+ binds to His 146, it promotes the release of O2 (negative cooperativity)


Q #25 Would a decrease in pH promote the T or R conformation of hemoglobin?

The T conformation. Decreases in pH mean increases in H+ concentration leading to the production of the T state and the release of O2 in whatever region.


Q # 26 Carbon Dioxide also binds to hemoglobin. Do Co2 and O2 bind at the same site? Do they both cause the same changes in conformation?

CO2 and O2 do not bind to the same site. O2 binds to heme groups, while CO2 binds as a carbamate group to the alpha amino group at the amino terminal end of each globin chain, forming carbaminohemoglobin. This reaction produces H+ and also produces salt bridges that stabilize the T state.


Q # 27 How are the binding of H+, CO2, and O2 interdependent?

We have proved this in previous cards. Binding of CO2 and H+ are needed specifically because CO2 needs a carrier protein and H+ needs to be taken to the kidneys and lungs. These interactions help stabilize pH (especially when we talk about carbonic anhydrase). The interactions between these three molecules becomes important when we think about where O2 needs to travel and where the other molecules need to travel. We need to think of this molecule in the light of cellular environments.


What is the Bohr Effect?

The effect of pH and CO2 concentration on the binding and release of oxygen by hemoglobin. H+ and CO2 binding is inversely proportional to the binding of O2


What role does 2,3 bisphosphoglycerate (BGP) play in the binding of oxygen to hemoglobin?

BGP is known to reduce the affinity of hemoglobin for oxygen. This is especially important at altitude and conditions of low oxygen concentration. BGP promotes the removal of oxygen from tissues.



Lowered oxygenation of peripheral tissues due to inadequate functioning of the lungs or circulatory system.



To prevent from happening, to make impossible


Q # 28 Where in the hemoglobin molecule does 2,3 BGP bind?

2,3 BGP Binds to the space between ß subunits in in a hemoglobin tetramer. Only one BGP molecule can bind to a single tetramer. BGP binds to the positively charged amino acid residues in hemoglobin. This binding stabilizes the T state of hemoglobin. As T->R, the pocket narrows for BGP binding and BGP is less likely/able to bind to hemoglobin.


Q # 29 The equation HbBPG + O2 HbO2 + BPG indicates that oxygen and BGP can bind to hemoglobin. Do these two ligands bind at the same site? Do both cause the same changes in conformation?

BGP binds to positively charged residues in between ß subunits, while oxygen binds to heme groups. They do not bind to the same site. They also do not cause the same changes in conformation. BGP stabilizes the T state of hemoglobin reducing its affinity for oxygen, while oxygen increases the affinity for other oxygen molecules to join.


Q # 30 How would the oxygen binding curve for fetal hemoglobin compare to that of maternal hemoglobin? Does this make physiological sense?

Fetal hemoglobin would have a greater affinity for oxygen, a steeper curve, because its replacement of gamma subunit with the beta subunit does not bind BGP with the same affinity as beta subunits. This leads to a greater affinity of oxygen. This makes physiological sense, because the fetal environment is surrounded by fluid, and not readily available oxygen from the air.


Q # 32 What protein purification techniques might be useful in separating HbS from HbA?

Column Chromatography. We should separate based on charge. Valine is not charged, while glutamate has a negative charge at pH 7.4


Q # 33 Would it be better for patients with sickle-cell disease to live at sea level or at high altitude?

Sea level. At altitude, there is less available oxygen from red blood cells to bind, and also there would be an increased amount BGP to promote the release of oxygen to the already oxygen deplete tissue. Those two events would cause sickle-cells to mis shape, become insoluble in the circulatory system, and cause clots. Also, these cells rupture easily in less available cells for oxygen transport.


Q # 34 Part A: What are the defense responsibilities of the humoral immune system?

The Humoral immune system defends the body against bacterial infections, and extracellular viruses, but can also fight against individual foreign proteins.


Q # 34 Part B: What are the defense responsibilities of the cellular immune system?

Destroys cells infected by viruses and also destroys some parasites and foreign tissues



a.k.a immunoglobins (Ig); bind bacterial viral, and large molecules; mark them as foreign, target them for destruction. Antibodies are made by b-lymphocytes or B cells



Small molecules that must be bound to large proteins in order to cause an immune response.


Q # 35 Which type of white blood cell produces antibodies? What are the analogous recognition proteins produced by the cells of the cellular immune system?

B cells/Lymphocytes. T cells have receptors on their cell surface and produce helper T cells and cytotoxic T cells.


Q # 36 Would a single amino acid be an effective antigen? A dipeptide? Hemoglobin?

Antigens must have a molecular weight greater than 5,000 to be antigenic. If they are less to that, they must be bound by a protein.


Q # 37 what is the role of Th cells in the protection of the organism under viral attack?

Th cells produce cytokines that direct the proliferation of other lymphocytes, and make macrophages more lethal


Q # 38 What is the role of B cells in the defensive strategy?

B Cells have antibodies on their cell surface and produce free floating antibodies that can bind to antigens. If an antigen binds to cell receptors on B cells, that B cell produces other B cells and free floating plasma cells.



Constant region. Crystallizes readily.



Antigen binding fragment


Immunoglobin fold

A well conserved structural motif in all the ß class proteins


Q # 39 What forces hold light and heavy chains together in antibodies?

non covalent and disulfide bonds.


Q # 41 How are basophils and mast cells conscripted into the army of defensive cells of the immune response?

IgE binds to Histamine secreting mast cells and basophils, which cause vasodilation of vessels so that other immune responding cells can migrate to tissue. IgE is the immunoglobulin most tied with allergies.


On what basis is specificity conferred between an antigen and its binding site?

shape, charge, non-polar, and hydrogen binding groups


Q # 43 How does induced fit increase the strength of the interaction between antibody and antigen?

Conformational changes that occur when an antigen binds to its antibody causes the complex to change into a more stable form by full interaction.


Polyclonal antibodies

many different B lymphocytes producing different antibodies in response to one antigen


Monoclonal antibodies

Identical B cell produce one type of antibody to respond to an antigen


What are the applications of antibodies?

Scientists use antibodies to separate proteins. Proteins bound to antibodies stay in the column, while unbound proteins elute.



remove, especially with solvent



Enzyme-Linked immunosorbent assay: Step 1 coat surface with sample antigens; Step 2 Block unoccupied sites (to block proteins in subsequent steps from absorbing to unoccupied sites; Step 3 Incubate primary antibody against specific antigen Step 4 Treat with secondary antibody. (antibody against the primary antibody linked to an enzyme catalyzes the reaction that causes the formation of color.


Western Blot

Immunoblot Essay 1) proteins separated by gel electrophoresis is transferred electrophoretically to a nitrocellulose membrane 2) The membrane is blocked, then treated with primary antibody, secondary antibody linked to enzyme, and substrate. A colored precipitate forms to indicate binding.


Q # 46 What are the important protein-protein interactions among motor proteins?

ionic, hydrogen bonding, hydrophobic interactions and van der waals interactions.


What are the two major proteins of muscle?

Actin and Myosin


Characteristics of Myoglobin

6 subunits (2H 4L); at carboxyl terminus, they form tight left handed alpha helixes. At the amino terminus, each heavy chain occurs where ATP is hydrolyzed


Q # 48 What is the role of ATP in actin filament assemblyt?

Actin molecules bind to ATP, then hydrolyzes it to ADP. Every actin molecule is complexed to ATP.


A band

region contains thick filaments


I band

region contains thin filaments


Z disk

Anchors I band


M line

Anchors A band


How does Gibbs free energy relate to the affinity of a protein for its ligand?

∆G˚ = -RT lnKa or RTlnKd


What are the to theories of protein/ligand interaction?

1) Complementary model - lock and key 2) Induced Fit - both protein and ligand change their conformations "inducing" a perfect fit.

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