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The interaction between proteins and other molecules/proteins is many times key to what?
The function of the protein
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What is a ligand?
- Molecule (protein or otherwise) that is bound by a receptor (proteins) in a reversible manor
- Smallest component of the two
- Whatever is floating around freely
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What is a binding site?
- Site in the receptor to which the ligands bind
- Akin to the active site in enzymes
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What is induced fit?
Structural adaptation/conformational change induced by binding, that increases the affinity of the binding interaction
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Describe ligand-protein interactions
- Highly specific
- Depend on charge, shape, size, hydrophobic or hydrophilic character
- Receptor can have different affinities for each ligand
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What do ligand-protein interactions depend on?
- Shape
- Size
- Hydrophobic or hydrophilic character
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What is affinity in terms of receptors and ligands?
Measure of the strength of the interaction between receptor and each ligand
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What are the members of the family of oxygen binding proteins?
- Myoglobin
- Hemoglobins
- Neuroglobin
- Cytoglobin
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Why are globins called globins?
They look like globes/balls
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What is myoglobin?
- Oxygen binding proteins in muscles
- Fascilitates oxygen diffusion and storage
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What is hemoglobin?
- Tetrameric oxygen binding proteins in blood
- For transport of oxygen
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What is the structure of globins?
- 8 α-helical segments connected by bends
- Each segment is labeled A to H
- Bends are labeled in between
- Heme group in the middle
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What does the prosthetic group of heme consist of?
- Protoporphyrin
- Fe2+ (iron in its ferrous state)
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What is protoporthyrin?
Complex ring structure of prosthetic heme group
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What does the heme group transport?
- Oxygen
- (Along with CO2, H+, CO, NO)
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What is the structure of the heme group?
- Fe2+ is bound to 4 nitrogen atoms in the flat porphyrin ring (coordination bonds)
- Has two additional perpendicular coordination bonds available
- One perpendicular bond is bound to nitrogen of a histidine residue
- Other perpendicular bond will bind to oxygen
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What binds to free heme with an affinity 20,000 higher than oxygen?
Carbon monoxide
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What happens to the affinity of the heme group when part of a protein like myoglobin?
Drops to 200
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Why does the affinity of the heme group drop when part of a protein?
Due to steric effects
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Why is the heme group more stabley bound to CO than O2?
- O2 is based at an angle bound to Fe
- CO is more linear
- Its linearness makes it more stable than bent
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What is myoglobin?
- Globin protein that provides oxygen to hard-working muscles
- Binds oxygen
- Insensitive to small changes in [O2]
- Functions as a storage protein
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What are a some examples for myoglobin providing oxygen to hard-working muscles?
- Deep-diving marine animals
- Marathon runners
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Where is myoglobin found?
Key component of muscles found in muscle fiber cytoplasm
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What's another name for muscle fiber cytoplasm?
Sarcoplasm
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How is the structure of myoglobin depicted?
- Ribbon depiction
- Cylinder depiction
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What does the binding of oxygen to myoglobin depend on?
- Molecular motions
- Breathing
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Myoglobin binds to oxygen in what manor?
In a reversible manor
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What does the function of myoglobin depend on?
The protein's ability not only to bind oxygen but also to release it when and where it's needed
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How can myoglobin's ability to bind and release oxygen be expressed?
In a quantitative manner
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How is myoglobin's ability to bind and release oxygen expressed?
P + L ⇌ PL (ka on top; kd on bottom)
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What does ka on top of an arrow stand for?
Rate of association
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What does kd under an arrow stand for?
Rate of dissociation
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How is the equilibrium constant for the binding of oxygen to myoglobin written?
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What is the equilibrium constant for the binding oxygen to myoglobin also called?
Association constant
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What is dissociation constant?
- Reciprocal of Ka
- Equivalent to the molar concentration of ligand at which half of the available ligand-binding sites are occupied (50% saturation)
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It's more intuitive to use which constant to describe the interactions between protein and ligand?
Dissociation constant Kd
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What does a low value of Kd mean?
Higher affinity for the ligand
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The lower the value of Kd, the ______ the affinity for the ligand
higher
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What is θ in terms of dissociaton constant?
- Percentage of binding?
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What are the axis for the dissociation constant graph?
θ vs. [L]
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How does the graph for myoglobin binding (dissociation constant) change when binding oxygen?
- Oxygen is a gas
- So the expression for saturation must be changed to consider the partial prressures of oxygen instead of concentration
- [L] on the x-axis changes to pO2 (kPa)
- Kd changes to P50
- Reaches saturation at lower pressures
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How does the theta equation change when considering oxygen binding to myoglobin instead?
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What is hemoglobin?
- The oxygen carrier protein in erythrocytes (RBCs)
- Pretty sensitive to changes in [O2]
- Job is to transport and release oxygen
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Describe RBCs
- Cells that don't have a nucleus
- Survive for about 125 days w/o the nucleus
- Nucleus gets kicked out during the last maturation phase
- Goes from ball shape to curvy
- Gets rid of nucleus to increase surface area per volume ratio
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Hemoglobin subunits are structurally similar to what other molecules?
Myoglobin
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What does the myoglobin binding curve/graph look like when binding to oxygen?
- Y/θ on y-axis (fractional saturation)
- pO2 (partial pressure)
- Hyperbolic binding curve for O2
- Insensitive to small changes in [O2]
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What does the hemoglobin binding curve/graph look like when binding to oxygen?
- Y/θ on y-axis (fractional saturation)
- pO2 (partial pressure)
- Sigmoidal curve
- Much more sensitive to changes in oxygen
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What does the sigmoidal hemoglobin curve tell us?
- It's not just a ligand binding
- It's more complicated
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What permits hemoglobin's high sensitivity to small [O2] changes?
Interactions between the 4 subunits containing 4 heme groups
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What is the structure for hemoglobin like?
- Similar to myoglobin
- But has 4 subunits containing 4 heme groups (2α, 2β)
- Each α interacts with 2 β's and each β interacts with 2 α's (like a circle)
- Strong interactions between α1 and β1 (and α2/β2) subunits hold them together even in the presence of urea
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What is the hemoglobin quaternary structure called?
α2β2 heterotetramer
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What are the 4 hemoglobin subunits individually called?
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What holds the hemoglobin subunits together, even in the presence of urea?
Strong interactions btwn α1 and β1 (and α2/β2)
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Why are the hemoglobin subunit interactions so strong?
β subunit has many places of contact for non-covalent interactions
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How many different conformational states does hemoglobin exist in?
2
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What two conformational states does hemoglobin exist in when crystalized?
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When is the R state of hemoglobin stable?
Stabilized by oxygen
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When is the T state of hemoglobin stable?
More stable form in the absence of oxygen
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Oxygen has a higher affinity for which hemoglobin state?
R state
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Where is hemoglobin in its T state found more?
Found where there's not a lot of oxygen
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Where is hemoglobin in its R state found more?
Found where there's a lot of oxygen
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What's the difference between the structures for hemoglobin in its R state vs. T state?
- Histidine groups (His HC3) point toward outside for T
- Points toward inside for R
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Binding of oxygen to a hemoglobin triggers what kind of shift in state?
Shift from T state to R state
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What is the hemoglobin shift from T to R state called?
Allosteric transition
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Why did Max Perutz suggest a structural change in the crystal molecules from T to R?
T state cracked when oxygen was added
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What are the molecular changes when transitioning from T to R state?
- When oxygen binds, the α1β1 contact changes a little
- Large change at the α1β2 contact, with several ion pairs broken
- Changes in inter and intrachain hydrogen bonds
- The His KC3 residues at the carboxyl termini of the β subunits (involved in ion pairs in the T state) rotate in the R state toward the center of the molecule
- There, they are no longer in ion pairs
- Narrowing of the pocket between the β subunits
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R state tends to be more flexible as a result of what?
Molecular changes, including Histidine groups shifting inwards and narrowing of the pocket btwn β subunits
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What is an allosteric protein?
One in which the binding of a ligand to one site affects the binding of proteins to another site on the same protein
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What are the two kinds of allostery?
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What is homotropic allostery?
Normal ligand and modulator are identical
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What is heterotropic allostery?
Normal ligand and modulator are different molecules
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What is cooperativity?
- Ligand binding by one subunit affects subunit assembly and/or subunit-ligand interaction in a multi-subunit complex
- Pretty much binding of one subunit affects the other subunits
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Where is cooperativity seen?
Only in proteins with multiple subunits
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What is an example of cooperativity?
Oxygen binding cooperatively to hemoglobin
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How does the affinity of the states change as more O2 molecules are bound to hemoglobin?
- Low-affinity state (T) to high affinity state (R)
- Binding of second, third, and fourth oxygens is easier than first
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What can cause a change in affinity of O2 for other subunits in Hb?
O2 binding in one Hb subunit can affect the affinity of other subunits
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Why can't a single-subunit protein with a single ligand binding site produce a sigmoidal curve, even if binding elicits a conformational change?
Because each ligand molecule binds independently and cannot influence the binding of other molecules
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What does the MWC model show?
Subunits change conformation from T to R with oxygen/ligand binding
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What does the Koshland model show?
Each subunit can change independently of binding
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How does binding of O2 to individual hemoglobin subunits affect the affinity of adjacent subunits?
- Initial O2 binding is weak (T state) but binding to the 4th subunit is stronger (R state)
- Affinity increases going from T to R
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Which equation describes cooperative binding?
Hill equation
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What is the equilibrium of a protein with n binding sites?
P + nL ⇌ PLn
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What is Ka for a protein with n binding sites?
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What is the fractional saturation of heme group with oxygen for a protein with n binding sites?
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What is the Hill equation?
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What does Kd stand for in the Hill equation?
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What is the Hill plot for Hb binding to O2?
- Gives a straight line with slope n
- Mb slope = 1
- Hb slopes at low and high affinity states is 1
- Hb slope at intermediate oxygen concentrations is 3
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When can you tell there's cooperativity in ligand binding based on the hill plot?
Hill coefficient (n) > 1
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What is the Hill coefficient?
Slope, or n value, of a hill plot
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What is the hill coefficient used for?
Way to identify if there's cooperativity or not
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n in a hill plot is always _____ than the actual number of binding sites in the protein.
less
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What is the physiological advantage of cooperativity?
Makes protein more malleable and inducible
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What is the difference in saturation and pO2 btwn Mb and Hb in tissues and in lungs?
- pO2 in tissues- 4 kPa; Hb about 60% saturated; rest of oxygen is released; Mb traps whatever O2 Hb releases
- pO2 in lungs- 13 kPa; Hb pretty much saturated with O2
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What is the Bohr Effect?
- Affinity of Hb for Oxygen changes according to pH
- H+ binds to Hb and stabilizes the T state
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How does H+ binding to Hb stabilize the T state?
- Protonates His146
- This forms a salt bridge with Asp94
- Leads to the release of O2 (in the tissues)
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What did Christian Bohr discover?
- H+ and CO2 concentrations affect oxygen bindnig and release by Hb
- Bohr measured the partial saturation of Hb as a function of the partial pressure of O2
- Binding is best at high pH
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At what pH is oxygen binding the best?
High pH
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High CO2 concentrations means ____ pH
low
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Why does our affinity change with higher CO2 concentrations?
- high metabolic activity = High CO2 concentration = low pH
- Low pH = low affinity = not so good oxygen binding
- Affinity changes so we could release more oxygen
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Describe Hb and CO2 export
- CO2 is produced by metabolism in tissues and must be exported
- 15-30% of CO2 is exported in the form of a carbamate on the amino terminal residues of each of the polypeptide units
- Formation of carbamate yields a proton which can contribute to the Bohr effect
- Carbamate forms additional salt bridges stabilizing the T state
- Rest of CO2 is exported as dissolved bicarbonate
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What is carbamate?
Salt or ester containing the anion NH2COO- or the group -OOCNH2
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What is 2-3 Biphosphoglycerate?
- Negative heterotropic regulator of Hb function
- Small negatively charged molecule
- Binds to positively charged central cavity of Hb
- Stabilizes T states (when less oxygen is present)
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Where is 2-3 Biphosphoglycerate located?
Present at mM concentrations in erythrocytes (RBCs)
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How is 2-3 Biphosphoglycerate produced?
Produced from an intermediate in glycolysis
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Why is 2-3 Biphosphoglycerate important?
Very important in the physiological adaptation to high altitudes
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What is 2-3 BPG like in fetuses?
Has very low affinity for fetal Hb, which results in higher oxygen affinity for fetal Hb
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What happens to oxygen at high altitudes?
- Partial pressure of oxygen is lower
- Reduces overall amt of oxgyen for lungs
- Higher BPG at higher altitudes?
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What is fetal Hb?
- Hbf
- The main Hb during fetal life
- About 60% of normal Hb at birth
- Disappears gradually
- Composed of 2 α and 2 γ chains
- Babies don't have β chains
- Hbf has greater affinity for O2 than HbA to ensure O2 transfer from maternal circulation to fetus RBCs through placenta
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What is Hbf composed of?
- 2 α chains
- 2 γ chains
- No β chains
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What is significant about the γ chains?
Not sensitive to BPG
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Why does Hbf have greater affinity for O2 than HbA?
To ensure O2 transfer from maternal circulation to fetus RBCs through placenta
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How much oxygen in blood is transported bound to hemoglobin?
98%
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What is the most common form of hemoglobin?
HbA
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What is anemia?
A reduction in the oxygen transporting capacity of blood
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What is an example of anemia?
Sickle cell anemia
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What is sickle cell anemia?
- Characterized by RBCs acquiring abnormal, rigid, sickle shape
- Found in 1/600 African americans
- No sign till 6 months
- Life expectancy of someone with SCA is reduced
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What causes sickle cell anemia?
- Point mutation
- Leads to a change in one of the 287 AAs in the β chain from glutamic acid to valine (HbS)
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What kind of mutation provides resistance against malaria?
- Heterozygous mutations in hemoglobin that causes sickle cell anemia
- HbAS
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What happens to abnormal hemoglobin when blood O2 is low?
- Hb crystalizes (forms a chain)
- Causes RBCs to become sickle shaped
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