Topic 4

What is protein phosphorylation

• reversible

• post-translational modification

• phosphate group is added to a protein to alter it

What are the three most frequently targeted residues for protein phosphorylation

Serine, Threonine, and Tyrosine.

What are three examples of covalent modifications of proteins?

1. Linking fatty acid to side chain of cys

2. adding sugar to side chain of asn

3. adding phosphoryl group to side chain of ser

Why do we want to ever change the the function of a protein?

• To be able to regulate protein func.

• make it active or inactive by changing the side chain of the residue which will change conformation of the protein

Describe what ligands are

any molecule that binds to a target protein at designated binding site to form a protein ligand complex

How do ligands bind to proteins

• multiple weak interactions (e.g., hydrogen bonds, van der Waals interactions)

• or a few strong interactions

What are three types of ligand types and protein binding examples

1. Protein + small molecule

2. Protein + peptide

3. Protein + protein

What is a ligand defined by?

ligand is defined by function (binding), not by size or molecular type

What does the dissociation constant (Kd) measure?

binding strength or affinity between a protein and ligand

Can the equation of PL →← P + L be reversible?

Yes, because it has an equilibrium

explain the formula for Kd

P = molar conc. of the free protein

L = molar conc. of the free ligand

PL = molar conc. of the protein-ligand complex

What does a low Kd mean

Low Kd = high affinity

→ little free protein and ligand is required to keep them bound together

What does a high Kd mean?

high Kd = low affinity

→ Lots of free ligand is needed to stay bound

What is Fractional Saturation (Y):

the total protein sites bound to the ligand

What is the relationship between these elements

L/Kd → 0 = Free ligand concentration is negligible compared to Kd

• almost no protein is bound (Y = 0%)

L/Kd = 1 = Free ligand concentration is exactly equal to Kd

• Exactly half of all protein binding sites are occupied (Y = 50%)

L/Kd → infinity = Free ligand concentration is overwhelmingly high compared to Kd

• The system hits a plateau; protein binding sites are fully saturated (Y = 100%)

What is the relation of dissociation constants and their affinity?

High affinity = tight binding = small Kd

Name the 7 prefixes from low affinity to high affinity:

What is the primary role of myoglobin

• Oxygen binding protein

• facilitates oxygen diffusion through muscle tissue

What is the historical significance of myoglobin

• first protein whose structure was determined by x ray crystallography

What structural quaternary classification does myoglobin have

Monomeric — a single polypeptide chain

How many α-helices does myoglobin contain, and how are they labeled?

8 α-helices, labeled A through H

What prosthetic group does myoglobin contain, and why is it essential?

• Heme group (shown in purple in the diagram) tucked within a hydrophobic pocket

• essential because its the site of oxygen binding.

How many coordination bonds does the Fe(II) in heme form, and what are they?

• the central Fe atom can form up to 6 coordination bonds

→ Bonds 1-4: Bonded with 4 nitrogen atoms

→ Bond 5: Bonded w/ N from side chain of His 8

→ Bond 6: Reserved for O2, which reversibly binds to the Fe atom

What is the role of His F8?

• forms a direct coordination bond to Fe which anchors the heme group to the protein

What is the role of the His E7

• does not coordinate directly w/ iron atom

• Instead, it forms hydrogen bond with the bound O2 and stabilizes it

• this also hinder CO binding

Why is O₂ binding to heme reversible?

Because it binds via a coordination bond at the 6th coordination site, not a permanent covalent bond.

Why does the hydrophobic pocket around heme matter?

It shields Fe(II) from oxidation to Fe(III), which cannot bind oxygen.

What does this eq tell us

• Mb: Unbound (deoxygenated) myoglobin.

• O2 Free oxygen (the ligand).

• MbO2: Bound (oxygenated) myoglobin complex.

→ a reversible rxn

What is the dissociation constant for the eq. of myoglobin?

Free [Mb] and Free [O] in numerator

Bound complex [MbO2] in denominator

K is a measure of the affinity of Mb for O2

For this eq, what is the relationship between affinity and K?

As affinity increases, K decreases (almost all protein is bound to oxygen)

As affinity decreases, K increases (the protein drops oxygen easily)

ex. If Protein A has K = 10^-9 and Protein B has K = 10^-6, Protein A has the higher affinity (smaller K)

What does each variable represent

Y = fractional saturation of myoglobin (0 to 1)

pO₂ = partial pressure of oxygen

K = equilibrium constant (reflects myoglobin's binding affinity for O₂)

What happens to Y as pO₂ increases toward very high values?

Y approaches 1 — myoglobin becomes fully saturated with oxygen (the pO₂ term dominates over K in the denominator).

What happens to Y as pO₂ approaches 0?

Y approaches 0 — myoglobin releases essentially all its oxygen (little to no O₂ available to bind).

What is the physical/biological meaning of K in this equation?

a lower K means higher affinity (myoglobin saturates at a lower pO₂), and a higher K means lower affinity.

If pO₂ = K, what is Y?

Y = 0.5 (50% saturation)

What shape does a graph of Y vs. pO₂ produce for myoglobin, based on this equation?

A hyperbolic curve — Y rises steeply at low pO₂ and gradually plateaus toward 1 at high pO₂.

What two factors determine how much O₂ is bound to myoglobin?

The oxygen partial pressure (pO₂) and myoglobin's affinity for O₂

What is P₅₀?

The partial pressure of O₂ (pO₂) at which myoglobin is 50% saturated (Y = 0.5)

• i.e., half of the binding sites are occupied.

What is the relationship between K (from the equation Y = pO₂/(K + pO₂)) and P₅₀?

K = P₅₀ — they are the same value.

Can P₅₀ be a low number? What would that mean?

Yes.

A low P₅₀ means the protein only needs a small amount of oxygen pressure to become half-saturated

• this indicates high affinity for O₂

What would a high P₅₀ indicate about a protein's O₂ affinity?

Low affinity — it takes a lot of oxygen pressure to reach half-saturation.

What is hemoglobin's function, and where is it found?

• oxygen-transport protein

• found in red blood cells (transports O₂ through the bloodstream).

What is hemoglobin's quaternary structure?

A tetramer composed of 2 α subunits and 2 β subunits.

How does hemoglobin's structure differ from myoglobin's?

Myoglobin is monomeric (1 chain, 1 heme, 1 O₂ site); hemoglobin is tetrameric (4 chains, 4 hemes, 4 O₂ sites).

At what level of protein structure are myoglobin and hemoglobin similar? At what level do they differ?

• Similar in secondary and tertiary structure (individual chain folding)

• different in quaternary structure (myoglobin = monomer; hemoglobin = tetramer of similar subunits).

If myoglobin and hemoglobin's subunits have different amino acid sequences, why do they fold into nearly the same 3D structure?

They are homologous proteins, evolved from a common ancestral gene

• only critical residues (essential for function) need to be conserved; the rest of the sequence can vary substantially while the fold and function are maintained.

What percentage sequence identity do myoglobin and hemoglobin share, despite their near-identical 3D structure?

Only ~18% identical in primary sequence.