Week 3
Protein-Ligand binding
o How do protein binding pockets accommodate ligands?
Ligands are small molecules that can form a complex with a larger biomolecule using non-covalent interactions
Ligands bind to characteristic binding sites that are highly conserved and very specific
These binding sites have complementary size, shape, and charge (use opp charges)
Examples of ligands include: substrates, inhibitors, activators, metals, hormones, toxins, and neurotransmitters.
o Understand the basics of ligand binding equilibrium
Ligand binding is reversible and as such can be described as an equilibrium (P + L ↔ PL)
The equilibrium state can be described with equilibrium association constant (Ka ) and equilibrium dissociation constant (Kd )
▪ Be able to assess binding strength using Kd
Low Kd = stronger binding; higher affinity (overall better interaction)
High Kd = weaker binding; lower affinity
▪ Know the meaning of Y, and how to use [L] vs Y plots to assess Kd
Y represents the fraction of binding sites occupied
0 no ligand bound
1 means a lot of ligand bound approaching saturation
o Understand the concept of allosteric regulation
Allosteric regulation is the regulation of protein function by the binding of an effector molecule at a site other than the impacted sit
Homotropic regulation is when the substrate also regulates function
Heterotropic regulation is when a different molecule than the substrate regulates function
Positive regulation increases activity/binding, negative decreases activity/binding
o Understand the concept of cooperativity Hemoglobin ONLY
Cooperativity is when the shape of one subunit (in a multisubunit protein) is altered by the ligand, impacting the shape of a neighboring subunit
Positive cooperativity is when this effect increases affinity for the ligand at the other subunits - Sigmoidal binding curve is the tell
▪ What is the Hill coefficient? What does its value tell you?
Hill coefficient (n) determines the degree of cooperativity of a protein-ligand system
Cannot exceed the number of ligand binding sites on the protein
N= 1 non cooperative
0 < n < 1 negatively cooperative
N > 1 positively cooperative
- Globins
Know the general structure and function of the heme group
Heme is a nonprotein prosthetic group that gives myoglobin and hemoglobin their oxygen-binding ability.
Heme is responsible for reversible oxygen binding
Myoglobin and hemoglobin are conjugated proteins because they have both a polypeptide chain and a nonprotein prosthetic group
Globins are hemoproteins (proteins with heme prosthetic groups)
Heme contains ferrous iron (Fe2+)
What amino acids surround the heme group and how do they assist in its proper function?
Proximal Histidine – occupies 5th coordination site, holds heme in place
Distal Histidine – Stabilizes oxygen binding and acts as a gate for ligand entry
Other Heme stabilizers – Val and Phe for MB and Val and Leu for HB: all of these are nonpolar so like associating with like and stabilizing the ring
Which atoms/molecules bind to Fe2+? How do they affect its function?
Fe2+ has 6 coordination sites; 4 of these site occupied by N of pyrrole rings
5th site occupied by proximal Histidine
Why can’t heme carry oxygen on its own (what does the globin do)?
Heme provides the oxygen-binding iron; the globin environment makes that binding more controlled and specific. Free heme is too strong at binding oxygen
The globin is responsible for :
Holds and stabilizes the heme group.
Uses proximal histidine to anchor the heme.
Uses distal histidine to stabilize oxygen and control ligand entry.
Reduces the relative preference for carbon monoxide compared with free heme.
Why is carbon monoxide dangerous? What globin design features make it less dangerous?
A competitive inhibitor is a compound that binds to the same site as the intended molecule (substrate or ligand), blocking the site
Carbon monoxide acts as a competitive inhibitor to oxygen with respect to Hb
o Myoglobin
What is the general structure and function of myoglobin?
Single subunit heme containing protein that can bind a max of one oxygen molecule
Acts as oxygen storage and facilitates diffusion in muscular cells
How do myoglobin’s properties serve its physiological purpose?
Myoglobin binds oxygen very tightly and saves it until you start exercising which drops the kPa in your cells greatly triggering myoglobin to release O2
Useful for oxygen storage in muscle
Facilitated Diffusion
Why can’t myoglobin serve as an oxygen carrier?
Myoglobin tightly binds to O2 at low pO2 and high pO2
What makes a protein an efficient oxygen carrier?
An efficient carrier of O2 has to let go of it when exposed to lower pO2
Has multiple binding sites
Communication between binding sites called cooperativity. Cooperativity allows for the transition between low and high affinity
Flip behavior sigmoidally when moving from low to high affinity
o Hemoglobin
What is the general structure and function of hemoglobin?
Four subunit: two alpha-globin and 2 Beta-globin subunits. Each contains a heme group
Transports oxygen from lungs to peripheral tissues
Can bind four oxygen molecules
Understand cooperativity in the context of hemoglobin
Hemoglobin is positive cooperativity
As oxygen binds, hemoglobin becomes more likely to transition from the low-affinity T state to the high-affinity R state.
What are the states of hemoglobin, what are their properties, and how are their transitions mediated by oxygen binding?
T State: low affinity
R State: high affinity
When two O2 are loaded you are equally likely to be in T or R state
Be able to describe the structural changes moving from T→R
In the T state, Fe²⁺ sits slightly outside the plane of the protoporphyrin ring. When oxygen binds to Fe²⁺, it causes the iron to move toward the plane of the porphyrin ring, pulling the proximal histidine (His F8) along with it. Because proximal histidine is part of alpha helix F, this movement shifts helix F and initiates a conformational change that promotes the transition from the low-affinity T state to the high-affinity R state.
As oxygen binds, Fe2+ is pulled downward along with the proximal His
This shifts the alpha Helix (Helix F) and that alters spatial arrangement of subunits
Shifting causes salt bridges between adjacent subunits to break.
As O2 binds, bridges break which smooths transition from T to R
Downstream processes allowing the T → R transition
Movement of helix F changes the spatial arrangement of hemoglobin's subunits, particularly at the α/β interfaces. These conformational changes disrupt salt bridges between adjacent subunits that stabilize the T state.
As more salt bridges break, the T state becomes less stable and hemoglobin transitions toward the relaxed R state. The R state has a higher affinity for oxygen, allowing additional oxygen molecules to bind more easily and promoting positive cooperativity.
Know all of the allosteric regulators of hemoglobin and their functions
Oxygen – Homotropic positive
2,3-BPG: heterotropic negative regulator of O2 binding that’s present in human red blood cells at high concentration. It binds to the central cavity of hemoglobin, stabilizing the T state. Without BPG, hemoglobin would hold on to O2 too tightly in hypoxic condition. Helps hemoglobin release oxygen under low pO2 conditions.
PH and CO2 lower oxygen binding affinity (negative allosteric regulators)
What physiological “purpose” do they serve and how is it achieved?
tunes hemoglobin's binding curve so oxygen can be delivered to tissues rather than remaining bound too tightly.
How, structurally, do they regulate hemoglobin’s behavior?
Pure hemoglobin couldn’t release O2 at low pO2 , which would create a low efficiency delivery system - BPG attunes hemoglobin’s binding curve for maximal delivery
Fetal Hemoglobin:
Fetal hemoglobin binds 2,3-BPG less effectively and therefore maintains higher oxygen affinity.
Higher affinity allows for transfer of O2 from the gestational parent’s blood to the fetus
Bohr Effect:
Bohr Effect: Regulation of oxygen binding and release by pH and CO2
The “intent” – Rapidly metabolizing tissues (such as contracting muscle) generate CO2 and lower pH; by lowering O2 affinity, these effectors release O2 where the need is greatest . Opposite occurs in lungs (lower pCO2 , increased O2 affinity)
1. The Bohr Effect – pH
Histidine 146 becomes protonated
Lysine 40 can form a salt bridge with the C-terminus
This locks protonacoted histidine 146 into a position where it can interact with aspartate 94
These interactions stabilize the T state.
Oxygen affinity decreases, promoting oxygen release.
2. The Bohr Effect – CO2
CO2 stimulates oxygen release by two mechanisms
CO2 reacts with water to form carbonic acid (H2CO3 ) – accelerated by carbonic anhydrase
Carbonic acid has a pKa of 3.5
This feeds into our pH mechanism for impacting O2 affinity.
Carbon dioxide also interacts directly with hemoglobin as part of CO2 export
Hb + CO2 forms carbaminohemoglobin, which has a lower affinity for O2
Carbaminohemoglobin:
CO2 reacts with terminal amino groups to form carbamate groups, which are negatively charged - Four terminal amino groups, four CO2 molecule capacity - Carbamate groups participate in salt-bridge interactions that stabilize the T state