Protein-Ligand Interactions and Hemoglobin

• Explores protein function and mechanisms of protein-ligand interactions
• Assesses oxygen binding properties of myoglobin and hemoglobin
• Interprets mechanisms of physiological regulation of oxygen delivery
• Identifies mechanisms of the allosteric regulation of hemoglobin
• Summarizes the molecular basis of sickle cell anemia and its relation to malaria

Static vs Dynamic Protein Functions

• Some proteins display structural flexibility and can change conformation for dynamic physiological functions.
• These structural and functional changes are often influenced through interaction with other molecules.
• Hemoglobin serves as a useful model for studying dynamic protein structure and function.

Ligands

• Many proteins undergo reversible interactions with other molecules.
• These interactions serve to regulate protein function.
• A ligand is a molecule that is reversibly bound by a protein.
• A ligand can be any molecule, including another protein.

Protein Ligands Specificity

• A ligand binds at a specific site called the binding site on the protein.
• The binding site is usually complementary to the ligand’s shape, charge, hydrophobicity, and hydrogen bonding potential.
• A given protein are able to have multiple binding sites for multiple ligands
• Hemoglobin's ligands include oxygen and 2,3 bisphosphoglycerate (2,3 BPG).

Protein Ligands Induced Fit

• Ligand binding may cause a conformational change in the protein.
• The "induced fit" changes the properties of the protein.
• Changes in protein structure often relate to changes in function.

Oxygen Delivery and Storage Overview

• Every cell needs a constant oxygen supply, which determines organism size.
• The solubility of oxygen is too low for many multicellular organisms to meet oxygen demands through passive diffusion.
• Amino acid side chains don't facilitate reversible oxygen binding
• Transition state metals bind oxygen strongly but produce damaging free radicals.
• Specialized proteins for oxygen storage and delivery are the solution to these problems.
• Heme groups are utilized to harness iron's oxygen-binding properties safely.

Oxygen Delivery and Storage Systems

• Myoglobin and hemoglobin play distinct but complementary physiological roles
• Myoglobin and hemoglobin share structural and functional features
• Myoglobin (Mb) is a monomeric protein that facilitates oxygen storage in peripheral tissue.
• Hemoglobin (Hb) is a tetrameric protein in red blood cells that carries oxygen from lungs to the periphery.

Oxygen as a Limiting Resource

• Oxygen has poor solubility in aqueous solutions
• The emergence of larger, multicellular organisms depended on proteins for oxygen transport and storage
• The amount of available oxygen which can be delivered within the organism it's able to limit size
• Insects grown in elevated oxygen presence can grow to greater sizes.

Reversible Oxygen Binding - Heme Prosthetic Groups

• Cellular iron gets bound in forms that sequester it and/or reduce reactivity.
• Heme includes a protoporphyrin ring system bound to a single (Fe2+) iron atom.
• Fe2+ binds O2 reversibly, while Fe3+ does not bind O2.
• The ring system results in four coordinating interactions with the iron atom.
• The electron-donating characteristic of nitrogen prevent conversion of Fe2+ to Fe3+.
• Myoglobin and hemoglobin both utilize heme.
• Heme is bound within discrete pockets of myoglobin and hemoglobin.

Reversible Oxygen Binding

• Fe2+ seeks six coordinating interactions.
• There are four interactions with heme.
• A fifth interaction occurs with an imidazole group of a proximal histidine residue.
• The sixth ligand position is for O2 binding.
• Distal histidine offers a stabilizing interaction for bound O2.

Heme Prosthetic Groups - Carbon Monoxide Poisoning

• Carbon monoxide (CO) has a similar molecular structure as oxygen (O2).
• Carbon monoxide exerts its deadly effects by competing with oxygen to heme.
• Carbon monoxide binds to heme with 200 times greater affinity than O2.

Oxygen Binding Proteins - Myoglobin vs Hemoglobin (Structures)

• Myoglobin, having a single subunit, shows a tertiary structure.
  • With one heme group, it binds to one oxygen molecule.
• Hemoglobin, has four subunits, is an example of quaternary structure.
  • With four heme groups, hemoglobin can bind four oxygen molecules.
• Every subunit of hemoglobin closely resembles myoglobin.

Oxygen Binding Proteins - Myoglobin vs Hemoglobin (Functions)

• Myoglobin has a higher affinity for oxygen than hemoglobin.
• Myoglobin has a hyperbolic curve of oxygen binding.
• Binding of oxygen by hemoglobin displays sigmoidal behavior, an indication of cooperativity of oxygen binding.

Myoglobin - Structure

• Myoglobin is a small globular protein
  • Myoglobin consists of a single polypeptide of 153 residues arranged in eight α-helices.
  • Contains a heme (iron porphyrin) prosthetic group.
• Sperm whale myoglobin has protein structure was determined first by x-ray crystallography

Myoglobin - Oxygen-Saturation Curve

• Myoglobin's oxygen saturation curve is hyperbolic, showing a single O2 binding capacity.
• P50 quantifies the amount of O2 required to half-saturate the protein.
• The P50 of myoglobin is 3 torr.
• In a physiological context, pO2 in the lungs (with high O2 concentrations) is 100 torr; in the periphery (low O2 concentrations), it is 20 torr.

Myoglobin - Fraction Saturation

• Myoglobin's fraction saturation with oxygen at a given oxygen partial pressure is calculated by: θ= [pO2] / ([pO2] + [P50])
• In peripheral tissues, with O2 partial pressure around 20 torr, saturation is: θ = 20/(20 + 3) = 87%.
• In the lungs, with O2 partial pressure at 100 torr, saturation is: θ = 100/(100 + 3) = 97%.
• Myoglobin features increased oxygen affinity, remaining nearly saturated with oxygen through the body.

Oxygen Transport in the Blood - Hemoglobin.

• Hemoglobin resides within erythrocytes (red blood cells).
• Hemoglobin is a quaternary protein (α2β2) with four subunits, allowing each subunit to bind one oxygen molecule.
• Hemoglobin has an allosteric nature: physiological signals regulate regulate oxygen affinity

Allosteric Proteins-General

• The term "allosteric" comes from the Greek words allos (other) and stereos (structure), signifying "Other Structure."
• They have active (R) and (inactive) T forms
• These forms are in rapid equilibrium.

Allosteric Proteins - Hemoglobin

• Proteins with a continuous high affinity for oxygen would saturate with oxygen in lungs but not release it to tissue.
• A protein equipped with reduced oxygen affinity has the ability to release oxygen to tissues, but does not feature the necessary affinity in order to saturate in the lungs.
• Hemoglobin addresses by the problem by transitioning between high and low affinity states.
• Proteins with a single ligand-binding site (such as myoglobin) can't attain this this cooperative effect.

Allosteric Proteins - Modulators (Effectors)

• Allosteric effectors (modulators) attach to specific binding allosteric proteins at specific positions
• Allosteric modulators can act both as activators and inhibitors
• Allosteric activators are responsible stabilizing the R state; allosteric inhibitors are responsible for stabilizing the T state.
• The normal ligand and modulator are identical, the interaction tends to be homotropic.
• If the modulator differs from the normal ligand, the interaction is heterotropic.

Allosteric Properties of Hemoglobin

• Hemoglobin binds and releases O2 allosterically.
• As an example O2 is a homotropic allosteric activator of hemoglobin.
• Hemoglobin's binding of the first O2 causes a conformational change, causing greater binding for subsequent O2.
• O2 binding promotes and stabilizes the R state of hemoglobin, which has higher O2 affinity.

Allosteric Properties of Hemoglobin - T to R Transition

• The iron atom rests a short distance outside; outside the plane of the heme ring within T state hemoglobin.
• When transitioning to the R state (O2 is bound), iron re-enters the plane of the ring
• Minor movement within the single subunit is responsible for structural changes within the protein's quaternary structure.

Hemoglobin - Oxygen-Saturation Curve

• The hemoglobin saturation with oxygen over the physiological O2 range is notable
• Largely saturating with oxygen at partial pressures found within the lungs
• Releasing over half of its O2 load at partial pressures measured at in the periphery
• Hemoglobin's P50 tightly coordinates to levels of O2 at the periphery
  • Hemoglobin's sensing and responding to levels of Oxygen at parts of the body at a greater hypoxia risk

Allosteric Inhibition of Hemoglobin: 2,3-Bisphospho-D-glycerate

• Early testing on hemoglobin noted high affinity for the oxygen
  • This would slow the release of protein oxygen to parts of the body
  • 2,3 BPG lowers decreased hemoglobins' affinity for oxygen; it is a heterotropic allosteric inhibitor.

Allosteric Inhibition of Hemoglobin: 2,3 Bisphospho-D-glycerate

• 2,3 BPG carries five units of negative charge.
• The subunit interface of deoxyhemoglobin contains six positively charged residues with the pocket that is formed
• The pocket has a nature is unique to deoxyhemoglobin

Fetal Hemoglobin -2,3 Bisphospho-D-glycerate

• Fetal hemoglobin has higher oxygen affinity over adult hemoglobin
• Because a fetus needs to strip or pull Oxygen from the mother's blood in the womb by stripping
• Adult Hb has six (+) residues at the 2,3BPG binding site, fetal Hb has four. (His143 replaced by Ser)
• Decreased affinity for 2,3 BPG translates into higher O2 affinity for fetal Hb.
• Lower fetal hemoglobin affinity bestows a greater affinity for oxygen

BPG - High Altitude Adaptation

• At altitudes with lesse oxygen in the atmopshere:
  • Adaptation occurs through increased 2,3 BPG production
  • This results in an decreased Oxygen afinity
  • This allows for good peripheral delivery.

The Bohr Effect

• This describes dependence of hemoglobin's oxygen affinity to PH.
• Hemoglobin has lowered affinity when PH decreases.
• With its' raised muscle activity raising CO2 producion, active tissues lower the PH
  • Muscles that are exposed to extreme activity increases PH futher when lactic acid is released.

Coordination of O2 Delivery and CO2 Removal

• There are two primary challenges to cellular respiration and metabolism:

1. Sufficient tissue O₂ delivery
2. Eliminating CO2 (the "exhaust" of metabolism) from periphery

• Increased muscle activity increases both oxygen need carbon dioxide production
• Through these adaptations, the body coordinates these events.
• O2 is used throughout process, with constant CO2 output.

Coordination of O2 Delivery and CO2 Removal - Mechanism #1

• CO2 converted to carbonate and a proton within red blood cells by the enzyme carbonic anhydrase

1. The CO2 converts into a soluble substance that can be transported to the lungs
2. O2 releases promoted to active tissues due to decreased affinity of Oxygen due to hemoglobin

Coordination of O2 Delivery and CO2 Removal - Mechanism #2

• CO2 produce a covalent carbamate that binds to the N terminus of chains in hemoglobin to produce carbaminohemoglobin.
• The reaction three significant results:
  • Convert Carbon Dioxide into soluble substance that assists flow to lungs
  • Hemoglobin that contains carbamino experiences decreased oxygen affinity that enhances O2 to release
  • Oxygens release gets encouraged thorough release proton

Sickle Cell Anemia

• Results from one amino change. (Glu6Val).
• Fibers form HbS deoxy forms.
• Fibers appear in capillaries and cuts off extremities blood flow
• T state = peripheral of body.

Sickle Cell Anemia - Malaria

• This affects African America and Africa, and those regions have malaria SCA has shown to be linked to the regions of malaria.
• Theory individuals heterozygous for Malaria demonstrate resistance to malaria
  • red blood cells get infected by Malaria
  • infection decreases pH. in and around red bleed cells
• In turn decrease pH cause the release of oxygen by the HB.
• For people with sickle cell, hb's deoxy chains end up deforming the blood cells, so, The the spleen destroys those red blood cells

Other Oxygen Transport Proteins - Hemocyanin

• Some invertebrates, like horseshoe crabs, use hemocyanin instead of hemoglobin to transport oxygen.
• Copper used with this that makes blood appear clue not red.
• Two oxygen molecules bind at a single atom
• With the atoms of copper connected to the histidine without any kind of heme.
• Not confined within specialized oxygen transport cells.

Explore the dynamics of protein function through hemoglobin, focusing on ligand binding site characteristics and induced fit. Understand the impact of pH and the effects of deoxy HbS in sickle cell anemia. These concepts are crucial for understanding protein behavior and function.