Chapter 5 Notes: Binding, Molecular Recognition, and Allostery
5.1 Binding Is a Fundamental Process in Biochemistry
Molecular recognition: the ability of specific molecules to bind to one another in the face of many alternatives.
Sets of molecules stick together and thus spend more time together than they would without binding.
Occurs despite many other potential binding partners around them.
Receptor (R) and ligand (L) form a complex:
R + L \rightleftharpoons RL
Complex binding depends on concentrations of binding partners (three qualitative regimes):
If binding is infinitely strong, every added L binds an R to form RL until all R are bound (green panel).
If binding tendency is very weak, most of the time no complex forms because thermal motion dominates (blue panel).
In between, some L binds to R to form RL while others remain free (red panel).
As more L is added in the intermediate regime, the probability that a given receptor is bound to L increases.
L1/2 (half-maximum binding): the concentration of L at which half the receptors are bound to L and half are free.
L1/2 indicates the concentration range around which the receptor transitions from mostly free to mostly bound.
The tighter the binding, the smaller the value of L1/2.
L1/2 serves as an inverse measure of binding affinity.
Quantitative perspective on binding: the binding affinity is often summarized by a dissociation constant.
Kd = [R][L] / [RL].
Concept recap:
Binding is a function of ligand and receptor concentrations and their intrinsic affinity.
The RL population at equilibrium reflects the balance of association and dissociation processes.
5.2 Myoglobin and Hemoglobin Bind Oxygen in Heme Groups
Two ligand-binding proteins:
Hemoglobin (Hb): a component of red blood cells; efficiently binds and carries oxygen from lungs to tissues; helps transport CO2 and H+ back to lungs.
Myoglobin (Mb): found primarily in muscle tissue; facilitates diffusion of oxygen to cellular sites requiring oxygen; serves as an oxygen reserve.
Heme group is essential for both Mb and Hb oxygen binding.
Heme consists of an organic component, protoporphyrin, and a central iron atom (Fe).
The iron is coordinated to four pyrrole nitrogens (the porphyrin plane).
Two additional coordination sites (fifth and sixth) are available for ligands on either side of the heme plane.
Under normal (deoxy) conditions, the iron is in the Fe^{2+} state.
The sixth coordination site becomes available for O2 binding when conditions favor it.
Structural context:
Deoxyhemoglobin and deoxymyoglobin: the sixth coordination site is unoccupied in these states.
The iron atom lies ~0.4 Å outside the porphyrin plane in the deoxy state due to its size.
Binding of O2 at the sixth coordination site rearranges the electrons within iron, effectively making the ion smaller and allowing it to move into the plane of the porphyrin.
The heme group contains important surrounding residues:
Proximal histidine bonds to the iron on the side of the heme that is closer to the protein core.
Distal histidine (on the opposite side) plays crucial roles in selectivity and stabilization (see 5.2–5.3).
The distal histidine (a key factor in ligand selectivity):
Helps globins select O2 over larger/dangerous ligands (e.g., CO).
It blocks larger molecules from approaching the iron and provides a hydrogen bond that stabilizes O2 binding in the proper orientation.
Additional notes on CO binding:
CO can also bind to the central iron, but the orientation differs and the distal histidine hydrogen bond is missing.
The distal histidine simultaneously increases O2 affinity while decreasing affinity for other ligands like CO.
Summary of the heme-based oxygen binding:
Oxygen binding depends on the presence of heme and the appropriate alignment of the iron within the porphyrin ring.
The distal histidine and proximal histidine coordinate with the iron to control binding specificity and stability.
5.3 Hemoglobin Binds Oxygen Cooperatively
Hemoglobin (Hb) structure:
Hb is an allosteric protein composed of four polypeptide chains: two identical α chains and two identical β chains.
The Hb tetramer (HbA) can be viewed as a homodimer of heterodimers: (α1β1) and (α2β2).
Oxygen-binding curve characteristics:
HbA displays a sigmoid (S-shaped) oxygen-binding curve, indicating cooperativity.
Binding of O2 at one site increases the likelihood of O2 binding at the remaining sites; unloading at one site facilitates unloading at others.
This cooperative behavior arises from inter-subunit communication rather than independent sites.
Functional consequence:
Cooperativity enables efficient oxygen transport from lungs (high O2) to actively metabolizing tissues (lower O2).
Practical observation:
In the lungs, Hb becomes nearly saturated with O2 (≈98% occupied).
In tissues, Hb releases O2, leading to saturation around ≈32%.
The difference (≈66% of total potential binding sites) contributes to the overall oxygen delivery.
Structural basis for cooperativity (qualitative):
Quaternary structural changes accompany O2 binding, coordinating subunit interactions and shifting from a low-affinity T state to a high-affinity R state.
5.4 An Allosteric Regulator Determines the Oxygen Affinity of Hemoglobin
Allosteric effectors regulate enzyme or protein activity by altering conformation and affinity.
Hemoglobin allosteric regulator: 2,3-bisphosphoglycerate (2,3-BPG).
2,3-BPG is a highly anionic molecule present in red blood cells at roughly the same concentration as Hb (~2 mM).
It binds to a pocket that is present only in the T state (deoxy form) at the center of the Hb tetramer.
The interaction is primarily ionic between 2,3-BPG’s negative charges and three positively charged groups on each β chain.
Binding of 2,3-BPG stabilizes the T state, reducing Hb’s O2 affinity and promoting release of O2 (about 66% release under physiological conditions mentioned, rather than the higher baseline without 2,3-BPG).
Fetal hemoglobin (HbF) and 2,3-BPG binding:
HbF tetramers contain two α chains and two γ chains (α2γ2).
The γ chain is ~72% identical in sequence to the β chain.
A notable difference is a Ser residue replacing His at position 143 in the γ chain’s 2,3-BPG binding site, reducing 2,3-BPG affinity.
Lower 2,3-BPG affinity leads to higher O2 affinity for HbF, enabling efficient transfer of O2 from maternal Hb to fetal Hb in a low-oxygen environment.
5.5 Hydrogen Ions and Carbon Dioxide Promote the Release of Oxygen
Bohr effect: hydrogen ions (H+) and carbon dioxide (CO2) act as allosteric regulators that promote O2 release.
Metabolically active tissues release protons and CO2, reducing Hb’s O2 affinity and increasing oxygen delivery where it is needed most.
Hydrogen ion effect on Hb affinity:
Hb’s oxygen affinity decreases as pH decreases (more acidic conditions).
The combined effect of pH change and partial pressure can markedly increase O2 release (e.g., ~77% of total carrying capacity released under certain conditions, versus 66% without pH change).
Role of histidine residues in protonation-state changes (example provided):
Deoxyhemoglobin is stabilized by ionic interactions (salt bridges).
The protonation state of His146 varies with pH (high pH vs low pH) and contributes to stabilization of the T state during protonation.
Carbon dioxide’s direct effect on Hb affinity:
CO2 directly interacts with Hb by forming carbamate groups on terminal amino groups, stabilizing the T state and promoting O2 release in active tissues.
In the presence of CO2 at a partial pressure of about 40 torr and pH ~7.2, O2 release can approach ~90% of maximum carrying capacity.
CO2 and H+ transport in blood:
Hb carrying CO2 and H+ helps transport these species back to the lungs.
In total, Hb accounts for only a portion of CO2 and H+ transport (roughly 14% of total); the rest is transported as bicarbonate (HCO3–) via spontaneous formation or through the enzyme carbonic anhydrase in red blood cells.
5.6 Mutations in Genes Encoding Hemoglobin Subunits Can Result in Disease
Sickle cell anemia (HbS) is caused by a single amino acid substitution in one Hb chain.
Blood smears show red blood cells with a sickle shape.
Inheritance: HbS is autosomal recessive (homozygous individuals show disease); heterozygotes (sickle cell trait) are usually asymptomatic.
Molecular basis of sickling:
HbS differs from HbA by a single substitution: Valine replaces Glutamate at position 6 of the β chains (Glu6Val).
The exposed Val side chain interacts with a complementary hydrophobic patch formed by Phe β85 and Leu β88 on another Hb molecule.
This hydrophobic interaction is exposed preferentially in the deoxygenated form, promoting polymerization of deoxygenated HbS into long fibrous aggregates.
Consequences:
Polymerization and aggregation of HbS lead to distortion of red blood cells (sickling), especially under low oxygen tension.
The fundamental properties of Hb’s oxygen affinity and allosteric regulation are not substantially altered by this mutation, but the polymerization of deoxygenated HbS causes cellular deformation.
Clinical relevance:
Sickle cell disease is a clinically important example illustrating how a small genetic change can have large physiological and pathological consequences.
Connections to foundational principles and real-world relevance:
Binding and recognition rely on thermodynamics and equilibrium constants (Kd) encapsulating affinity and specificity.
Allostery demonstrates how distant sites communicate structural changes to alter function (e.g., T to R transitions in Hb, effectors like 2,3-BPG, H+, CO2).
The Bohr effect links metabolic state (pH, CO2) to oxygen delivery, illustrating how physiological conditions tune protein function in tissues.
The Hb system integrates structure, energetics, and regulation to achieve efficient oxygen transport and distribution, with clinical relevance in diseases like sickle cell anemia and in fetal development via HbF.
The heme prosthetic group shows how cofactors extend protein function and how protein environment (distal/proximal histidines) modulates ligand binding.
Quantitative descriptors (L1/2, Kd, P50) provide a framework to compare binding across ligands, receptors, and allosteric states.