Hemoglobin and Myoglobin
Page 1: Myoglobin and Hemoglobin
Overview
B₂ Chemistry 237: A course reflecting on oxygen-binding proteins.
Focus on O2 binding proteins such as Myoglobin and Hemoglobin.
Page 2: Introduction to Topics
What is Coming
General protein-ligand interactions.
Definition and description of binding including thermodynamics.
Oxygen binding properties of Myoglobin and Hemoglobin.
Structural-function correlation for Myoglobin and Hemoglobin.
Study of Hemoglobin mutants and the correlation of changes in their structure and function.
Page 3: Protein-Ligand Interactions
Key Concepts
Reversible and Transient Process: Chemical equilibrium represented as A + B ⇌ AB.
Ligand: A small molecule that binds to the protein.
Binding Site: The specific region on the protein where the ligand binds.
Binding Mechanism: Ligand binds via non-covalent forces allowing transient interactions.
Page 4: Binding Quantification
Equilibrium Composition
Ligand (L) binding to a protein site (P) characterized by the equilibrium constant (K):
Formula: K_A + k_a & k_b 𝑃𝐿 ⇌ 𝑃 + 𝐿
A large value of K_A indicates tight binding:
[PL] is the concentration of the bound form.
Page 5: Binding Characteristics
Important Definitions
Equilibrium Composition characterized by the Equilibrium Dissociation Constant (K_d):
K_d = [P]×[L] / [PL]
K_d small indicates tight binding.
Page 6: Bound Fraction Analysis
Analysis of Binding Fraction
Bound Fraction (Θ): Can be determined practically to simplify the binding equation.
Substituting [PL] and rearranging leads to formulas based on equilibrium constants.
Page 7: Binding Curves
Key Concepts
K_d indicates ligand concentration at half saturation.
Typically a plot of the fraction of bound sites vs ligand concentration.
Page 8: Analysis of Binding Curves
Characteristics of Binding Curves
Binding strength is contingent on free ligand concentration and K_d.
K_d determinable graphically and may indicate the half-occupancy of binding sites.
Page 9: Thermodynamics of Binding
Thermodynamic Connections
Association constant (K_a), dissociation constant (K_d) are crucial for describing binding.
Relationship: K_d = 1/Ka
Free energy (∆G°) equation: ∆G° = ∆H° - T∆S°.
Strong binding: K_d < 10 nM; weak binding: K_d > 10 µM.
Page 10: Examples of Binding Strength
Protein Dissociation Constants
Protein | Ligand | Kd (M) |
|---|---|---|
Avidin | Biotin | 1 × 10^-15 |
Insulin receptor | Insulin | 1 × 10^-10 |
Anti-HIV immunoglobulin | gp41 | 4 × 10^-10 |
Nickel-binding protein | Ni²+ | 1 x 10^-7 |
Calmodulin | Ca2+ | 3 × 10^-6 |
Sequence-specific Protein-DNA | - | 10^-16 |
Page 11: O2 Transport in Organisms
Importance of O2
Active cells require O2 for efficient energy conversion through oxidative metabolism.
O2 needs to diffuse readily through tissues to sustain cellular functions.
CO2 produced by active cells must also be removed efficiently.
Page 12: O2 and Iron Interaction
Binding Dynamics
Iron interactions related to O2 binding.
Page 13: Myoglobin and Hemoglobin Overview
Key Features
Each subunit binds one iron-containing heme group.
Alpha subunit has 7 alpha helices; Beta has 8.
Heme group is nestled in a hydrophobic pocket formed by C, F, and E helices.
No covalent attachments between the heme and the protein exist.
Myoglobin is monomeric; Hemoglobin is tetrameric.
Page 14: Function and Structure of Myoglobin and Hemoglobin
Myoglobin
Located in muscle tissue; composed of 153 amino acids in a single polypeptide.
Functions to store and transport O2 within muscle tissue, enhancing solubility and diffusion rates.
Hemoglobin
Found in red blood cells; a tetrameric protein (α2β2) with 141 amino acids in α and 143 in β subunits.
Transports O2 from the lungs to tissues and CO2 from tissues to lungs.
Page 15: Iron Binding Sites
Binding Characteristics
Interaction of iron with heme and its related dynamics.
Page 16: Measuring O2 Binding
Spectrophotometric Analysis
Heme group is a strong chromophore absorbing UV and visible light.
Electronic state changes of Fe affect absorbance during O2 binding, monitored through UV-Vis spectrophotometry.
Page 17: O2 Binding Curve for Myoglobin
Curve Characteristics
X-axis: Partial pressure of O2 (pO2).
Y-axis: Degree of saturation (Θ).
Myoglobin binding curve is hyperbolic, contrasting with the sigmoidal curve of Hemoglobin.
Page 18: Function of Partial Pressure of O2
Protein Functionality
Myoglobin aids O2 movement within the muscle; Hemoglobin facilitates O2 delivery from lungs to tissues.
Partial pressure of O2 measures O2 solubility in blood or cellular fluid.
Page 19: Definition of Saturation Degree
Degree of Saturation
Often referred to as 'θ' or Y_O2:
θ = [O2 bound]/[total binding sites].
P50 defined as the pO2 at θ = 0.5.
Page 20: O2 Binding Curve Analysis
Effects at Varying pO2 Levels
Low pO2: Reduced binding.
High pO2 (lungs ~13 kPa): Effective binding occurs.
Page 21: Myoglobin Functions
Key Functions
Increases solubility of O2 in water (in muscles).
Enhances O2 diffusion.
Critical for O2 storage in deep-diving mammals.
Knockout mice can survive without substantial natural function of Myoglobin.
Page 22: Myoglobin as a Transport Protein
Transport Limitations
Myoglobin's tight O2 binding is advantageous for storage, but it is not an efficient transport protein.
Page 23: Requirements for Cooperative Binding
Cooperative Mechanism
Cooperative proteins with multiple binding sites enhance binding interactions.
Positive Cooperativity: First binding increases affinity for remaining sites.
Page 24: Hemoglobin Structure
Characteristics
Available in red blood cells and is a tetramer (α2β2) with specified subunit structures.
Functions mainly for O2 transport and CO2 removal in circulation.
Page 25: Cooperative Binding in Hemoglobin
Binding Curve Properties
Binding is cooperative resulting in the sigmoidal shape of the curve.
Models: Hill Model, Adair Model, MWC symmetry model.
Page 26: T and R States Defined
Structural States
T-State: Low affinity, associated with deoxy state.
R-State: High affinity, associated with oxy state.
Definitions based on studies by Jacques Monod, Jeffrey Wyman, and Jean-Pierre Changeux.
Page 27: T-State vs R-State
Binding Characteristics
T-state shows weak binding at all sites.
R-state allows for strong binding at all sites. Interaction between T and R states determines binding affinity.
Page 28: Importance of Conformational States
Physiological Relevance
Hb binds O2 efficiently in lungs (pO2 ~100 torr) and releases it in the body (pO2 ~30 torr).
Page 29: T-State & R-State Structures
Visualization
Structural representations of T-state and R-state in visual studies.
Page 30: Positive Cooperativity Concept
Reinforcing Binding Dynamics
Binding of one O2 influences other binding sites leading to easier O2 binding as saturation progresses, resulting in a sigmoidal curve.
Page 31: Effectors Influencing O2 Binding
Concept of Effectors
Effectors modify protein/enzyme activity:
Positive effectors: Enhance binding.
Negative effectors: Decrease binding affinity.
Example: O2 acts as a positive effector for itself in Hemoglobin.
Page 32: Influence of Blood Acidity on O2 Affinity
Acidity Impact
Increased H+ and CO2 stabilizes T-state, reducing O2 affinity.
CO2 binds at Hb’s N terminus reducing affinity.
Blood stream molecules alter O2 affinity, BPG stabilizes T-state.
Page 33: The Bohr Effect
Description and Observation
Observed by Christian Bohr in 1904, increased acidity causes a right shift in Hb binding curve.
Page 34: Physiological pH Conditions
Metabolism Effects
Metabolic processes generate CO2 and protons affecting blood pH.
Equilibrium: CO2 + H2O ⇌ HCO3- + H+.
Page 35: pH Impact on O2 Affinity
Correlations
Higher levels of H+ reduce O2 affinity, facilitating release when metabolism increases.
Page 36: Binding of Protons in T-State
Proton Binding Dynamics
Protons bind to stabilize T-state H-bonds during O2 binding dynamic.
Page 37: CO2 Binding Mechanism
Binding Dynamics
CO2 binds to beta subunit N-termini leading to increased blood acidity; thus enhancing O2 release.
Page 38: Summary of Effects
Cellular Metabolism Summary
O2 consumption generates H+ which destabilizes Hb-O2 bond, increasing O2 release.
H+ binding leads to stabilizations in T-state.
In lungs, high pO2 drives R-state formation, releasing H+.
Page 39: The BPG Effect in Hemoglobin
Behaviors of Purified vs In-Blood Hemoglobin
Carbamate formations and effects of H+ and BPG binding need to be distinguished.
Page 40: BPG Properties
Biochemical Role
BPG (bis-phosphoglycerate) is a small molecule with five negative charges binding to Hb under certain states, decreasing O2 affinity.
Page 41: BPG Binding Dynamics
Binding Site / States
BPG binds only in the T state's central region; high O2 concentrations disallow such binding in R state.
Page 42: Physiological Influence of BPG
Implications at High Altitudes
Increased [BPG] raises P50, resulting in better O2 offloading in low oxygen environments.
Page 43: BPG Effect at High Altitude
Adaptive Mechanism
Adaptation to lower oxygen uptake leads to increased tissue O2 delivery despite decreased atmospheric O2 levels.
Page 44: Changes Over Time at High Altitudes
BPG and Altitude Prevalence
Correlation of P50 changes with duration at high altitudes.
Page 45: Myoglobin Structure
Overview of Myoglobin
Key component in muscles aiding O2 diffusion, structured from 153 amino acids with globin regions.
Page 46: Structural Composition of Myoglobin
Key Elements
Presence of 8 alpha helices and 1 heme, stabilized by hydrophobic interactions and ionic interactions.
Page 47: Heme Binding Mechanics
Heme Functional Dynamics
Bound between E and F helices, crucial interactions prevent oxidation of Fe2+ and allow reversible O2 binding.
Page 48: Important Residues in Heme Interaction
Residue Interactions
Various amino acids play roles in the heme functional activity and binding properties.
Page 49: Salt Bridges in Myoglobin
Summary of Interactions
Key salt linkages contributing to Myoglobin stability.
Page 50: General Structural Properties of Hemoglobin
Overview of Structure
Composed of 4 subunits and specified contacts crucial for full functionality.
Page 51: Similarities and Differences in Structures
Sequence Identity
Myoglobin and Hemoglobin show structural similarities despite low sequence homology.
Page 52: Structural Identity of Heme
Comparative Overview
Myoglobin and Hemoglobin consist of similar frameworks, showing approximately 20% sequence identity.
Page 53: Interface Dynamics of Hemoglobin
Hemoglobin Dimer Interactions
Recognition of contacts and structural changes during transitions.
Page 54: Detailed Interactions
Residue Changes and Conformational Switches
Key residues involved in transitions between T and R states.
Page 55: Visualizations of Dimer Structure
Color-Coded State Illustrations
Detailed structural changes among the subunits depicted in color-coded forms.
Page 56: Structural Changes During O2 Binding
Geometric Adjustments
Observations of heme geometry alteration upon O2 binding.
Page 57: Movement Correlation in Heme and Helices
Movement Dynamics
Micro-level movements resulting from O2 binding seen within the helical structures.
Page 58 & 59: X-Ray Structure of Deoxy and Oxy Hemoglobin
Structural Insights
Visualization of hemoglobin structures in both deoxygenated and oxygenated forms.
Page 60: T and R-State Interface Changes
Interaction Changes
Notable hydrogen bond alterations arising during O2 binding.
Page 61: C-Termini Changes in Hemoglobin
Influence of Binding
O2 binding affects C-terminal stability positively impacting R-conformation.
Page 62 & 63: Structural Profiles of Deoxy and Oxy Hemoglobin
Reiteration of Structural Changes
Similar reiteration of key structural information in underlying diagrams.
Page 64: BPG Binding Sites
Binding Dynamics
Collage of structure information detailing the binding affinity themes.
Page 65: Hemoglobin Stabilization Factors
Summary of Stabilization Forces
Recognizable interactions holding hemoglobin together during its functional activities.
Page 66: Hemoglobin Variants Origin
Gene Relationships
Overview of structural and evolutionary connections among globin genes.
Page 67: Fetal Hemoglobin Structure
Comparison with Adult Hemoglobin
Composition noted for fetal hemoglobin contrasting with adult forms.
Page 68: Affinity of Fetal Hemoglobin
Binding Properties
Fetal hemoglobin displays higher O2 affinity at lower pO2 due to its binding stability.
Page 69: His143 Substitution Effects
Impact on Binding Affinity
Role of Ser143 in decreasing BPG binding, influencing O2 affinity.
Page 70: Overview of Abnormal Hemoglobin Variants
Variant Characteristics
Highlight of various abnormal hemoglobin states with potential clinical manifestations.
Page 71: Sickle-cell Hemoglobin Description
Characteristics and Consequences
Sickle-cell variant characterized by altered red blood cell morphology leading to transport difficulties.
Page 72: Mechanism of Sickle Cell Formation
Structural Alteration
Glu6 to Val mutation leading to fiber formation under T-state, causing physical red blood cell deformities.
Page 73: Sickle Cell Fiber Formation
Insights on Fiber Contacts
Detailed understanding of intermolecular contacts causing fiber formations in sickle-cell hemoglobin.
Page 74: Trait vs Disease in Sickle Cell
Prevalence and Advantage
Exploration of the heterozygous advantage against malaria and persistence in populations despite health issues from the homozygous state.
Page 75: Malaria and Sickle-cell Gene Correlation
Visual Insights
Graphical representation showcasing the overlap of malaria prevalence with sickle-cell gene distribution.
Page 76: Table of Hemoglobin Variants
Variant Names and Effects
Name | Mutation Description | Effect |
|---|---|---|
Hammersmith | Phe CD1(42)β → Ser | Weakens heme binding |
Bristol | Val E11(67)β → Asp | Weakens heme binding |
... | ... | ... |
Page 77: Disruption of Binding by Variants
Effects of Variants
Impact on heme binding and overall functionality of affected variants from external influences.
Page 78: Further Variants Effects
Similar Observations and Impacts
Comprehension of structural disruptions stemming from specific mutations and their subsequent impacts.
Page 79: Methemoglobins
Overview
Mutations leading to oxidation of Fe2+ to Fe3+ nullifying O2 binding capability.
Page 80: Mutation Dynamics
Structural Implications
Detailed overview of mutations that influence stability leading to altered functional dynamics in hemoglobin.
Page 81: Effects of Switching Region Mutations
Stabilization Consequences
Analysis of how mutations in key regions stabilize either T or R state configurations.
Page 82: Summary of Mutation Effects
Overview of Cooperativity and Affinity
Understanding of how alterations enhance or inhibit O2 affinity and cooperativity.