BIOC 460 Study Notes
Introduction
This document serves as study notes for BIOC 460/660 Foundations of Biochemistry and Molecular Biology I, focusing on the three-dimensional structure and function of proteins as detailed in Chapters 4 and 5 of the lecture slides.
Chapter 4: The Three-Dimensional Structure of Proteins
Overview
Amino acid sequences convey crucial information regarding:
Three-dimensional (3D) structure
Function
Cellular location
Evolution
Proteins function as polymers of amino acids; structure heavily influences function.
Amino Acid Sequence and Importance
Primary amino acid sequence comparisons: The direct comparison of the linear order of amino acids between different proteins helps to deduce relationships, functional similarities, and evolutionary origins. Such comparisons are fundamental for understanding protein behavior.
Consensus sequence: Represents the most common amino acid at each position across a set of aligned proteins, highlighting residues essential for maintaining structure or function. Its presence indicates high selective pressure on certain positions.
Signature sequence: Specific protein segments or motifs that are highly conserved within a particular protein family or taxonomic group, allowing for the identification of remote homologs and assisting in constructing evolutionary trees based on protein sequences.
Homologs: Proteins sharing a common ancestry and biochemical function. Identified through sequence comparison against protein databases using algorithms like BLAST.
Paralogs: Homologs within the same species, often arising from gene duplication events. They may have evolved new functions or subfunctionalized.
Orthologs: Homologs found in different species, usually retaining the same functional roles. They are often studied to infer the function of an unknown protein from a known one.
Conserved substitutions of amino acids: Replacement of an amino acid with another that has similar physiochemical properties (e.g., Leu to Ile, Asp to Glu), which is less likely to significantly alter protein structure or function. Indicates evolutionary pressure to maintain certain properties.
How amino acid sequence conservation equates to relationships between organisms: Greater sequence similarity between proteins from different organisms indicates a more recent common ancestor. Examination of multiple protein families allows insights into evolutionary relationships and the history of life.
Bioenergetics and Protein Structure State
The folding of a protein into its native 3D structure (native fold) is a spontaneous process, driven by favorable energetic interactions within the protein and with its solvent environment, leading to a net decrease in the system's free energy (\Delta G).
This conformation arises from numerous favorable interactions within the protein, despite an entropy cost resulting from the polypeptide chain becoming more ordered.
Example: Folding is likened to organizing a messy room, where energy is required to arrange a disordered state into organized form, but the organized state (native fold) is more stable and functional.
Favorable Interactions in Proteins
These non-covalent interactions stabilize the native fold and are also crucial for ligand binding and protein interactions:
Hydrophobic effect: The most significant driving force in protein folding. Increased entropy due to the release of water molecules from around nonpolar regions as they coalesce in the protein's interior, minimizing their contact with water.
Hydrogen bonds: Form between N-H and C=O groups in peptide bonds (stabilizing secondary structures) and between polar side chains or side chains and the polypeptide backbone. These are weaker individually but collectively contribute significantly to stability.
van der Waals forces: Weak, transient attractive forces between electron clouds of closely approaching atoms. They are crucial for tight packing in the protein's interior, contributing to overall stability, especially in the hydrophobic core.
Electrostatic interactions (Salt Bridges): Strong attractive forces between oppositely charged groups (e.g., Lys and Asp side chains). These are particularly strong and stabilizing when located within the hydrophobic interior of a protein, where water cannot screen the charges.
Where in a protein can these forces predominate?
Hydrophobic effect and van der Waals forces predominantly stabilize the protein's interior core, where nonpolar residues are packed together away from water.
Hydrogen bonds and electrostatic interactions can occur on the surface (interacting with water or other molecules) and in the interior (forming structural elements like salt bridges).
Protein Primary Sequence and Structure
Proteins exhibit four hierarchical levels of structure:
Primary structure: The linear sequence of amino acids, specified from the amino (N)-terminus to the carboxyl (C)-terminus. It is determined by the genetic code and dictates all subsequent structural levels.
Secondary structure: Regular, recurring local structures formed through hydrogen bonding between amide and carbonyl groups of the polypeptide backbone. The most common types are α helices and β sheets.
Tertiary structure: The overall three-dimensional arrangement of all atoms in a single polypeptide chain, including side chains. It is stabilized by various interactions between side chains (hydrophobic, H-bonds, electrostatic, disulfide bonds).
Quaternary structure: The complex of multiple polypeptide subunits (each a tertiary structure) forming a functional protein assembly. Stabilized by non-covalent interactions and sometimes disulfide bonds between subunits.
Resonance within a peptide affecting peptide bond: The peptide bond (C-N) between amino acid residues has partial double-bond character due to resonance stabilization. This makes the peptide bond rigid, planar, and unable to rotate freely.
Which bonds in a polypeptide show rotation and which do not?: The peptide bond itself does not show free rotation. Rotation is restricted to the bonds connecting the α-carbon to the amino nitrogen (N- ext{C}oldsymbol{\alpha} bond) and the α-carbon to the carbonyl carbon ( ext{C}oldsymbol{\alpha} -C bond).
Dihedral angles (torsion angles): These describe the rotations around the bonds in a polypeptide chain.
\Phi (phi) angle: Rotation around the N- ext{C}oldsymbol{\alpha} bond.
\Psi (psi) angle: Rotation around the ext{C}oldsymbol{\alpha} -C bond.
Protein Secondary Sequence
Ramachandran plot (what can they tell you?): A graphical representation plotting the \Phi and \Psi angles for each amino acid residue in a protein. It illustrates sterically allowed combinations of these angles, revealing common secondary structure regions (e.g., α-helices, β-sheets) and identifying disallowed conformations due to steric hindrance, thus predicting potential secondary structures.
What role does phi/psi angles play in secondary structure?: The specific combinations of \Phi and \Psi angles define the distinct geometries of secondary structures. For example, specific ranges of angles are characteristic of α-helices and β-sheets.
Common secondary structures:
α-Helix:
Structure: A right-handed coiled structure where the polypeptide backbone winds around a central axis.
Handedness: Almost always right-handed in proteins, due to the L-amino acid chirality.
Stabilization: Primarily stabilized by hydrogen bonds formed between the carbonyl oxygen of an amino acid residue (n) and the amide hydrogen of the residue four positions ahead (n+4).
Approximate diameters/length per turn: Has approximately 3.6 residues per turn, a pitch (rise per turn) of 5.4 \mathring{A}. The diameter is about 5.0 \mathring{A} (not including side chains).
Amino acid sequence and alpha-helix stability: Certain amino acids (e.g., Ala, Leu, Met) are strong helix formers, while others (e.g., Pro, Gly) are helix breakers. Proline introduces a kink and lacks an N-H for H-bonding. Glycine's flexibility allows too many conformations.
Alpha-helix dipoles and amino acids at ends: Due to the alignment of peptide bond dipoles, an α-helix possesses a significant macroscopic dipole moment, with a partial positive charge at the N-terminus and a partial negative charge at the C-terminus. Negatively charged amino acids are often found at the N-terminus, and positively charged at the C-terminus, to neutralize these partial charges.
β-Sheet:
Structure: Consists of multiple polypeptide strands (β-strands) connected laterally by hydrogen bonds. The backbone is extended and zig-zagged.
These H-bonds occur between the carbonyl oxygens of one strand and the amide hydrogens of an adjacent strand.
Can be parallel (adjacent strands run in the same N- to C-terminal direction) or antiparallel (adjacent strands run in opposite directions). Antiparallel sheets are generally more stable due to linear H-bonds.
β-Turns (or reverse turns):
Function: Often found in protein structure to reverse the direction of the polypeptide chain abruptly, allowing the protein to fold into a compact globular shape.
Characteristics: Typically involves four residues (Type I and Type II are common types) and is stabilized by a hydrogen bond between the carbonyl oxygen of residue n and the amide hydrogen of residue n+3 (for Type I and II).
Common amino acids: Proline (at position 2) and glycine (at position 3) are common within β-turns. Proline's rigid ring structure facilitates the sharp turn, and glycine's small side chain provides flexibility.
Circular dichroism (CD): A spectroscopic technique used to determine the secondary structure content (e.g., % α-helix, % β-sheet, % random coil) and folding state of proteins. It measures the differential absorption of left and right circularly polarized light by chiral molecules. Distinct CD spectra are observed for different secondary structures.
Protein Tertiary/Quaternary Structure
Tertiary structure: The unique overall 3D shape of a single polypeptide chain, resulting from interactions between amino acid side chains, as well as between side chains and the polypeptide backbone. It is stabilized by hydrophobic interactions, hydrogen bonds, salt bridges, van der Waals forces, and covalent disulfide bonds (between cysteine residues).
Major classes of protein (structure-wise):
Globular proteins: Compact, roughly spherical proteins, typically water-soluble, with diverse functions (e.g., enzymes, transport proteins, regulatory proteins). They often contain a mixture of α-helices and β-sheets.
Fibrous proteins: Extended, elongated proteins, typically insoluble in water, serving structural and protective roles (e.g., collagen, keratin). They usually consist of a single type of secondary structure (e.g., α-helical coiled-coils or β-sheets).
Coiled-coil structure: A supersecondary structure where two or more α-helices are wrapped around each other, forming a stable structure. It is characterized by a heptad repeat of amino acids (abcdefg) where positions 'a' and 'd' are typically hydrophobic, forming a hydrophobic strip that mediates the interaction between helices.
Structures of three fibrous protein examples (similarities and differences):
α-Keratin: (Hair, nails, skin) Two right-handed α-helices twisted together to form a left-handed coiled-coil. Rich in Cys, allowing disulfide bonds to form between intermediate filaments, contributing to its strength.
Collagen: (Connective tissue) Consists of three left-handed helical polypeptide chains (not α-helices) woven together to form a right-handed superhelical triple helix. Rich in Gly, Pro, and 4-hydroxyproline. High tensile strength.
Silk Fibroin: (Silk fibers) Composed predominantly of antiparallel β-sheets. Glycine and alanine residues alternate, allowing for tight packing of sheets, giving it flexibility and strength.
Similarities: All are structural proteins, exhibit high tensile strength, and are typically insoluble. They derive their strength from repetitive secondary structures and their hierarchical assembly.
Differences: Differ in their constituent secondary structures (α-helices, triple helix, β-sheets), amino acid composition, and the type of interchain interactions (disulfide bonds in keratin, H-bonds in collagen/silk).
How are protomers of alpha-keratin and collagen held together?
α-Keratin: Protofilaments (dimers of coiled-coils) associate to form protofibrils, which then assemble into intermediate filaments. Disulfide bonds between cysteine residues in adjacent chains significantly cross-link and strengthen the keratin structure, especially in harder keratins (e.g., horns).
Collagen: Three polypeptide chains form a triple helix, which is stabilized by interchain hydrogen bonds (often involving 4-hydroxyproline). Collagen fibrils are further strengthened by covalent cross-links (aldol condensations) between lysine and hydroxylysine residues in adjacent triple helices.
4-hydroxyproline, vitamin C, and the relationship between collagen structure and scurvy:
4-hydroxyproline: A modified amino acid crucial for collagen stability. Hydroxylation of proline residues (catalyzed by prolyl hydroxylase) allows for additional hydrogen bonding within the collagen triple helix.
Vitamin C (Ascorbate): A required cofactor for prolyl hydroxylase. Without vitamin C, 4-hydroxyproline synthesis is impaired.
Scurvy: A disease caused by vitamin C deficiency. In scurvy, collagen is improperly hydroxylated, leading to unstable collagen fibrils. This results in symptoms like fragile blood vessels (bleeding gums, bruising) and connective tissue weakness.
Protein Data Bank (PDB): A public repository that archives experimentally determined 3D structures of biological macromolecules (proteins, nucleic acids). It is an invaluable resource for researchers in structural biology and drug discovery to study protein architecture, function, and interactions.
Motifs (folds) and domains:
Motifs (supersecondary structures): Recurring combinations of secondary structure elements (e.g., β-α-β loop, hairpin β-motif, helix-loop-helix) that often have a specific functional role or structural purpose.
Domains: Independent, compact globular units within a single polypeptide chain, typically comprising 50-200 amino acids. Domains often fold independently and may have distinct functions (e.g., DNA-binding domain, catalytic domain). Proteins can have multiple domains.
Intrinsically disordered proteins/regions (IDPs/IDRs):
Properties: Proteins or regions within proteins that lack a stable, defined 3D structure under physiological conditions. They are highly flexible and dynamic.
Benefits: Their flexibility allows them to bind to multiple different partners with high specificity but low affinity (known as 'promiscuous binding'). They are often involved in signaling, regulation, and molecular recognition.
SCOP2 database (Structural Classification of Proteins - extended): A hierarchical database that classifies proteins based on their evolutionary and structural relationships. It helps to understand protein evolution, predict protein function, and analyze structural diversity.
Usefulness and what it tells us about motifs, structure, and relationships: Organizes proteins into main classes (all α, all β, α/β, α+β), then into folds (defined by major secondary structures and their arrangements), superfamilies (evolutionarily related proteins with similar folds), and families (clearly homologous proteins). It highlights how motifs combine to form distinct folds and how evolution conserves structural scaffolds even with diverse sequences.
X-ray crystallography (advantages and limitations):
Advantages: Provides high-resolution atomic 3D structures of proteins, detailing every atom's position. Applicable to a wide range of protein sizes and complexes.
Limitations: Requires protein crystallization, which can be difficult and time-consuming. The crystal environment might not perfectly reflect physiological conditions. Does not easily capture dynamic processes.
NMR (Nuclear Magnetic Resonance) spectroscopy (advantages and limitations):
Advantages: Determines protein structures in solution, which is closer to physiological conditions, allowing for studies of dynamic processes and conformational changes. Can characterize intrinsically disordered regions.
Limitations: Typically limited to smaller proteins (<~30-40 \text{kDa}). Requires highly concentrated samples. Resolution is generally lower than high-resolution X-ray crystallography.
Proteostasis: The cellular process of maintaining protein homeostasis, ensuring that proteins are correctly folded, trafficked, and degraded when necessary. It involves a complex network of protein synthesis, folding, refolding, and degradation machinery.
Potential outcomes of a partially-folded protein:
Aggregation: Partially folded proteins often expose hydrophobic regions normally buried in the interior, leading to aggregation and formation of insoluble protein clumps. These aggregates can be toxic to cells.
Misfolding-related diseases: Accumulation of misfolded proteins can lead to neurodegenerative diseases (e.g., Alzheimer's, Parkinson's) and other protein conformation disorders.
Loss of function: A partially folded protein may not be able to achieve its native, functional conformation, leading to a loss of its biological activity.
Chaperones and their roles: A class of proteins that assist in the proper folding of other proteins by preventing misfolding and aggregation. They do not become part of the final functional protein structure.
Hsp70 (chaperone) function: Heat shock protein 70 (Hsp70) binds to nascent polypeptide chains emerging from the ribosome and to unfolded proteins, using ATP hydrolysis to facilitate their proper folding and prevent aggregation. It can also help refold misfolded proteins or target them for degradation.
GroEL/GroES (chaperonins) function and mechanism: GroEL/GroES are barrel-shaped chaperonin complexes found in bacteria that provide an isolated environment for protein folding. An unfolded protein enters the GroEL central cavity; GroES then caps the cavity, creating a folding chamber. ATP hydrolysis drives conformational changes, promoting correct folding and preventing aggregation. After ATP hydrolysis, the protein is released (either folded or available for another folding cycle).
Protein denaturation: The loss of a protein's native 3D structure (secondary, tertiary, and quaternary, but not primary) due to external stresses like heat, pH changes, chemical denaturants (urea, guanidinium chloride), or detergents. Denaturation often leads to loss of biological function.
How measured and features of, including Tm: Denaturation can be measured by changes in spectroscopic properties (e.g., UV absorption, circular dichroism, fluorescence) as the protein unfolds. The melting temperature (Tm) is the temperature at which 50% of the protein is denatured, indicating its thermal stability.
Ribonuclease refolding experiment (Anfinsen's experiment):
How performed: Christian Anfinsen denatured bovine pancreatic ribonuclease A (a protein with four disulfide bonds) using urea and a reducing agent (mercaptoethanol) to break disulfide bonds. This resulted in a completely unfolded, inactive enzyme.
Why important: When the denaturants were removed, the enzyme spontaneously refolded into its native, biologically active conformation, with the correct disulfide bonds reforming. This demonstrated that the primary amino acid sequence contains all the necessary information for a protein to fold into its correct 3D native structure.
Protein folding follows a distinct path and does not occur by random sampling - Why?: If proteins folded by randomly sampling all possible conformations, it would take an astronomically long time (Levinthal's paradox). Instead, folding follows specific pathways, often involving intermediate states, guided by the formation of local secondary structures and collapse of hydrophobic regions to rapidly narrow conformational space and reach the native fold.
Consequences of protein misfolding: Misfolded proteins can lose their function, become toxic to cells, or aggregate to form insoluble deposits. These aggregates are associated with various diseases, including:
Amyloidosis: A group of diseases where insoluble protein aggregates (amyloid fibrils) deposit in tissues and organs.
Neurodegenerative diseases: Such as Alzheimer's disease (amyloid-β, tau), Parkinson's disease (α-synuclein), and Huntington's disease (huntingtin), where misfolded proteins form plaques or inclusions in the brain.
Prions and their mechanism of action:
Prions: Infectious proteinaceous particles that cause transmissible spongiform encephalopathies (TSEs), such as mad cow disease (BSE) and Creutzfeldt-Jakob disease. Prions are composed solely of a misfolded host protein (PrPSc) that can induce the misfolding of the normal cellular form (PrPC).
Mechanism of action: The abnormally folded PrPSc acts as a template, converting normal PrPC proteins into the pathogenic PrPSc form. This conformational change is autocatalytic, leading to a chain reaction of misfolding and aggregation, forming amyloid plaques that damage neuronal tissue.
Chapter 5: Protein Function
Overview
Learning Goals
Understand ligand binding methods and protein interactions.
Quantitatively model these interactions.
Explore the interactions of globins with oxygen and mechanisms of muscle contraction.
Ligand Binding
Some functions of globular proteins:
Enzymatic catalysis: Accelerate biochemical reactions (e.g., hexokinase, chymotrypsin).
Transport and storage: Bind and carry specific molecules (e.g., hemoglobin, myoglobin, albumin).
Immune protection: Recognize and neutralize pathogens (e.g., antibodies).
Mechanical support: Provide structure (e.g., actin).
Signal transduction: Transmit signals across membranes or within cells (e.g., hormone receptors, G proteins).
Regulation: Control protein activity or gene expression (e.g., transcription factors, kinases).
Ligand: Any molecule that binds reversibly to a protein, typically smaller than the protein.
Binding site: The specific region on a protein where ligands attach. This binding often involves non-covalent interactions (hydrogen bonds, ionic interactions, hydrophobic interactions, van der Waals forces) similar to those governing protein structure, ensuring specificity and reversibility.
Why is reversible binding of ligands important?: Allows proteins to carry out their functions dynamically and respond to cellular needs. For example, transport proteins can pick up and release cargo, and signaling proteins can be activated and deactivated, maintaining cellular homeostasis and responsiveness.
What types of interactions facilitate ligand binding?: Primarily non-covalent interactions (hydrogen bonds, van der Waals forces, electrostatic interactions/salt bridges, and hydrophobic effects) which allow for transient, specific, and reversible interactions.
Quantitative Description of Binding:
For the binding of ligand (L) to protein (P) forming a protein-ligand complex (PL):
The association rate constant (k_a) describes the rate at which ligand binds to the protein.
The dissociation rate constant (k_d) describes the rate at which ligand releases from the protein.
At equilibrium: Rate of association = Rate of dissociation
ka[P][L] = kd[PL]
Association equilibrium constant (Ka): Measures the affinity of a protein for its ligand. A higher Ka indicates strong binding.
Ka = ka / k_d = [PL]/([P][L])
Dissociation equilibrium constant (Kd): Measures the tendency of a complex to dissociate into protein and ligand. A lower Kd indicates strong binding affinity.
Kd = kd / k_a = ([P][L])/[PL]
How are Ka and Kd related?: They are reciprocals of each other: Ka = 1/Kd .
How does one calculate Ka (or Kd)?: Ka is calculated as the ratio of the concentration of the protein-ligand complex to the product of free protein and free ligand concentrations at equilibrium. Kd is its inverse.
How does one calculate \theta (fraction of occupied binding sites) and how is it related to K_d?:
The fraction of occupied binding sites, \theta, represents the proportion of total protein binding sites that are bound by ligand.
\theta = [PL] / ([P][\text{total}])
For a single ligand and single binding site, \theta can be expressed in terms of ligand concentration and Kd: \theta = [L] / (Kd + [L])
Where [L] is the free ligand concentration. This indicates that when [L] = K_d, half of the binding sites are occupied (\theta = 0.5).
K_d measures that indicate strong and weak binding:
A very small K_d (e.g., nanomolar or picomolar range) indicates strong binding affinity, meaning the protein binds its ligand tightly.
A relatively large K_d (e.g., micromolar or millimolar range) indicates weak binding affinity, meaning the protein binds its ligand less tightly.
Relationships between \Delta G^o and Ka (or Kd):
The standard free energy change (\Delta G^o) for ligand binding is related to the equilibrium constants by:
\Delta G^o = -RT\ln Ka = RT\ln Kd \n
Where R is the gas constant and T is the absolute temperature. A negative \Delta G^o indicates a spontaneous binding process.
Binding specificity models:
Lock-and-key model: Proposed by Emil Fischer, suggesting that the binding site of a protein (the 'lock') is pre-formed and perfectly complementary to the ligand (the 'key') in shape and chemical properties, allowing for highly specific binding.
Induced-fit model: Proposed by Daniel Koshland, suggesting that both the protein and the ligand undergo conformational changes upon binding. The binding site is not perfectly complementary initially but adapts to the ligand, leading to a tighter fit and optimizing interactions. This model accounts for enzyme catalysis and allosteric regulation.
Binding and Transport of Oxygen
Why is prosthetic group (heme) necessary for O2 binding by proteins (globins)?: Amino acid side chains in proteins are not suitable for reversible oxygen binding. Oxygen binding requires a transition metal ion, and iron in the heme group provides the necessary coordination environment for reversible oxygen attachment.
Why would "free" heme be bad for cells for binding O2?: Free heme can bind oxygen but also readily form ferrous iron (\text{Fe}^{2+}) to ferric iron (\text{Fe}^{3+}) (oxidation), releasing reactive oxygen species (ROS) that can damage cellular components. Binding heme within a protein (like myoglobin or hemoglobin) prevents this oxidation and ROS generation.
Key features of a porphyrin and how they coordinate \text{Fe}^{2+}:
Porphyrin: A complex organic ring system (protoporphyrin IX) that forms a planar structure. It contains four pyrrole rings linked by methine bridges. The center of the ring contains four nitrogen atoms.
Coordination of \text{Fe}^{2+}: The four nitrogen atoms of the porphyrin ring system act as coordinating ligands, binding to the central \text{Fe}^{2+} ion. This forms a square planar coordination sphere for iron.
How many potential coordinating groups can \text{Fe}^{2+} in heme have?: The \text{Fe}^{2+} ion in heme has six coordination sites. Four are occupied by the nitrogen atoms of the porphyrin ring. The fifth site is occupied by a histidine residue from the globin protein (proximal His). The sixth site is available for reversible binding of oxygen or other ligands (like CO).
Why doesn't heme bind to two O2 molecules?: The spatial arrangement of the heme within the globin protein's binding pocket only allows for one molecule of \text{O}_2 to bind to the available sixth coordination site of the ferrous iron. Steric hindrance prevents a second oxygen molecule from binding.
Which helices and residues are important for heme-protein association and heme-O2 binding?:
The F helix (specifically the proximal histidine, His F8 at the 8th position of the F helix) is crucial for coordinating the fifth ligand site of the heme iron, anchoring the heme to the globin protein.
The E helix contains the distal histidine (His E7), which doesn't directly bind iron but influences \text{O}_2 binding affinity and reduces CO affinity by steric hindrance.
Why don't we all die of CO poisoning, despite CO binding to heme over 20K times more efficiently than O2?:
While CO has a much higher affinity for free heme than \text{O}2 , the presence of the distal histidine (His E7) in globins sterically hinders the linear binding of CO to the heme iron. It forces CO to bind at an angle, reducing its effective affinity to about 200 times that of \text{O}2 , rather than 20,000. This slight reduction, coupled with low atmospheric CO levels, prevents widespread CO poisoning.
What residue helps position O2 in the binding pocket and how?: The distal histidine (His E7) helps position the bound \text{O}_2 molecule by forming a hydrogen bond with one of the oxygen atoms. This H-bond stabilizes the complex and also contributes to reducing CO's affinity.
How can binding of O2 to heme be measured?: The binding of \text{O}_2 to the heme iron causes changes in the electronic structure of the heme, leading to distinct color changes. This can be quantitatively measured using spectrophotometry, specifically observing changes in the absorbance spectrum of hemoglobin or myoglobin in the visible light range (e.g., at 540 nm and 577 nm for oxyhemoglobin).
Fraction of oxygen bound to myoglobin:
Myoglobin has a single oxygen-binding site, and its oxygen-binding curve is hyperbolic.
\theta{\text{Mb}} = pO2 / (p{50} + pO2)
Relationship between \theta , pO2 , and p{50}:
\theta is the fraction of binding sites occupied. pO2 is the partial pressure of oxygen. p{50} is the partial pressure of oxygen at which half of the binding sites are occupied (\theta = 0.5). A lower p_{50} indicates higher oxygen affinity.
Why is myoglobin bad for releasing O2 to tissues, but hemoglobin is not?:
Myoglobin: Has a very high oxygen affinity (low p{50}) and a hyperbolic binding curve. It binds \text{O}2 strongly even at low pO2 found in tissues, making it an excellent oxygen storage protein (e.g., in muscle) but poor at releasing oxygen to active tissues, except under extreme conditions of very low pO2 .
Hemoglobin: Exhibits cooperative binding and a sigmoidal binding curve, allowing it to efficiently load \text{O}2 in the lungs (high pO2) and release it in the tissues (lower pO2). Its affinity changes with pO2 and other effectors, making it ideal for oxygen transport.
Cooperative Binding and Function
Cooperative binding: A phenomenon where the binding of one ligand molecule to a multi-subunit protein complex influences the binding affinity of subsequent ligand molecules for other sites.
Positive cooperativity: Binding of the first ligand molecule increases the affinity of the remaining binding sites for the ligand (e.g., hemoglobin's \text{O}_2 binding).
Negative cooperativity: Binding of the first ligand molecule decreases the affinity of the remaining binding sites for the ligand (less common, but occurs in some enzymes). This ensures that the protein does not become oversaturated too quickly.
Hill coefficient (n_H) and relationship for indicating cooperativity:
The Hill coefficient is a measure of the degree of cooperativity in ligand binding. It is derived from the Hill equation, which describes fractional saturation as a function of ligand concentration:
\theta = [L]^n / (KA' + [L]^n) Where n is the Hill coefficient (often written as nH).
If n_H = 1: Non-cooperative binding.
If n_H > 1: Positive cooperativity.
If n_H < 1: Negative cooperativity.
Shape of a Hill plot for a non-cooperative and a cooperative binder (and why?):
A Hill plot graphs \log(\theta / (1 - \theta)) versus \log[L] . The slope of this plot is the Hill coefficient.
Non-cooperative binder (n_H = 1): The Hill plot will be a straight line with a slope of 1. This represents independent binding events.
Cooperative binder (n_H > 1): The Hill plot will have a slope greater than 1 in the physiological range, indicating positive cooperativity. This reflects the increased affinity upon initial ligand binding.
Allosteric regulation: Regulation of a protein's activity or binding affinity by the binding of an effector molecule at a site other than the active or binding site.
Homotropic allosteric regulation: The ligand itself acts as an allosteric effector, promoting conformational changes that alter its own binding affinity (e.g., \text{O}_2 binding to hemoglobin).
Heterotropic allosteric regulation: A molecule other than the primary ligand binds to an allosteric site to modulate the protein's activity or binding affinity (e.g., 2,3-BPG, \text{CO}_2 , and \text{H}^+ binding to hemoglobin).
General structure of hemoglobin (subunits and as complex):
Hemoglobin is a heterotetramer, meaning it consists of four polypeptide subunits. Adult hemoglobin (HbA) is composed of two identical α-subunits and two identical β-subunits ( \alpha2\beta2 ), each containing one heme group and capable of binding one oxygen molecule. It forms a compact, roughly spherical complex.
Generally how the subunits interact and how pH alters these:
Subunits interact through extensive non-covalent interactions (hydrophobic, hydrogen bonds, salt bridges) at their interfaces. These interactions change upon oxygen binding.
pH alterations (Bohr effect): A decrease in pH (or increase in \text{H}^+ concentration) reduces hemoglobin's affinity for oxygen. This is because specific amino acid residues (like His residues) become protonated at lower pH, forming salt bridges that stabilize the T (tense) state, promoting \text{O}_2 release. This effect is crucial for oxygen delivery to metabolically active tissues which produce acid.
T (tense) and R (relaxed) states of hemoglobin (why?, effect?, how regulated?):
T state (tense state): The deoxy (unoxygenated) form of hemoglobin. It is characterized by numerous ion pairs (salt bridges) at the \alpha1\beta2 and \alpha2\beta1 interfaces, making it a low-affinity state for \text{O}2 . It is stable in the absence of oxygen and promotes \text{O}2 release in tissues.
R state (relaxed state): The oxy (oxygenated) form of hemoglobin. Binding of \text{O}2 triggers conformational changes, breaking some of the ion pairs and shifting the protein to the R state, which has a higher affinity for \text{O}2 . This state is stable in the lungs and promotes \text{O}_2 uptake.
Regulation: The transition between T and R states is influenced by \text{O}2 binding (homotropic allosteric effector) and by heterotropic allosteric effectors like \text{H}^+ , \text{CO}2 , and 2,3-bisphosphoglycerate.
How can CO2 be removed from tissues (hint: buffer and hemoglobin itself)?:
As bicarbonate (buffer system): The majority of \text{CO}2 from tissues is converted to bicarbonate (\text{HCO}3^{-}) by carbonic anhydrase in red blood cells. \text{H}^+ ions produced acidify the blood, contributing to the Bohr effect and promoting \text{O}_2 release.
Direct binding to hemoglobin: Hemoglobin can directly transport \text{CO}2 by forming carbamate groups. \text{CO}2 reacts with the N-terminal amino groups of hemoglobin's polypeptide chains, forming carbaminohemoglobin. This reaction also releases protons, further contributing to the Bohr effect and stabilizing the T state.
2,3-bisphosphoglycerate (2,3-BPG) is what type of regulator of hemoglobin and why?:
2,3-BPG is a heterotropic allosteric effector of hemoglobin. It binds to a central pocket formed by the four subunits in the T (deoxy) state of hemoglobin, stabilizing this low-affinity conformation. It is negatively charged and interacts with positively charged residues in the central cavity.
How does 2,3-bisphosphoglycerate affect O2 binding?:
2,3-BPG reduces hemoglobin's affinity for oxygen by preferentially binding to and stabilizing the T state. This makes it easier for hemoglobin to release \text{O}_2 in peripheral tissues. Its concentration increases at high altitudes or in conditions of chronic hypoxia, serving as an adaptive mechanism to enhance oxygen delivery.
Sickle cell anemia (how and why despite being bad for red blood cells is it selectively advantageous for some populations?):
Mechanism: Caused by a single point mutation in the \beta -globin gene (Glu6Val), leading to a change in a single amino acid in the hemoglobin protein (HbS). At low oxygen concentrations, deoxygenated HbS molecules aggregate to form long, insoluble fibers that distort red blood cells into a sickle shape, leading to anemia, blood vessel blockages, and pain crises.
Selective advantage: Despite its severe consequences, the sickle cell trait (heterozygous for HbS) confers resistance to malaria. The distorted red blood cells are less hospitable to the malaria parasite, and infected cells are prematurely removed from circulation, providing a survival advantage in regions where malaria is endemic.
Antibody-Antigen Interactions
Two types of immune system:
Innate immune system: Provides immediate, non-specific defense against pathogens. It involves physical barriers (skin), chemical defenses (pH), and specialized cells (phagocytes, NK cells) that recognize general pathogen-associated molecular patterns (PAMPs).
Adaptive (acquired) immune system: Provides highly specific, memory-driven defense against pathogens. It involves lymphocytes (B cells and T cells) that recognize specific antigens and generate a targeted, long-lasting immune response.
Antibody composition, structure, and domain function/interaction:
Antibody (Immunoglobulin, Ig): A Y-shaped protein produced by B lymphocytes that specifically recognizes and binds to antigens, facilitating their removal.
Structure: Composed of four polypeptide chains: two identical heavy chains and two identical light chains, linked by disulfide bonds. It has two identical antigen-binding sites.
Domains: Each chain consists of variable (V) and constant (C) domains.
Variable domains (\text{V}H and \text{V}L): Located at the tips of the Y, these form the antigen-binding site and exhibit high sequence diversity to recognize a vast array of antigens.
Constant domains (\text{C}H and \text{C}L): Determine the antibody class (e.g., IgG, IgM) and mediate effector functions, such as binding to immune cells or activating complement.
What is an antibody, antigen, and epitope?:
Antibody: A protein produced by the immune system in response to an antigen, specifically recognizing and binding to it.
Antigen: Any molecule (protein, carbohydrate, lipid) that can be recognized by the immune system and elicit an immune response (e.g., a viral coat protein, bacterial toxin).
Epitope: The specific, small structural region on an antigen that is directly recognized and bound by an antibody. An antigen can have multiple different epitopes.
Monoclonal vs. polyclonal antibodies:
Polyclonal antibodies: A heterogeneous mixture of antibodies produced by different B cell clones, each recognizing a different epitope on the same antigen. Generated by immunizing an animal.
Monoclonal antibodies: A homogeneous population of antibodies, all produced by a single B cell clone, recognizing a single specific epitope on an antigen. Produced via hybridoma technology for high specificity.
ELISA (Enzyme-Linked Immunosorbent Assay) (process and uses):
Process: A plate-based assay used to detect and quantify proteins, peptides, antibodies, or hormones. Typically involves coating a plate with an antigen or antibody, adding a sample, and then detecting binding using an enzyme-linked antibody that produces a colored product.
Uses: Diagnostic tool for detecting infections (e.g., HIV, Lyme disease), pregnancy tests, screening for certain cancers, and quantifying protein levels in research.
Western blotting of proteins separated by SDS-PAGE:
Process: A technique used to detect specific proteins in a complex mixture. Proteins are first separated by size through SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis), then transferred from the gel to a membrane. The membrane is then probed with a specific antibody that binds to the target protein, and the antibody is detected using a secondary antibody linked to an enzyme or fluorophore.
Uses: Verifying protein expression, identifying post-translational modifications, and diagnosing certain diseases.
Interaction Producing Motion
Myosin (structure):
Myosin is a motor protein primarily associated with muscle contraction and cell motility. It is a large protein, typically composed of two heavy chains (each with a globular head domain and a long coiled-coil tail) and several light chains associated with the head. The head domain contains the actin-binding site and the ATPase activity.
Myosin interaction with actin:
Myosin's globular head domain binds to actin filaments, which are long polymers of actin protein. The interaction is a cyclical process where myosin binds, pulls, and then detaches from actin, consuming ATP in the process.
Muscle fibers (thick and thin filaments, Z disk, M line, A band, and I band):
Muscle fibers: Elongated cells containing myofibrils, which are structural units of muscle contraction.
Sarcomere: The basic contractile unit of a myofibril, delimited by Z disks.
Thick filaments: Composed mainly of myosin molecules, located in the center of the sarcomere.
Thin filaments: Composed mainly of actin, along with troponin and tropomyosin, anchored to the Z disk.
Z disk: Defines the boundaries of the sarcomere and anchors the thin filaments.
M line: Midline of the sarcomere, anchoring the thick filaments.
A band: The dark region corresponding to the length of the thick filaments (includes overlapping thin filaments).
I band: The light region containing only thin filaments.
How the process of muscle contraction occurs molecularly (sliding filament model):
ATP binding: Myosin head binds ATP, causing it to detach from actin.
ATP hydrolysis: Myosin hydrolyzes ATP to ADP + Pi, cocking the head into a high-energy conformation.
Cross-bridge formation: Myosin head binds weakly to a new site on the actin filament.
Power stroke: Release of Pi triggers the power stroke, where the myosin head pivots, pulling the actin filament towards the M line. ADP is then released.
New ATP binding: A new ATP molecule binds to myosin, causing it to detach from actin, and the cycle repeats.
Troponins and tropomyosin:
Tropomyosin: A coiled-coil protein that lies along the actin filament, covering the myosin-binding sites on actin in a relaxed muscle, preventing contraction.
Troponin: A multimeric protein complex associated with tropomyosin. It has three subunits: troponin C (binds \text{Ca}^{2+}), troponin I (inhibits actin-myosin interaction), and troponin T (binds to tropomyosin).
Role in contraction: When calcium ions (\text{Ca}^{2+}) are released (e.g., from sarcoplasmic reticulum), they bind to troponin C. This binding causes a conformational change in the troponin complex, which moves tropomyosin away from the myosin-binding sites on actin, allowing myosin to bind and initiate muscle contraction.
How myosin acts not only as a structural protein, but also as an enzyme:
Structural protein: Myosin forms the thick filaments of muscle, providing the core structure for muscle contraction.
Enzyme: Myosin possesses intrinsic ATPase activity in its globular head domain. It hydrolyzes ATP to ADP and Pi, releasing the energy required to drive the conformational changes that power the movement along actin filaments, making it a mechanochemical enzyme.
Conclusion
The study of protein structure and function is essential for understanding biochemical processes. Significant insights can be gained by analyzing relationships between these aspects, paving the way for advancements in fields ranging from molecular biology to medicine.
Here's a detailed daily study guide to help you prepare for your BIOC 460/660 exam on Monday, incorporating your TA's advice on active recall and utilizing the provided notes.
General Approach for Each Day:
Active Recall First (Without Notes): For each topic listed, try to explain it out loud or write down everything you know about it, focusing on:
Definition of the term/concept.
How it was used in the lecture/context.
How it might be applicable to a potential problem or question.
Consult Notes to Fill Gaps: After attempting active recall for a topic, then refer to your comprehensive notes and lecture slides to correct any inaccuracies, add missing details, and deepen your understanding.
Identify Weak Areas: Keep a running list of topics you consistently struggle with. These will be your priority for review.
Take Breaks: Aim for 45-60 minute study blocks followed by 5-10 minute breaks to maintain focus and prevent burnout.
Wednesday: Foundations & Primary/Secondary Structure
Focus Areas:
Amino Acid Sequence and Importance
Primary amino acid sequence comparisons
Identification of a consensus sequence
What is a signature sequence?
Identification of homologs (paralogs and orthologs)
Conserved substitutions of amino acids
How amino acid sequence conservation equates to relationships between organisms
Protein Primary Sequence and Structure
Four levels of protein structure
Bioenergetics and protein structure state
What forces stabilize protein structure and how?
Where in a protein can these forces predominate?
Resonance within a peptide affecting peptide bond
Which bonds in a polypeptide show rotation and which do not?
What are dihedral angles? (\Phi and \Psi)
Which dihedral angles are associated with which bonds in a polypeptide chain?
Protein Secondary Sequence
Ramachandran plots (what can they tell you?)
Common secondary structures
What role does phi/psi angles play in secondary structure?
Alpha-helix (structure, handedness, stabilization, approximate diameters/length per turn)
Amino acid sequence and alpha-helix stability
Alpha-helix dipoles and amino acids at ends
Beta-strands and beta-sheets (parallel vs. antiparallel)
Beta-turns (types; common amino acids in which positions)
Circular dichroism (what can it tell you about secondary structure within a protein/complex?)
Activities:
Go through each item, actively recalling information. Write down your explanations or speak them aloud.
Compare your recall with the provided notes. Fill in gaps and reinforce correct information.
Pay special attention to the formulas discussed in the notes, for example, the definition of heta and its relation to K_d . While not in these sections, keep an eye out for how quantitative descriptions are presented.
Draw out an alpha-helix and a beta-sheet, labeling key features like hydrogen bonds and residue positions.
Thursday: Tertiary/Quaternary Structure & Proteostasis
Focus Areas:
Protein Tertiary/Quaternary Structure
Tertiary structure (what is it?; how is it stabilized?)
Major classes of protein (structure-wise: globular vs. fibrous)
Coiled-coil structure
Structures of three fibrous protein examples (α-Keratin, Collagen, Silk Fibroin: similarities and differences)
How are protomers of alpha-keratin and collagen held together?
4-hydroxyproline, vitamin C, and the relationship between collagen structure and scurvy
Protein Data Bank (PDB) - use and significance
Motifs (folds) and domains
Intrinsically disordered proteins/regions (IDPs/IDRs) - properties and benefits of
SCOP2 database, usefulness, and what it tells us about motifs, structure, and relationships
X-ray crystallography (advantages and limitations)
NMR (advantages and limitations)
Proteostasis
What are some potential outcomes of a partially-folded protein?
Chaperones and their roles
Hsp70 (chaperone) function
GroEL/GroES (chaperonins) function and mechanism
Protein denaturation (how measured and features of, including T_m )
Ribonuclease refolding experiment (how performed and why important)
Protein folding follows a distinct path and does not occur by random sampling - Why?
Consequences of protein misfolding (e.g., Amyloidosis, Neurodegenerative diseases)
Prions and their mechanism of action
Activities:
Continue with active recall for each topic. Focus on describing mechanisms and distinguishing between similar concepts (e.g., X-ray vs. NMR).
For fibrous proteins, create a comparison table highlighting structures, key amino acids, and stabilization mechanisms.
Review the chaperonin mechanisms. Perhaps try drawing the GroEL/GroES cycle.
Understand the significance of Anfinsen's experiment and Levinthal's paradox.
Pay close attention to disease relevance in the proteostasis section (Scurvy, Misfolding-related diseases, Prions).
Friday: Protein Function & Oxygen Binding
Focus Areas:
Protein Function and Ligand Binding
Some functions of globular proteins
Ligand, Binding site
Why is reversible binding of ligands important?
What types of interactions facilitate ligand binding?
Quantitative Description of Binding: ka, kd, Ka, Kd
How are Ka and Kd related?
How is Ka calculated (or how is Kd calculated)?
How does one calculate heta (fraction of occupied binding sites) and how is it related to Kd ? ( heta = [L] / (Kd + [L]) )
K_d measures that indicate strong and weak binding
Relationships between riangle G^o and Ka (or Kd ): ( riangle G^o = -RT ext{ln }Ka = RT ext{ln }Kd )
Binding specificity models (lock-and-key and induced-fit)
Binding and Transport of Oxygen
Why is prosthetic group (heme) necessary for O2 binding by proteins (globins)?
Why would "free" heme be bad for cells for binding O2?
Key features of a porphyrin and how they coordinate ext{Fe}^{2+}
How many potential coordinating groups can ext{Fe}^{2+} in heme have?
Why doesn't heme bind to two O2 molecules?
Which helices and residues are important for heme-protein association and heme-O2 binding?
Why don't we all die of CO poisoning, despite CO binding to heme over 20K times more efficiently than O2?
What residue helps position O2 in the binding pocket and how?
How can binding of O2 to heme be measured? (Spectrophotometry)
Fraction of oxygen bound to myoglobin ( heta{ ext{Mb}} = pO2 / (p{50} + pO2) )
What type of binding curve is observed for one protein-one ligand? (Hyperbolic)
Relationship between heta , pO2 , and p{50}
Why is myoglobin bad for releasing O2 to tissues, but hemoglobin is not?
Activities:
Focus heavily on the quantitative descriptions of binding. Practice manipulating the equations for Ka, Kd, heta and their relationship to riangle G^o . Understand what each constant signifies.
Distinguish clearly between the lock-and-key and induced-fit models, providing examples if possible.
Study the details of heme and its interaction with iron and oxygen. Understand the roles of proximal and distal histidines.
Compare myoglobin and hemoglobin's oxygen binding curves and their functional differences. Try to sketch them.
Saturday: Cooperative Binding & Immune/Motion Interactions
Focus Areas:
Cooperative Binding and Function
Cooperative binding (positive and negative)
Hill coefficient ( nH ) and relationship for indicating cooperativity ( heta = [L]^n / (K{ ext{A}}' + [L]^n) )
Shape of a Hill plot for a non-cooperative and a cooperative binder (and why?)
Allosteric regulation (homotropic and heterotropic)
General structure of hemoglobin (subunits and as complex, ext{ }\alpha2\beta2 )
Generally how the subunits interact and how pH alters these (Bohr effect)
T (tense) and R (relaxed) states of hemoglobin (why?, effect?, how regulated?)
How can CO2 be removed from tissues (hint: buffer and hemoglobin itself)?
2,3-bisphosphoglycerate (2,3-BPG) is what type of regulator of hemoglobin and why?
How does 2,3-bisphosphoglycerate affect O2 binding?
Sickle cell anemia (how and why despite being bad for red blood cells is it selectively advantageous for some populations?)
Antibody-Antigen Interactions
Two types of immune system (Innate vs. Adaptive)
Antibody (Immunoglobulin, Ig) composition, structure, and domain function/interaction
What is an antibody, antigen, and epitope?
Monoclonal vs. polyclonal antibodies
ELISA (Enzyme-Linked Immunosorbent Assay) (process and uses)
Western blotting of proteins separated by SDS-PAGE (process and uses)
Interaction Producing Motion
Myosin (structure)
Myosin interaction with actin
Muscle fibers (thick and thin filaments, Z disk, M line, A band, and I band)
How the process of muscle contraction occurs molecularly (sliding filament model)
Troponins and tropomyosin (structure and role in contraction)
How myosin acts not only as a structural protein, but also as an enzyme
Activities:
Deep dive into hemoglobin's allosteric regulation. Understand how ext{H}^+ , ext{CO}_2 , and 2,3-BPG affect its affinity and the T/R state transition.
Understand the Hill coefficient and how to interpret a Hill plot.
For antibody-antigen interactions, clarify the roles of each component (antibody, antigen, epitope) and the difference between monoclonal/polyclonal.
Review the processes of ELISA and Western blotting step-by-step; they are highly applicable practical questions.
Thoroughly understand the sliding filament model of muscle contraction, including the roles of myosin, actin, troponin, and tropomyosin, and the role of ATP. Try to draw the cycle.
Sunday: Comprehensive Review & Rest
Activities:
Review Weak Areas: Go back to the list of topics you identified as challenging earlier in the week. Focus your last study session on these.
High-Level Overview: Take about 1-2 hours to quickly skim through all your notes and the study guide one last time. Don't try to memorize new things, just reinforce existing knowledge.
Simulate Exam Conditions (Optional but Recommended): If you have any mock exams or a comprehensive set of practice questions, try to do a timed session to gauge your pacing and recall under pressure.
Self-Explain Key Concepts: Pick 3-5 major overarching concepts (e.g., protein folding stability, allosteric regulation, molecular motors) and try to explain them from start to finish without notes.
Final Check of Formulas: Ensure you are confident with all relevant equations and their applications.
Prioritize Rest: Stop studying early in the evening. Get a full night's sleep to allow your brain to consolidate information and be fresh for the exam.