Protein Interactions, Antibodies, and Cytoskeletal Dynamics – Key Concepts

Flashcards cover core concepts of protein-ligand and protein-protein interactions, antibody structure and binding, methods for studying interactions, and key cytoskeletal dynamics (actin and microtubules), drawn from the lecture notes.

In a cell where a biomolecule is present at ~1 mM, approximately how often will it collide with a given protein surface per second?

About 10^6 (one million) collisions per second.

Write the basic equilibrium expression for protein–ligand binding (P + L ⇌ PL) and define kon and koff.

Association rate = kon[P][L], dissociation rate = koff[PL]; at equilibrium, kon[P][L] = koff[PL]. The equilibrium constants are Ka = kon/koff and Kd = koff/kon.

What does affinity measure and how is it commonly reported in binding studies?

Affinity measures the strength of the interaction; commonly reported as the equilibrium dissociation constant (Kd) or the association constant (Ka).

How is binding specificity different from affinity, and what structural feature typically defines specificity?

Specificity is the ability of a molecule to preferentially bind particular partners, often defined by the shape and chemistry of the binding site formed by tertiary and quaternary structures; affinity is the strength of binding at that site.

Which antibody regions form the antigen-binding site and what are the key variable components called?

The antigen-binding site is formed by the variable regions of the heavy and light chains (VH and VL); hypervariable loops (CDRs) within these regions determine antigen recognition.

What is the Ig fold and how are antibodies structurally organized around it?

The Ig fold is a beta-sheet–rich immunoglobulin domain; antibodies contain constant and variable Ig domains that stack to form the overall Y-shaped molecule.

For small antigens like phosphocholine and larger antigens like lysozyme, what are typical binding-pocket sizes observed in antibodies?

Small antigens ~160 Å^2 buried surface; larger antigens ~750 Å^2 buried surface.

Name common methods to determine antibody binding and a brief note on each.

ELISA (enzyme-linked immunosorbent assay); ITC (isothermal titration calorimetry); Analytical ultracentrifugation (AUC); Fluorescence quenching/anisotropy; Filter binding assays; Surface plasmon resonance (SPR).

Differentiate affinity and avidity in the context of antibodies.

Affinity is the strength of a single binding interaction; avidity is the overall functional strength when multiple binding sites interact (multivalency), often roughly the product of the individual affinities.

What cleavage products result from papain digestion of an antibody, and what are their components?

Papain cleavage yields Fab fragments (the antigen-binding regions) and Fc fragment (the constant region of the heavy chain).

What is the typical range for the antibody–antigen association constant (Ka)?

Ka is in the range of approximately 10^7 to 10^8 M^-1.

Why is there extreme hypervariability in antibodies, and how is it balanced with maintaining a stable core fold?

Antibodies possess hypervariable loops (CDRs) in the Fab that confer diversity, while the overall Ig fold and core framework remain conserved to maintain structural stability.

What is the significance of the two conformational states of an antibody’s antigen-binding region (bound vs unbound)?

Antibodies can undergo conformational adjustments upon antigen binding; the antigen-bound state may stabilize the interaction while the unbound state is the resting conformation.

Why do antibodies present different pocket sizes for small versus large antigens, and what is the practical implication?

Binding pockets are shaped to accommodate antigen size/shape; smaller pockets fit small antigens (e.g., ~160 Å^2), larger antigen surfaces fit bigger antigens (e.g., ~750 Å^2), affecting specificity and affinity.

List several experimental approaches to determine antibody binding and briefly indicate what each measures.

ELISA (binding detection); ITC (thermodynamics and Ka); AUC (size/shape and binding); Fluorescence quenching/anisotropy (binding dynamics); Filter binding (binding); SPR (kinetics and affinity).

Explain the difference between affinity and avidity with an everyday analogy.

Affinity is like the strength of a single grip; avidity is like how many hands are gripping at once, increasing overall hold even if individual grips aren’t perfect.

What is a Fab fragment and how does it relate to antibody function?

Fab contains the antigen-binding sites (VH and VL with CDRs); it binds antigen, while Fc mediates effector functions.

What stabilizes the heavy and light chains and their disulfide connections in an antibody?

Disulfide bonds (S-S) stabilize the pairing of heavy and light chains and the overall quaternary structure of the antibody.

Name the four major types of noncovalent interactions that stabilize biomolecular complexes.

Hydrogen bonds; Ionic (electrostatic) interactions; Hydrophobic interactions; Van der Waals interactions.

What is Yeast Two-Hybrid (Y2H) used for, and what are its common advantages and drawbacks?

Y2H is used to detect protein–protein interactions. Pros: powerful screen, detects transient/weak interactions, reporter readout. Cons: false positives, extensive validation needed, labor-intensive.

In Yeast Two-Hybrid, what are the terms ‘bait’ and ‘prey’?

Bait is the protein of interest fused to a DNA-binding domain; prey is a potential interacting protein fused to an activation domain.

Outline the basic workflow and purpose of Co-immunoprecipitation (Co-IP) and tag pull-down assays.

Co-IP uses an antibody to pull down a protein complex from lysate to identify interactors; tag pull-down uses immobilized tags/antibodies to capture tagged proteins and associated partners.

List the major advantages and caveats of Co-IP/Tag pull-down experiments.

Pros: confirms complex formation; versatile with purified or tagged proteins. Cons: not inherently quantitative; cannot distinguish direct vs indirect interactions; may create non-physiological interactions after lysis.

What thermodynamic information does ITC provide and what are typical material requirements?

ITC provides binding enthalpy, entropy, Ka (and n); requires relatively large amounts of material (often millimolar concentrations).

What information does SPR provide and what are its practical benefits?

SPR measures binding kinetics (Kon and Koff) and affinity (Ka/Kd) by detecting refractive index changes; requires relatively small amounts of material and works well for low nanomolar to micromolar interactions.

What is dynamic instability in microtubules?

Frequent transitions between growth and shrinkage driven by loss of the GTP cap, called catastrophe, with occasional rescue events.

What is treadmilling in actin filaments?

A cycle where actin adds at the plus end and disassembles at the minus end, maintaining length while monomers are recycled through ATP/ADP turnover.

Name the two major cellular roles of myosin movement along actin filaments.

Myosin powers cargo transport and muscle contraction by hydrolyzing ATP as it steps along actin filaments.

Describe the typical architecture and dynamics of actin (G-actin vs F-actin) and the role of ATP/ADP.

G-actin is the globular monomer bound to ATP; polymerization forms F-actin; ATP is hydrolyzed to ADP-Pi upon incorporation, and Pi is released after incorporation, affecting filament stability.

What is the typical diameter of microtubules and what subunits compose them?

Microtubules are about 25 nm in diameter and are built from α/β-tubulin heterodimers arranged in protofilaments.

What is the role of tubulin’s nucleotide state in microtubule dynamics (GTP cap, catastrophe, rescue)?

GTP-bound β-tubulin at the plus end stabilizes growth (GTP cap); hydrolysis to GDP promotes catastrophe and shrinkage; rescue can re-stabilize growth.

What features help explain why actin-binding proteins (ABPs) are tightly regulated and can be targeted by pathogens?

ABPs regulate polymerization/depolymerization, capping, severing, and cross-linking of actin; their abundant yet regulated activity makes them targets for pathogens aiming to disrupt the cytoskeleton.

Where are antigen-binding sites located on the antibody, and what is their structural basis?

Antigen-binding sites are formed by the variable regions of heavy and light chains (VH and VL) with hypervariable CDRs creating a unique binding surface.

What is the “lock and key” model and how does it compare to “induced fit” in protein–substrate interactions?

Lock and key: the binding site and substrate are already complementary without change. Induced fit: binding induces conformational changes in the protein to achieve a proper fit.

If you were to run an SDS-PAGE on an antibody under reducing conditions, what would you expect to see? And under non-reducing conditions?

Reducing: two bands corresponding to heavy (~50 kDa) and light (~25 kDa) chains. Non-reducing: a single band around ~150 kDa representing the intact antibody.