Protein Interaction Measurements Lecture Notes

Surface Plasmon Resonance (SPR) Spectroscopy

  • Principle: Detect mass change on a gold-labeled optical chip using a laser.
  • Components:
    • Chip with gold film and glass port.
    • Incident light beam (laser).
    • Prism for reflection.
    • Detector to measure intensity drop (shadow).
  • Key Advantage: Detect on and off rates of molecular interactions (rate of association and dissociation).
  • Key Drawback: Requires immobilization of one interaction partner (ligand).
  • Nomenclature: In SPR literature, the ligand is often the protein of interest, and the analyte is its interactor.

SPR Applications

  • Versatile: Can study interactions involving various molecules.
    • Organic molecules (drugs, lead molecules).
    • Protein-protein interactions.
    • Protein-nucleic acid interactions.
    • Glycoproteins (carbohydrate interactions).
    • Viruses and whole cells.
  • Response and Molecular Weight: Response per molar unit is proportional to the molecular weight.
  • Detection Limit: Lower limit for detection of small molecules is around 100 Daltons.

SPR Measurements and Practical Aspects

  • Measured Parameters:
    • KAK_A (affinity or association constant).
      • Derived from k<em>ak<em>a (on rate/association rate) and k</em>dk</em>d (off rate/dissociation rate).
    • Units for KAK_A: Molar (M).
  • Response and Mass Concentration: Measured response reflects change in mass concentration.
  • The smaller your molecule, the harder it is to measure
    • Below 100 Daltons, measurement becomes difficult.
  • Immobilization Strategy: Consider immobilizing the smaller partner first to get a more significant response from the larger molecule's binding, assuming molecular crowding and mass transport effects are minimized.

SPR Assay Modes

  • Direct Binding Assay: Immobilize ligand, measure analyte binding.
  • Direct Binding Assay with Enhancement: Use an enhancer molecule that binds tightly and rapidly to the analyte to boost the signal.
  • Inhibition Assay (In-Solution Competition):
    • Immobilize ligand.
    • Detecting molecule binds to analyte.
    • Measure response based on how much analyte saturates the detecting molecule's binding site.
    • Determines IC50 value (inhibitory constant at 50% saturation).
  • Surface Competition Assay: Two competing analytes compete for the same ligand.

SPR Data Interpretation

  • Classical SPR Signal Curve:
    • Baseline.
    • Association phase (increase in mass deposition until saturation).
    • Plateau (saturation of immobilized ligand).
    • Dissociation phase (reduction in mass as buffer washes over).
    • Regeneration phase (returning fully to baseline) to prepare the ligand for another interaction measurement after stopping amalli injection. Need conditions that effectively remove analyte without denaturing ligand.

SPR Immobilization Techniques

  • Common Method: BIAcore sensor chips (CM5) with a dextran matrix on top of a gold layer on a glass chip.
    • Covalently attach ligand molecules to the dextran matrix.
    • Alternative: Immobilize an antibody to capture the ligand and then detect the analyte.
  • Amine Coupling: Use amine technology for covalent binding to functional amines on the ligand (N-terminus or lysine residues).
  • Process:
    1. Use EDC and NHS to prepare and prime the surface.
    2. Wash on the ligand to achieve a desired response rate.
    3. Wash with buffer.
    4. Use ethanolamine to quench the reaction, yielding the final amount of immobilized protein.

SPR Immobilization Considerations

  • Impact: Immobilization significantly affects the ability to measure protein interactions.
  • Examples:
    • 10,000 response units (RUs) immobilized via amine coupling with no activity.
    • 12,000 RUs immobilized via thiol coupling (3-SH groups) with only 5% activity.
    • 2,000 RUs immobilized via polyhistidine tag capture with 20% activity (better due to higher signal relative to immobilized protein).
  • Conclusion: Carefully consider the immobilization method.

SPR Flow Rate

  • Importance: Flow rate of ligand and interactors over the chip affects measurements differently depending on the specific interaction being studied.
  • Optimization: Empirically evaluate different flow rates to identify the best conditions for the assay.

SPR Sample Data Analysis

  • Affinity Differences: Interactions can range from very tight (e.g., 1 nanomolar) to weaker (e.g., 6 nanomolar).
  • KD Difference: A sixfold KD difference can result from variations in the off rate rather than the on rate.
  • On and Off Rates: Similar KDs can arise from different on and off rates, affecting mechanism and downstream applications.
  • Example 1: 1 nanomolar interaction has a very slow off rate.
  • Example 2: 6 nanomolar interaction has a much faster off rate.
  • Threefold difference in affinity can occur with different on rates but nearly identical off rates.

SPR Kinetics in Drug Discovery

  • Importance: Kinetics are crucial in drug discovery.
  • Example: Three molecules with the same affinity can have vastly different kinetic profiles.
    • Fast on, fast off.
    • Intermediate on, intermediate off.
    • Slow on, very slow off.
  • Application: Depends on the desired drug action – quick but short-lived, or long-lasting.
  • Business Implications: Drug discovery executives might manipulate on/off rates for financial gain (selling more pills).

Fluorescence Polarization (FP)

  • Principle: Based on molecular tumbling or rotational motion of a fluorescently active molecule.
  • Mechanism:
    • Polarized light interacts with the molecule.
    • Small molecules rotate faster; large molecules rotate slower.
    • Slower rotation gives a larger signal.
    • Fluorescently tagged molecule rotates slower when bound to an interactor due to increased mass.

FP Signal and Binding Affinity

  • Polarized Light: Obtained by passing depolarized excitation light through a polarization filter.
  • Signal Conversion: Change in signal due to slower rotation is converted into a binding affinity.
  • Fluorescence Parameters:
    • Fluorescence intensity, color, and anisotropy are measured.
    • Anisotropy (A) is the difference between vertically and horizontally polarized emissions.
  • Focus: FP interaction measurements focus on anisotropy and polarized fluorescence intensity.
  • Information Provided: Indicates size of fluorescent species via rotational correlation time.
  • Basis for Interaction Measurement: Anisotropy depends on molecular volume, making it sensitive to changes in size upon complex formation.

FP Design Considerations

  • Fluorescence Probe: Required for measuring fluorescence anisotropy.
  • Data Measurement: Measure IC50 values with different amounts of interactor.
  • Probe Options:
    • Intrinsic tryptophan residues.
    • Site-directed mutagenesis to add a tryptophan.
    • Fusion to GFP (green fluorescent protein).
    • Covalent linkage of a fluorescent dye via cysteine residues.

FP Assay Modes

  • **Direct Binding Assays.
  • Competitive Binding.
  • Enzyme Assays.
  • Protease Assays (size reduction after degradation).
  • Incorporation of fluorescence polarizing labels.

MicroScale Thermophoresis (MST)

  • Principle: Quantitative analysis of protein interactions in free solution with low sample consumption.
  • Mechanism:
    • Based on thermalphoresis – directed motion of molecules in temperature gradients.
    • Laser light shone onto sample capillary causes molecule movement due to heat.
    • Movement speed depends on size and binding to an interactor.
  • Advantages:
    • No immobilization required.
    • No labeling needed if GFP fusion or intrinsic tryptophans are present; can also be fully label-free.

Recap of Protein Interaction Measurements

  • Measurements: Can be qualitative and quantitative.
  • Qualitative Methods:
    • Yeast two-hybrid system.
    • Pull-down assays.
    • Advantages: Wide screening, quick, low cost for identifying unknown interactions.
  • Quantitative Methods:
    • ITC (Isothermal Titration Calorimetry).
    • SPR (Surface Plasmon Resonance).
    • MST (MicroScale Thermophoresis).
    • Fluorescence Polarization.
    • Disadvantages: More expensive (hardware, consumables), require optimization, ITC consumes significant protein.
  • Quantitative Data Benefits: Affinity constants, entropy, on/off rates, stoichiometry.

Method Specifics

  • ITC: Measures affinities, stoichiometry, and entropy; provides binding mode data.
  • SPR: Measures affinities and on/off rates; valuable for drug discovery; often requires immobilization, which can be time-consuming.
  • Fluorescence Polarization: Quantitative analysis in solution, low sample consumption, high throughput screening (drug discovery); often requires sample labeling (GFP fusion or fluorescent dye), asset development required.
  • Overall Recommendation: Well-characterized and quantified protein interactions provide significant insight into biological problems and underlying mechanisms.