Protein Interactions Lecture Notes

Protein Interactions

Introduction

• Proteins are vital macromolecules that rarely function in isolation.

• Many cellular processes rely on macromolecular machines built from multiple proteins and, in some cases, nucleic acids.

• The ribosome is a prime example, composed of numerous proteins and RNA, facilitating tRNA translation into amino acids.

• Various multi-protein complexes exist, such as Rubisco, nucleosomes, and GroEL/ES chaperones, with varying numbers of protein subunits.

• Rubisco, crucial for carbon fixation in plants, may be the most abundant protein complex on Earth.

• Protein interactions can be obligate (always paired) or dynamic, influenced by post-translational modifications like phosphorylation/dephosphorylation.

BioGrid Database

• BioGrid is a database for protein, genetic, and chemical interactions, containing approximately 2,800,000 interactions.

• It includes data from biochemical, biophysical, and genetic experiments across major model organisms, including yeast and humans.

• The database contains over 1,300,000 mapped human protein-protein interactions.

• Example: Using PALB2 (partner and localizer of BRCA2) to demonstrate the network of interactions.

PALB2 and BRCA2 Interaction

• PALB2 is at the center of a network of interactions, including BRCA2. It is the partner and localiser of BRCA2

• The database allows filtering of interactions based on the number of times they've been observed, increasing confidence in the interaction.

• The thickness and color of the line connecting the proteins indicates method and frequency reported.

• Methods include affinity capture, mass spectrometry, and Western blotting.

Biophysical Assays for Protein-Protein Interactions

• Various biophysical assays can be used to study protein-protein interactions, including:

  • Nuclear Magnetic Resonance (NMR)

  • Cryo-Electron Microscopy (cryo-EM)

  • X-ray Crystallography

  • Electrophoretic Mobility Shift Assay (EMSA) for protein-DNA interactions

Limited discussion will occur on a few of these methodologies (IP and FP) in this lecture, while others will be covered in future lectures.

PALB2: A Case Study

• The structural basis for BRCA2 recruitment by PALB2 was determined to a 2009 paper.

• The C-terminus of PALB2 contains a 7-bladed beta-propeller domain (also known as WD40 repeat).

• WD40 repeats are characterized by a tryptophan (W) and aspartic acid (D) sequence within a 40 amino acid stretch.

• This region of PALB2 binds to the N-terminus of BRCA2.

• Mutations in BRCA2 predispose individuals to breast and cervical cancer; mutations in PALB2 cause Fanconi anemia.

• Fanconi anemia increases cancer risk later in life and causes bone marrow problems and skeletal abnormalities (e.g., bifurcated thumb).

BRCA2 N-Terminal Conservation

• Multiple sequence alignment reveals a conserved region in the N-terminus of BRCA2 across species (aspartic acid (D) to leucine (L)).

• Conservation suggests functional or structural importance.

• Mutations in tryptophan (W) within this region in Fanconi anemia patients further highlight its importance.

Immunoprecipitation (IP)

• Definition: Precipitation of a protein antigen from solution using a specific antibody.

• Requires a monoclonal antibody that recognizes a single antigen.

• Antibodies can target an amino acid motif within the protein of interest or an artificially introduced tag (His tag, Strep tag, FLAG, MYC).

• Monoclonal antibodies are produced from mouse hybridomas and are monospecific and monovalent.

IP Protocol

• Immobilize the monoclonal antibody on an affinity resin (e.g., Protein A or Protein G Sepharose).

• Prepare a cell lysate containing the protein of interest, washing the affinity resin with buffer to remove non-specific binding proteins.

• Mix the lysate with the resin, allowing the antibody to capture the protein.

• Wash the resin to remove unbound proteins.

• Analyze the remaining proteins using SDS-PAGE, Western blotting, or mass spectrometry.

• Direct and indirect interactions can be detected; buffer stringency affects which interactions are maintained.

• stringency is the

• High stringency retains only strong, direct interactions; low stringency may include nonspecific interactions.

• Stringency is controlled by salt concentration and pH.

Experimental Example (IP)

• HEK293T cells (human embryonic kidney cells) were used to express a double-epitope-tagged PALB2 (FLAG and another epitope).

• PALB2 was pulled down using an anti-FLAG monoclonal antibody.

• Western blotting was performed to probe for BRCA2 interaction using an anti-BRCA2 antibody or the second anti-epitope tag.

• An empty vector control ensured the experiment's cleanliness, confirming no pull-down without PALB2.

• This confirmed a robust interaction between PALB2 and BRCA2.

Fluorescence Polarization (FP) / Anisotropy

• A biophysical method used to observe the interaction of proteins.

• Introduction to Fluorescence Physics:

  • A fluorophore absorbs energy from light (photon) and is excited to a higher energy state.

  • After a short delay (fluorescence lifetime, $\tau$), the fluorophore loses energy and re-emits light at a longer wavelength.

  • Fluorescence lifetime ($\tau$) is the average time it takes for the absorbed energy to be released.

• Fluorescence Anisotropy:

  • Emitted light has a preferential direction.

  • Anisotropy means unequal in each direction.

FP Process Explanation

• Laser light is passed through a polarizer to ensure light waves are in the same plane.

• The light is shined onto a solution containing fluorophores.

• Only fluorophores in the correct orientation absorb the photons and become excited.

• These fluorophores then re-emit photons in a preferred direction.

• Example Fluorophore: Fluorescein

  • Excitation wavelength: 494 nm

  • Emission wavelength: 521 nm

  • Fluorescence lifetime ($\tau$): 3-4 nanoseconds

• Fluorescein can be attached to a polypeptide (amino acid sequence) at the N-terminus.

FP Experiment and Binding Curves

• An artificial peptide (amino acids 21-34 of BRCA2) with fluorescein label was created.

• The experiment measured increasing of signal as recombinant PALB2 protein added.

• Generated a binding curve to determine protein interaction.

• A fixed concentration of fluorescently labelled peptide was mixed with increasing amounts of recombinant protein.

Reason for Polarization Changes

• Light is passed through a polarizer and shined onto fluorophores.

• Only some fluorophores in the correct orientation absorb the light.

• Fluorophores tumble and rotate freely due to Brownian motion, resulting in a random mixture of orientations.

• Emitted light is in random directions; input signal coherence is lost.

• With protein binding, the rotation movement is slower.

• More emitted light is in the same plane as the excitation light.

• Input signal coherence is maintained.

Measuring Polarization

• A second polarizer is placed perpendicular to the first.

• Photomultiplier tubes (PMTs) convert light energy into electrical energy for measurement.

• The ratio of light passing through the parallel and perpendicular polarizers is measured.

• A rapidly tumbling peptide will result in roughly equal numbers of photons in parallel and perpendicular directions.

• Slowly tumbling peptides emit more photons in the parallel direction.

• Calculation:

  • Polarization is calculated from equation: $r = \frac{I<em>{parallel} - I</em>{perpendicular}}{I<em>{parallel} + 2I</em>{perpendicular}}$, where $r$ refers to anisotropy and the signal will be in millipolarization (mP).

  • Signal Values: 10-300 mP

  • Measurement is accurate to $\pm 2 \text{ mP}$

  • Concentration of ligand required is not needed but protein concentration must be very precise.

Applications of FP

• Determine if a protein binds to a particular peptide sequence.

• Generate a binding curve to confirm binding, which we can analyze the data to determine a binding constant

• Determine binding constants (Kd) using computer software like Prism or Origin.

• Fit a binding model to the experimental data; ensure the model agrees with the data (R2 > 0.7).

Dissociation Constant (Kd)

• Kd is the dissociation constant $(K_d)$, a value in molar units, typically micromolar range for protein-protein interactions.

  • Low Kd, tight interaction. High Kd, weak interaction.

  • Kd is the concentration at which 50% of the peptide is bound or the concentration in which 50% of the protein dissociates from the interaction.

• In FP, Kd calculation depends on knowing the concentration of the protein of interest.

• Kd is used to compare interactions of protein of interest with different ligands under the same experimental conditions.

• Can differ if factors like salt concentrations, and pH gradients change.

Crystal Structure and Validation

• The crystal structure of the WD40 domain in complex with the BRCA2 peptide was determined.

• The structure revealed the importance of specific amino acids in the interface, like tryptophan, that is mutated in patients.

• Mutating an alanine to an arginine sterically clashes with peptide binding.

• Artificially introduced alanine to arginine mutation in full-length PALB2 disrupted the interaction with BRCA2.

• Doing an IP expirement confirms the point which says the crystal structure is accurate.

IP Experiment Validation

• Artificially introduced the alanine to arginine mutation in full-length PALB2.

• The protein was expressed and folded correctly, but the interaction with BRCA2 was blocked.

Conclusion

• The IP experiment confirms both the hypothesis and validity of the observed interface.

• Techniques discussed IP and FP.

Next Steps

• Isothermal Titration Calorimetry (ITC) will be discussed. Its yet another biophysical method to look at protein protein interactions and will be the subject of the next lecture.