Protein-protein interactions

Proteins rarely act alone, but rather interact with each other to carry out various cellular functions. 

Studying these protein-protein interactions provides crucial insights into a wide range of biological processes. 

The importance of protein interactions

Proteins facilitate most biological processes in a cell. This includes gene expression, cell growth, proliferation, nutrient uptake, morphology, motility, intercellular communication, and apoptosis.

Protein expression is a dynamic process that responds to various stimuli. Specific proteins may not always be expressed or activated for certain tasks. Cells also vary in their protein expression, which can complicate the study of protein function in the appropriate biological context. However, with careful investigation and analysis, these challenges can be overcome.

Before the late 1990s, protein function analyses primarily focused on individual proteins. However, since most proteins must interact with other proteins to function, they should be studied in the context of their interacting partners. The publication of the human genome and the development of proteomics have made it essential to understand protein interactions and identify biological networks to understand their function within the cell.

Different types of protein interactions

Important types of protein interactions include:

  • Protein-ligand interactions
  • Protein-DNA interactions
  • Protein-protein interactions (PPIs)

In protein-protein interactions, proteins interact with each other to carry out specific functions in cells.

Reasons to study protein-protein interactions

Since nearly all biological processes involve one or more PPIs, studying these interactions helps us understand the molecular mechanisms interactions within these processes, including: 

Cell signaling
Many biological processes, such as cell growth, differentiation, and apoptosis are regulated by intricate signaling networks involving protein-protein interactions. By studying these interactions, researchers can understand the signaling pathways involved in diseases like cancer and develop targeted therapies that disrupt or modulate these pathways.
Enzymatic activity
Enzymes often function in complexes with other proteins, where protein-protein interactions are essential for catalytic activity. Understanding these interactions can shed light on enzyme regulation, substrate specificity, and metabolic pathways, aiding in drug discovery and metabolic engineering.
Gene regulation
Transcription factors and regulatory proteins often form complexes to control gene expression. Studying protein-protein interactions involved in gene regulation can reveal key players in transcriptional control and provide insights into diseases linked to dysregulated gene expression, such as diabetes and neurodegenerative disorders.
Protein trafficking and localization
Protein-protein interactions govern the intracellular trafficking and localization of proteins within cells. By deciphering these interactions, researchers can uncover the mechanisms underlying organelle targeting, vesicular transport, and protein sorting, which are crucial for cellular homeostasis and function.
Structural biology
Protein-protein interactions contribute to the assembly of macromolecular complexes with specific architectures and functions. Using techniques like X-ray crystallography and cryo-electron microscopy, it is possible to determine the structures of these complexes. These analyses provide atomic-level insights into their mechanisms of action and support rational drug design.
Disease mechanisms
Malfunctioning protein-protein interactions are implicated in numerous diseases, including neurodegenerative disorders, autoimmune diseases, and infectious diseases. By studying these interactions, researchers can identify potential therapeutic targets and develop drugs that disrupt harmful interactions or stabilize beneficial ones.
[shutterstock_362386109 2.jpg] shutterstock_362386109 2.jpg

How to measure protein-protein interactions

Protein-protein interactions can be studied with several experimental techniques, each with unique advantages and limitations. The insights that these studies provide depend on the selected analysis method. 

Some (but not all) of the most widely used PPI analysis methods include:

Method Description Malvern Panalytical instrument
Nuclear magnetic resonance (NMR) spectroscopy NMR spectroscopy provides atomic-level structural information, revealing details about protein conformational changes upon binding. --
Tandem affinity purification-mass spectroscopy (TAP-MS) TAP provides a purified protein complex which can be ran on a mass spectrometer (MS) to map protein-protein interactions. --
Grating-coupled interferometry (GCI) This label-free, real-time, and surface-based technology allows researchers to rapidly and accurately measure kinetic rates, determine affinity, and monitor the concentrations of even low-abundance interacting analytes in crude samples such as biofluids.

WAVEsystem, GCI technology

Surface plasmon resonance (SPR) SPR allows for real-time monitoring of protein interactions at the surface of a sensor chip, enabling precise determination of binding kinetics and affinities. SPR is a label-free technique that uses relatively small quantities of materials. This allows for precise, accurate analysis of protein interactions. --
Isothermal titration calorimetry (ITC) ITC measures the heat released or absorbed during binding events, providing crucial thermodynamic information for understanding interaction mechanisms.

PEAQ-ITC, MicroCal technology

Neighboring technologies:
Differential scanning calorimetry (DSC) DSC measures the thermal stability of proteins, which is useful for stability studies, bio-similarity assessments, and batch-to-batch comparability assessments. DSC measures thermal stability by monitoring the heat change of a molecule’s denaturation when heated at a constant rate.

PEAQ-DSC, MicroCal technology

Electrophoretic light scattering (ELS)
Dynamic light scattering (DLS)

ELS measures particle mobility and charge. DLS measures the particle size of dispersed systems from sub-nanometers to several micrometers in diameter. Combining these techniques gives a more comprehensive overview of protein-protein interactions – useful for developing interventions that target specific molecular interactions.

Zetasizer Advance

WAVEsystem

Next-generation bioanalytical instruments for drug discovery and life scien...
WAVEsystem

MicroCal ITC range

The "Gold Standard" for measuring binding affinity and stoichiometry
MicroCal ITC range

MicroCal DSC range

The "Gold Standard" for structural stability analysis of biotherapeutics, b...
MicroCal DSC range

Protein-protein interaction examples measured using Malvern Panalytical instruments

Malvern Panalytical instruments have been used in several studies that analyze PPIs. A few examples are below: 

Plant membrane receptor kinases

Grating-coupled interferometry (GCI) with the WAVEsystem was used to explore the binding between several plant receptors and their ligands, and the role of a coreceptor (SERK3). Where the individual receptors have significantly different binding affinities for their respective ligands, the SERK3 ectodomain binds to the ligand-associated receptors with similar binding kinetics. 

Read the full paper

Cytokines and their mimics

GCI with the WAVEsystem was used to analyze interactions with synthetically produced ‘receptor mimics’ of cell surface receptors, which are often of interest as drug targets. 

Read the full paper

Voltage-gated calcium channel (Cav) interactions

Isothermal Titration Calorimetry (ITC) on the MicroCal demonstrated that changing the backbone of the protein can change the protein-protein interaction in voltage-gated calcium channels. 

Read the full paper

Peptide-based inhibitors of protein-protein interactions (ITC)

This paper reviewed the uses of ITC alongside other technologies to investigate the characteristics of constrained peptides that inhibit protein-protein interactions. 

Read the full paper

WAVEsystem

WAVEsystem

Next-generation bioanalytical instruments for drug discovery and life sciences for both industry and academic research

MicroCal PEAQ-ITC

MicroCal PEAQ-ITC

The Gold Standard for measuring affinity and stoichiometry

MicroCal PEAQ-DSC

MicroCal PEAQ-DSC

Gold standard protein stability analysis for research applications

Zetasizer Advance Range

Zetasizer Advance Range

Light Scattering for every application

Technology
Grating-coupled interferometry (GCI)
Isothermal Titration Calorimetry (ITC)
Differential Scanning Calorimetry (DSC)
Electrophoretic Light Scattering
Dynamic Light Scattering