Biomolecular interactions are an essential component of many pathways and responses in living systems. These interactions include the binding of substrates and cofactors to enzymes, antigens to antibodies, proteins to proteins, sugars to lectins, and small molecules to transport proteins or receptors. The biomolecular interactions can determine the eventual activity, distribution, excretion, metabolism, and effects of biomolecules or other molecules in the body. As the basic unit of life, cells represent complex biological entities whose normal function revolves around interactions between multiple biomolecular systems. Proteins are large organic molecules with biological functions and are essential components of these systems, serving as molecular machines, sensors, transporters, and structural elements, among others.
Proteins usually interact with each other and interact with other molecules such as DNA and RNA, to perform their functions. Protein–protein interactions (PPIs) are dynamic in nature, adjusting in response to different stimuli and environmental conditions. This provides considerable flexibility in function and allows the cells to adapt to the changing environment in a measured manner. Proteins control the molecular and cellular mechanisms that determine the health and disease states of an organism. Even a subtle dysfunction of PPIs can have major effects throughout the body, perturbing interconnected cell networks and producing disease phenotypes. Therefore, the analysis of PPIs is essential to understand complex processes, as well as to identify molecules and PPIs suitable for therapeutic intervention.
A number of methods are available for large-scale studies of PPIs, each with its own strengths and weaknesses. When choosing an appropriate method to study the interaction partners of a protein of interest, factors that should be considered include clearly defining the objectives of the study, the different nature of the studied PPIs, time and cost, and specialized equipment.
Relying on professional team and advanced equipment, Amerigo Scientific provides cost-effective and efficient services for protein-protein interaction analysis to meet the diverse research needs of customers. Yeast one-hybrid (Y1H), yeast two-hybrid (Y2H), and yeast three-hybrid (Y3H) systems are widely used techniques to study protein-protein, protein-DNA, or protein-small molecule interactions.
Y1H, Y2H and Y3H technologies differ in their experimental systems, applications, and suitability for specific types of interactions. Y1H is ideal for investigating protein-DNA interactions, especially gene-centered Y1H for the identification of transcription factors that bind specific DNA segments. Nuclear Y2H is a widely used PPI screening method, which is suitable for the study of nuclear proteins. Unlike nuclear Y2H, membrane Y2H is specifically designed for studying membrane protein interactions. Y3H is an extension of the classic Y2H system, which is developed to study protein interactions with RNAs, small molecules, and natural ligands.
Comparison of Yeast Hybrid Techniques
| Technique |
Applications |
Advantages |
| Y1H |
- Detecting protein-DNA interactions
- Identifying transcription factors and their binding sites |
- Directly detects protein-DNA interactions
- Facilitates identification of novel transcription factors
- Simple and efficient, suitable for high-throughput screening |
| Nuclear Y2H |
- Detecting protein-protein interactions among soluble proteins localized in the nucleus
- Mapping protein interaction networks |
- Classical and well-established method
- Suitable for high-throughput identification of protein interactions |
| Membrane Y2H |
- Detecting interactions between membrane proteins or between membrane proteins and cytoplasmic proteins
- Studying membrane protein functions and signal transduction pathways |
- Specialized for membrane proteins, overcoming limitations of traditional Y2H
- Maintains native conformation and membrane environment of proteins
- Capable of detecting proteins difficult to solubilize or translocate to the nucleus |
| Y3H |
- Detecting interactions between proteins and RNA molecules or small molecules
- Investigating RNA-mediated regulation of protein interactions |
- Enables study of complex interactions, such as protein-RNA
- Reveals roles of RNA in regulating protein functions
- Useful for screening RNA-binding proteins and identifying small molecule modulators |
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