Under physiological condition, most protein-protein interactions are transient, and happen in a very short duration, which make them difficult to be studied. Crosslinking reagents, or crosslinkers, provide the analytical solution to capture protein-protein complexes by covalently binding them together as they interact, to freeze even transient, weak interactions for consequent isolation and characterization.
The crosslinking can be performed in vivo or in vitro. Whether to use in vivo or in vitro crosslinking depends on the specific needs of the project. Proteins are crosslinked in a native state during In vivo crosslinking, whereas, for in vitro crosslinking protein may be denatured.
In vivo crosslinking
For in vivo crosslinking, proteins are linked inside cells, hence the risk of false positive interaction or loss of protein complex stability can be eliminated. Hydrophobic and lipid-soluble crosslinking reagents are often used to react with proteins located within cell membrane, while hydrophilic, water-soluble crosslinking reagents are used to crosslink proteins located on the cell membrane, such as plasma-anchored receptors. Though proteins are remain in native status, due to the complexity of proteins in cells, optimization of in vivo crosslinking can be complicated as well, and possible nonspecific crosslinking may still occur. The nonspecific crosslinking problem can be eliminated by applying crosslinking reagents with shorter spacer arms.
In vitro crosslinking
During in vitro crosslinking process, cells are homogenized and lysed. Therefore, this method is more suitable for analyzing more stable protein-protein interactions. As cells are lysed in specific lysing buffer, the reaction conditions are easier to control, such as temperature, pH, ion strength, etc. Without cell boundary, many types of crosslinking reagents are suitable for this application. Nonspecific crosslinking is easier to be controlled.
Type of crosslinking methods:
Crosslinking reagents covalently link together interacting proteins, domains or peptides by forming chemical bonds between specific amino acid functional groups of two or more biomolecules that occur in close proximity because of their interaction. There are many commercial chemical crosslinkers to choose, homobifunctional or heterobifunctional, long and short length of arm, cleavable and non-cleavable, water-soluble and –insoluble, based on the specific requirements of the projects. Homobifunctional molecules target the same group on the protein, while heterobifunctional crosslinkers targets different functional groups on separate proteins for greater variability or specificity. Crosslinker molecule can also be designed to include cleavable elements, such as esters or disulfide bonds (diagrammed below), to reverse or break the linkage by the addition of hydroxylamine or reducing agents, respectively. Crosslinkers can be hydrophobic to allow passage into hydrophobic protein domains or through the cell membrane or hydrophilic to limit crosslinking to aqueous compartments.
As addition of crosslinkers to cell suspensions or cell lysates may cause many unspecific crosslinks to be formed, in addition to the target protein-protein interaction, more sophisticated crosslinker designs were created that incorporates photoreactive groups, which react at selected times and only in response to irradiation by UV light. Heterobifunctional crosslinkers with a chemical crosslinking group at one end and a photoreactive group on the other end can be selectively reacted to the target proteins through a two-step process.
Features of Crosslinking Protein Interaction Analysis
- Study transient in vivo protein-protein interaction
- Study low affinity in vitro protein-protein interactions
- Increase the detection sensitivity
Workflow of Crosslinking Protein Interaction Analysis
- Design the crosslinking method and type of crosslinking reagents based on your specific project needs: in vivo/ vitro
- Parameters optimization, such as binding and quenching time
- Cell lysis, CoIP
- WesternBlot/ mass spectrometry, depending on the project needs
- Data analysis & report delivery