Proteins are the building blocks of all organisms, and protein-protein interactions are the foundation for cellular structure, metabolism, and communication. Protein-protein interactions range from contacts of protein-complex subunits to dynamic connections among enzymatic signaling networks.
Two common techniques to study protein interactions include GST pull-downs and coimmunoprecipitations. Both methods, however, analyze proteins following cell lysis, which typically involves detergents that may cause aberrant protein interactions by disrupting hydrophobic interfaces of protein complexes or by mixing of cellular compartments.
Chemical crosslinking of protein complexes in live cells is a powerful way to capture both stable and transient protein-protein interactions in their native environment. Traditional amine-reactive crosslinkers such as formaldehyde or NHS-ester derivatives have been used to study protein-protein interactions in vitro but have varying degrees of success for studying protein complexes in their native environment.
Formaldehyde has been used extensively to reversibly crosslink proteins through primary amines with a zero-length bond distance. It, however, spontaneously forms polymers in solution and is not protein-specific as it also crosslinks proteins to DNA and other macromolecules.
Bifunctional NHS-ester crosslinkers (e.g., DSG and DSS) are more protein-specific than formaldehyde but only crosslink specific amino acid residues at defined spacer lengths. Additionally, formaldehyde and NHS esters must permeate cell membranes in order to crosslink intracellular proteins and must be quenched to prevent excessive crosslinking.