Technology and research progress on in vivo protein oligomerization detection

Proteins are large molecules of specific conformations, and their functions vary depending on their structure. As the key components involved in life processes, proteins often assemble into large complexes called oligomers to execute important activities. Oligomers are of two types: Homologous and heterologous. The homologous oligomers contain subunits of the same polypeptide chain, whereas the heterologous ones consist of two or more polypeptide chains with different amino acids or different biopolymers, such as nucleic acids. These oligomer complexes play significant roles in regulating enzyme activity, signal transduction, and cell adhesion. Oligomerization can increase protein concentration in local space to provide higher stability and enhance the specificity in a molecular recognition process. Oligomerization is influenced by factors such as the types of ligands, proteins, and temperature. For example, receptor proteins are often assembled into dimeric or higher-order oligomers mediated by their corresponding ligands, which promote downstream signaling cascades to transmit signals. The formation mechanisms of oligomeric proteins can be categorized into the following four types: Domain exchange, ligand-induced oligomerization, interfacial point mutation, and post-translational modification. Domain exchange allows functional conversion between protein monomers and oligomers. Ligands can induce oligomerization to activate protein activity, as is typically observed in receptor proteins. Interfacial point mutation involves changes in the amino acid sequence, insertion, or deletion of residues in an interfacial region, which distinguishes different oligomerization states. Post-translational modifications are essential for functional protein formation, although errors in modifications can result in the formation and accumulation of abnormal oligomers, which potentially contribute to disease development. Therefore, analyzing protein oligomerization is crucial for understanding biological processes such as protein-protein/ligand interactions, signal transduction, and disease-related mechanisms. Researchers have optimized the study of protein oligomerization from different perspectives, including predicting and identifying interacting proteins, labeling target proteins, and observing them through imaging. The conventional techniques such as co-immunoprecipitation (Co-IP) and yeast two-hybrid precipitation have some limitations, such as the inability to investigate directly in living cells as well as the possibility of giving false-positive or false-negative results. In recent years, however, the emergence of new technologies has enabled real-time, dynamic, and in vivo observation of protein-protein interactions, which has significantly contributed to the advancements in this field. For instance, the use of more stable and photobleaching-resistant fluorescent probes, combined with high-speed imaging techniques, has improved the temporal and spatial resolution in observing the dynamic behavior and interactions of proteins. In addition, the combination of emerging technologies and algorithms has expanded the scope of protein explored at both temporal and spatial scales, which has deepened our understanding of the relation between protein structure and function. This review summarizes the classification and formation mechanisms of oligomeric proteins, as well as highlights several major technologies for analyzing protein complex oligomerization, and provides an overview of the recent research progress in protein labeling and in vivo detection technologies. Finally, it offers a prospective outlook on the development of research in protein oligomerization, intending to provide theoretical references for researchers in selecting appropriate analytical techniques.