Probing Protein-Protein Interactions: Revolutionary Chemical Biology Techniques for Revealing Molecular Insights

Cheng Hongmei^*
*Correspondence: Cheng Hongmei, Department of Biochemistry, Slovak University of Technology in Bratislava, Bratislava, Slovakia, Email:

Keywords

Protein-protein interactions • Fluorescence resonance energy transfer • Mass spectrometry

Introduction

Fluorescence Resonance Energy Transfer (FRET) is a versatile technique widely employed to study protein-protein interactions in real-time. By tagging interacting proteins with suitable fluorophores, FRET enables the measurement of distance-dependent energy transfer between donor and acceptor molecules. This technique allows researchers to monitor dynamic changes in protein interactions, including binding affinity, conformational changes and kinetics, providing valuable insights into the underlying molecular mechanisms. Fluorescence Resonance Energy Transfer (FRET) and Fluorescence Correlation Spectroscopy (FCS) are commonly employed to study protein interactions [1]. FRET measures the transfer of energy between fluorescently labeled proteins in close proximity, providing information about their spatial arrangement and conformational changes. FCS analyzes the fluctuations in fluorescence intensity to determine the diffusion properties of interacting proteins, offering insights into their binding kinetics and local concentrations.

Cross-linking Mass Spectrometry combines chemical cross-linking with mass spectrometry to identify and characterize protein-protein interactions within complex mixtures. By introducing covalent cross-links between interacting proteins, followed by enzymatic digestion and mass spectrometric analysis, researchers can map protein interaction interfaces, determine protein stoichiometry and investigate dynamic changes in interactions under different conditions. XL-MS has revolutionized our ability to study protein complexes and elucidate their assembly pathways. Chemical cross-linking involves the introduction of covalent bonds between interacting proteins to stabilize their transient interactions [2]. This technique provides insights into protein complex assembly and structural dynamics. Cross-linking agents are used to create covalent bonds between amino acid residues in close proximity, preserving the interacting partners for subsequent analysis by mass spectrometry or other structural techniques. Chemical cross-linking offers valuable information about the spatial arrangement of interacting proteins and their conformational changes during the binding process.

Description

Surface Plasmon Resonance (SPR) is an optical technique used to measure the real-time binding kinetics and affinities of protein-protein interactions. By immobilizing one protein on a sensor surface and flowing its interacting partner over it, SPR detects changes in the refractive index at the surface, providing quantitative information about the binding events. SPR allows researchers to study protein interactions in a label-free manner, providing insights into binding kinetics, affinity constants and the influence of different conditions on the interaction [3]. Proximity-Dependent Biotinylation (BioID) is a proximity-based labeling technique that enables the identification of protein-protein interactions within the cellular context. A protein of interest is fused with a promiscuous biotin ligase, which biotinylates proximal interacting partners. Biotinylated proteins can then be selectively captured and identified using streptavidin-based affinity purification coupled with mass spectrometry. BioID allows for the identification of both stable and transient protein interactions, offering a comprehensive view of the protein interactome within living cells.

Cryo-Electron Microscopy (Cryo-EM) has revolutionized structural biology by enabling the visualization of protein complexes at near-atomic resolution. This technique has been instrumental in elucidating the three-dimensional architecture of protein-protein interactions, providing crucial insights into their mechanisms and dynamics. By flash-freezing samples and imaging them using an electron microscope, Cryo-EM allows researchers to capture transient or flexible protein complexes in their native states, overcoming the limitations of traditional crystallography [4]. The yeast two-hybrid assay is a widely used technique for investigating protein-protein interactions in a controlled environment. It relies on the modular nature of transcription factors, wherein the DNA-binding domain and the activation domain can be separated. By fusing the proteins of interest to these domains, their interaction leads to reconstitution of the transcription factor and subsequent activation of a reporter gene. The Y2H assay allows researchers to identify and study protein interactions, providing insights into their binding partners and the conditions that influence their association.

Co-immunoprecipitation is a classic technique used to detect and validate protein-protein interactions in complex biological samples. It involves the use of antibodies to selectively isolate a target protein and its interacting partners from a cell lysate. Following immunoprecipitation, the associated proteins are analyzed by techniques such as Western blotting or mass spectrometry. Co-IP provides valuable information about the direct or indirect interaction between proteins, their stoichiometry and potential dynamic changes under different physiological conditions [5]. A range of biophysical techniques offers valuable insights into protein-protein interactions at a structural and functional level. These techniques include Surface Plasmon Resonance (SPR), Isothermal Titration Calorimetry (ITC), Nuclear Magnetic Resonance (NMR) and X-ray crystallography. SPR measures the real-time binding kinetics and affinities of protein interactions, ITC quantifies the heat changes associated with binding events, NMR provides structural information about protein complexes and X-ray crystallography enables high-resolution visualization of protein interactions. These techniques contribute to our understanding of the atomic-level details of protein-protein interactions.

Conclusion

Revolutionary chemical biology techniques have significantly advanced our understanding of protein-protein interactions, providing unprecedented insights into their molecular mechanisms. Techniques such as FRET, XL-MS, SPR, BioID and Cryo-EM have enabled researchers to investigate protein interactions in real-time, map interaction interfaces, measure binding affinities and visualize complex structures. As these techniques continue to evolve and integrate with complementary approaches, they hold immense promise for unraveling the intricate web of protein-protein interactions, ultimately leading to novel therapeutic strategies and a deeper understanding of complex biological processes. Techniques such as yeast two-hybrid assays, co-immunoprecipitation, biophysical techniques, chemical cross-linking and fluorescence-based methods have revolutionized our ability to investigate and decipher the complex network of protein interactions. As these techniques continue to advance and new approaches emerge, we can look forward to uncovering deeper insights into the molecular mechanisms driving biological systems, paving the way for innovative solutions to various diseases and biological challenges.

References

1. Braun, Pascal and Anne‐Claude Gingras. "History of protein–protein interactions: From egg‐white to complex networks." Proteomics 12 (2012): 1478-1498.

Google Scholar, Crossref, Indexed at

2. Schenone, Monica, Vlado Dančík, Bridget K. Wagner and Paul A. Clemons. "Target identification and mechanism of action in chemical biology and drug discovery." Nat Chem Biol 9 (2013): 232-240.

Google Scholar, Crossref, Indexed at

3. Viani, Mario B., Lia I. Pietrasanta, James B. Thompson and Ami Chand, et al. "Probing protein–protein interactions in real time." Nat Struct Biol 7 (2000): 644-647.

Google Scholar, Crossref, Indexed at

4. Barsegov, V and D. Thirumalai. "Probing protein-protein interactions by dynamic force correlation spectroscopy." Phys Rev Lett 95 (2005): 168302.

Google Scholar, Crossref, Indexed at

5. Wang, Yong-Cui, Shi-Long Chen, Nai-Yang Deng and Yong Wang. "Computational probing protein–protein interactions targeting small molecules." Bioinform 32 (2016): 226-234.

Google Scholar, Crossref, Indexed at