Developing A Novel Quantitative Method For In Vivo Protein Interactome And Its Application

There are large number of in vivo protein-protein interactions(PPIs)that play the important roles in cellular functions.Researchers first pay attention to the protein-protein direct interactions and develop several related methods such as Yeast Two-Hybrid(Y2H)and Fluorescence Resonace Energy Transfer(FRET).On the other hand,there are not only the extensive pairwise interactions,but also the more complicated interaction networks that are called "interactome".Thus how to quantify the in vivo protein interactome is a major challenge in elucidating the molecular mechanisms of complex biological systems.Various protein interactome methods have been developed,such as High-throughput Y2H and Affinity purification/Mass spectrometry(AP/MS).Although these methods are widely used to study the PPIs in living cells,they have practical limitaions for quantitation,high-throughput or dynamic measurement.In the present study,we first developed a new method called Protein-dimerization Footprinting(PdF).The basic principle of PdF is that a DNA binding domain is fused to a target protein,and then the interaction between target proteins in living cells results in the dimerization of fusion proteins,which makes the DNA binding domains bind to the specific DNA sequence with high affinity,thereby protecting the DNA sequence against DNase I digestion.Through this process,a physical PPI is transcoded into a specific DNA sequence that can be detected by quantitative PCR,sequencing or other nucleic acid-based technologies using the copy number to quantify physical interaction intensity.Based on the PdF,we further developed a new method for systematically reveal the dynamic changes of in vivo protein interactome,which is called Protein-interactome Footprinting(PiF).The basic principle of PiF is that different orthogonal pairs of DNA binding domains and specific DNA sequences are constructed,and then applied for simultaneously detecting multiple PPIs in one cell and quantitatively measuring the correlations of PPI dynamics in the protein interactome for the first time.Using PiF method to measure correlations between different PPIs in the cell,we advanced and drew the PPI Correlation Network(PPICN)of in vivo protein interactome.In this special network,each vertex represents a physical PPI,and each edge connecting two vertices represents the correlation of two PPIs.Furthermore,we also combined microfluidic technology with PiF method to develop the microfluidic chip based PiF(mPiF)for further improving detection throughput.To further demonstrate the application of PiF and mPiF in biological complex systems,we used different approaches to study the PPICNs and related biological processes in E.coli chemotaxis protein complex and two-component signal transduction systems.Combining the mathematical models of PPI dynamics with the modular response analysis,we drew the PPICN of E.coli chemotaxis protein complex and revealed the intrinsic properties of complex assembly and disassembly within a protein network by giving the correlation of different physical PPIs.Combining the optogenetics,the transcriptome sequencing with the information theory,we measured the PPICN of E.coli two-component systems and described the relation between the information transfer abilities and the gene expression regulations of cellular signal transductions through the PPI correlations.Above all,we developed DNA sequence recognition based methods,i.e.PdF and PiF for studying in vivo protein interactome.The PPI-DNA transcoding principle provides a solution for the quantitative high-throughput detection of in vivo PPIs.The concept of the PPI correlation and the PPICN we proposed here can help us understand the roles of PPIs in biological complex systems.