The focus of our research is to develop novel technologies for protein manipulation in living cells, which include protein-based bioorthoganol chemistry and precise protein engineering. The aims of these research programs are to utilize the developed valuable tools to address fundamental questions in life sciences. The specific projects are summarized below:

Protein photo-cross-linking, via the introduction of non-natural amino acids, has emerged as a superior strategy for covalently trapping transient protein-protein interactions in living conditions, which is generally not feasible using other methodologies. This method would be particularly suitable for detecting protein-protein interactions in the periplasmic space of bacteria. We recently developed a highly efficient protein photo-cross-linking probe which allowed us to identify thein vivoclient proteins of an acid-stress chaperone, HdeA, in theE. coliperiplasm (Fig. 1). This probe exhibits a high adaptability to protein conformational changes and excellent cross-linking fidelity. It may be well-suited for profiling protein-protein interactions in various cellular compartments of living cells, particularly when only limitedstructuralinformation is available.

Fig. 1 The development of the pyrrolysine-derived diazirine-containing photo-cross-linking probe, DiZPK (a), for detecting protein-protein interactions in theE. colicell envelope (b).

[Reference]: Zhang M, et al. A genetically incorporated crosslinker reveals chaperone cooperation in acid resistance Nat. Chem. Biol.,20117, 671-677.

Currently, we are using this strategy to study the infectious and defense mechanisms of bacterial pathogens, which include: developing protein labeling and manipulation tools to trap protein-protein interactions upon bacterial infection of host cells; identify the host targets of bacterial toxic proteins; understand the antibiotic resistance mechanisms and the escaping strategy of bacteria from host immune responses; study the pH-mediated endocytosis pathway of bacterial pathogens and virulent toxins upon entry into host cells.

Organic Hydroperoxide (OHP) is a highly reactive form of ROS with distinct functional roles from other ROS species including H2O2, and it has also been implicated in the pathogenesis of a panel of chronic diseases such as neurodegenerative diseases and cardiovascular diseases. However, in contrast to the extensive study on the physiological roles of H2O2, the lack of a selective intracellular probe for OHPs has prevented us from fully elucidating their effects at the molecular level. In 2010, our group reported the first fluorescent probe for highly selective visualization of organic hydroperoxides (OHPs) such as CHP and TBHP against H2O2 in living cells (Fig. 2). This strategy replies on the insertion of a circularly permuted fluorescent protein Venus (cpVenus) into the oxidative-responsive region of OhrR, an OHP-specific regulatory protein. This precisely engineered construct, termed OHSer, allowed the selective imaging of OHP production in living cells. This probe will allow us to selective study the physiological and pathological roles of OHPs in a native cellular context.

Fig. 2.A Highly selective fluorescent probe for visualization of Organic hydroperoxides in living cells.

[Reference]: A Highly selective fluorescent probe for visualization of Organic hydroperoxides in livi6ng cells . Zhao, B, etal.J Am Chem Soc2010.132(48), 17065-17067.

Currently, we are investigating the virulence effects and the regulation mechanisms of reactive oxidative species (ROS) and transition metal ions. Develop biosensors for ROS and transition metal ions (e.g. copper and zinc) in living mammalian cells; study the virulent mechanism of organic hydroperoxides in neuronal cellsl; verify the beneficial effects of ROS on mammalian cells; examine the physiological and pathological roles of transition metal ions in neuronal cells; understand the consequence of misregulated ROS and heavy metal ions on human aging diseases.

One of the key challenges on using the chemical strategy for protein labeling is to selectively incorporate the bioorthogonal chemical groups into the target proteins, which allows the subsequent conjugation with probes bearing the complementary functionalities. These bioorthogonal groups have to be non-native, non-perturbing in order to satisfy the aforementioned stringent bioorthogonal requirements. A variety of strategies have therefore been developed in recent years towards installation of these chemical handles into proteins of interest, typically in the form of the substrates of protein modification enzymes or unnatural amino acids (UAAs).

A Pyrrolysine-based genetic-code expansion platform has recently been established in our lab at PKU, which allows the synthesis, evolution and encoding of UAAs in both prokaryotic and eukaryotic proteins. Recently, a concise and highly efficient route was developed for the synthesis of a cyclic pyrrolysine analogue bearing an azide handle (ACPK). Directed evolution enabled the encoding of this non-natural amino acid in both prokaryotic and eukaryotic cells with great efficiency. This offers a facile and cost-effective approach for the site-specific introduction of an azide group into target proteins that can be easily accessed by alkyne probes using click chemistry (Fig.3).

Fig. 3Bioorthogonal Protein Labeling in Living cells. (a) A two-step protein labeling in living systems, (b) The Pyrrolysine-based system serves as an One Stop-Shop for introducing UAAs into proteins in both Prokaryotic and Eukaryotic cells. (c) A facially synthesized Azide-pyrrolysine analogue for protein click labeling.

[References]:

Hao, Z.et al, Introducing Bioorthogonal Functionalities into Proteins in Living Cells .Acc. Chem. Res.,2011. DOI: 10.1021/ar200067r

Hao, Z.; Song, Y.; Lin, S.; Yang, M.; Liang, Y.; Wang, J.; Chen, P. A readily synthesized cyclic pyrrolysine analogue for site-specific protein click labeling .Chem Commun (Camb)2011.47(15),4502-4504.

Lin, SX.; Zhang, ZR.; Xu, H.; Li, L.; Chen, S.; Li, J.; Hao, ZY.; Chen, P*.; Site-Specific Incorporation of Photo-Cross-Linker and Bioorthogonal Amino Acids into Enteric Bacterial Pathogens ,J. Am. Chem. Soc.,2011, 133(50), 20581-20587

Yang, M.; Song, Y.; Zhang, M.; Lin, S.; Hao, Z.; Liang,Y.; Zhang, D. and Chen, P.*; Converting a solvatochromic fluorophore into a protein-based pH indicator for extreme acidity ,Angew. Chem. Int. Ed.,2012, DOI: 10.1002/anie.201204029