| The interactions between peptide-peptide and peptide-protein ubiquitously affect protein behaviors and play an essential role in drug discovery, protein engineering, biosynthesis and bionanomaterial design etc. Many strategies, such as Forster resonance energy transfer (FRET), Enzyme-linked immunosorbent assay (ELISA) and microarray, have been extensively applied to the study of this fundamental field in the past few decades. In this thesis, I briefly introduce our own methodology of investigating this kind of interaction, as well as its application in cell imaging and material science. The balance of specificity and reactivity has always been a big issue in targeted recognition. The conventional bulky tag or probe, such as SNAP tag and antibody, would probably, if not definitely interfere the structure and function of the parental protein. Besides, the low affinity and reversibility of many targeted models are not that robust to the ever changing biological environment. In this thesis, I'm going to elucidate the methodology we explored to develop an innovative covalent targeting system with high selectivity. As it is known, coiled coil, a well characterized secondary structure existing in abundant proteins and peptides, participates in many bioregulation processes. It is a super-helical motif formed by two or more alpha-helices peptides, each containing a primary repeating heptads of (abcdefg)n in either parallel or anti-parallel model. By mutating the two key amino acids in the dimeric parallel coiled coil system, the inter-molecular covalent linkage could be achieved specifically and irreversibly within tens of minutes, making it a good candidate of chemical probe and tag system in biorecognition and labeling aspects. Following this observation, genetically fusing one of the coiled coil peptide to the extracellular terminus of G-protein coupled receptors (GPCRs) and epidermal growth factor receptor (EGFR), would introduce the tag peptide to the membrane of mammalian cells and thus made the receptor recognizable and traceable to the artificially synthetic peptide probe containing a fluorescent moiety. In addition, I extended the application of peptide-protein interaction in nano particle surface fictionalization. Quantum dot (QD) is a novel fluorescent nanomaterial, which was widely used in transistors, solar cell and medical imaging, etc. With the assistance of the de novo designed dimeric peptide ligand, mCherry fused Tax-interacting protein-1 (TIP-1) could self-assembly on the surface of ZnS-CdSe quantum dots, dramatically enhanced the stability of QDs to imidazole replacement. Moreover, Histag-mCherry fused ubiquitin-like domain (ULD) protein would self-assembly into a tetramer due to the tetrameric propensity of ULD and bind to QDs with the controlled stoichiometry, which was not easy to achieve in the traditional surface chemistry. A novel protein mCherry-nanobelt was also designed to exhibit the predicted interaction with QDs in a controlled manner. |
