Study On Structure And Mechanism Of Npu DnaE Intein And Preparation And Application Of Protein Dual Mode Imaging Probes

This dissertation focuses on the protein research, including protein’s structure, protein’s mechanism, protein’s function and protein’s application. These investigations cover the following two parts:(1) Identification of the structure and splicing mechanism of Npu DnaE intein. The 3D structures of Npu DnaE intein have been solved by X-ray and NMR, respectively. However, both structures are cis-structures because the two split domains were artificially fused into a continuous sequence, which probably leads to subtle rearrangements of protein structure. We established a co-expression system to obtain the two splicing domains (NpuN and Npuc) simultaneously at 1:1 ratio in vivo. This allowed the two domains bind to each other and fold correctly into a native Npu DnaE intein protein complex. Guided by the structure information and a series of mutation assays, NArg50 and cSer35 are identified to be critical for protein splicing. (2) Characterization of the function and application of GFP36+-dLBT in tumor imaging. We designed a novel fluorescence/MR dual imaging probe by protein engineering. And the bimodal imaging efficiency of GFP36+-dLBT fusion protein was tested in vitro, in vivo and in mice. These results suggested that GFP36+-dLBT fusion protein retained the function of each imaing motif, hence is suitable for fluorescence and MR imaging in vivo. Additionally, GFP36+-dLBT fusion protein could passively target tumor by EPR effect to achieve specific imaging effect in mice.Chapter 1 provides the background knowledge of interns. Section 1 describes the discovery, occurence, nomenclature and classification of inteins. Section 2 focues on the intein’s strucuture (from amino acid sequence to 3D strucutures). Section 3 reviews the splicing mechanism of three different types of inteins. Part four is a summary of the latest application progress of inteins. This charpter emphasizes the stucutre and splicing mechanism of inteins.Chapter 2 is the investigations of structures and splicing mechanism of Npu DnaE intein. A co-expression system was established to obtain the N and C-terminal of Npu DnaE intein simultaneously. We solved the first trans-structure using this native Npu DnaE intein protein complex. The premise of trans-splicing is that the N and C-terminal domains could bind to each other and assembly into an efficient splicing precusor. Gudied by the structure, both electrostatic interactions and hydrophobic foces are observed in the asscoation regions, suggest that they are the driving forces for intein fragments recognization and protein complex assembly. By sequencing alignment, both NArg50 and CSer35 residues are well conserved in DnaE intein family that lacks a penultimate histidine residue. And the trans-structures show that NArg50 and CSer35 are folded into splicing center and formed H-bonding to conserved catalytic residues. The C-terminal asparagine CAsn36 of Npu DnaE intein exhibits two orientations of its side-chain and interacts with both NArg50 and CSer35 through H-bonding. The in vitro splicing assays demonstrate that mutation of either residue reduces intein activity, while double mutation of both NArg50 and CSer35 decreases the splicing rate even further. Hence, these results suggest that NArg50 and CSer35 synergistically modulate the protein trans-splicing efficiency. On the other hand, NArg50 also forms a H-bond with the highly conserved F-block aspartate CAspl7, thus influencing the N-S acyl shift during N-terminal cleavage. And in vitro cleavge assay indicates that the mutation of NArg50 would lead to the accumulation of the N-terminal cleavage side products, thus influencing the splicing efficiency. In conclusion, the conserved non-catalytic residues (NArg50 and CSer35) of Npu DnaE intein could modulate the efficiency of protein trans-splicing by H-bond interactions with the catalytic residues at the splice junction.Chapter 3 provides the brief introduction and recent progress of biology imaging. Part one summarizes the regular imaging techniques and compares the advantage and limitations between each imging methods. Part two describes the recent progress of dual modal imaging probes.Chapter 4 is about the preparation and function investigations of GFP36+-dLBT fusion protein. We propose a protein engineering-based strategy for the construction of a bimodal probe for fluorescence and magnetic resonance imaging. A recombinant protein was generated by fusion of supercharged green fluorescence protein (GFP36+) with a lanthanide-binding tag (dLBT) that can stably bind two Gd3+ions. The GFP36+-dLBT fusion protein showed strong fluorescence and exhibited efficient contrast enhancement in magnetic resonance imaging. This protein probe improves the MR relaxation more efficiently than the Gd-DTPA (gadopentetate dimeglumine). The superior cell-penetrating activity of GFP36+ allows efficient cellular uptake of this fusion protein and can thus be used as a cellular imaging probe. Dual imaging was conducted in vitro and in mice. This result indicates that the fusion of different functional domains is a feasible approach for making multi-modal imaging agents.