Structure And Function Of The UBX Domain Of UBXD2 Protein And NMR Investigation Of Interaction Of Dendrimer And Bioactive Surfactants And High Throughput Screening Of Dendrimer-binding Drugs

This thesis is divided into two portions. The first portion focuses on the structural and functional studies of human UBXD2 UBX domain by Nuclear Magnetic Resonance (NMR) method. This protein is cloned, expressed, purified and its solution structure is determined. Based on the NMR structure of UBXD2 UBX, the interaction between UBXD2 UBX and VCP VCP-N is preliminarily studied. And in the process of determination, a cis-proline is found which located in the key region contributed to the interaction to the VCP protein. The second portion is concentrated on the investigation of the interaction of dendrimer and bioactive surfactants and dendrimer-based high-throughput screening (HTS).Chapter 1 introduces the structural and functional studies of human UBXD2 UBX domain. It begins with a short summary on UBXD2 protein and UBX domain containing protein. UBXD2 which renamed erasin was first identified in the endoplasmic-reticulum-associated protein degradation (ERAD). Biological experiments confirm that UBXD2 binds the VCP protein by its UBX domain. UBX domain is the unique domain of UBXD2 protein. It is found that a R...FPR motif which is highly conserved in many other UBX domains is the key interaction region. This loop region is much shorter in ubiquitin protein. Recombinant UBXD2 UBX domain was cloned, expressed in E. coli and purified. The three dimensional solution structure was determined by traditional NMR experiments. UBX domain of UBXD2 is made up of twoαhelices and 4βsheets. Based on the UBX structure, two cis-prolines are found and one of them is located in the FP motif. The evidence for specific interaction between UBXD2 UBX domain and VCP VCP-N domain is provided by the chemical shift perturbation experiments. The binding interaction of UBXD2 UBX to VCP VCP-N is weak and in the fast-exchange limit on the NMR timescale. Because of the conserved three-dimensional structure of UBX domain homologies, we obtain the complex structure of UBXD2 UBX domain and VCP VCP-N domain by computer simulation, with the help of crystal complex structure of p47 UBX and VCP VCP-ND1. The interaction detail of UBXD2 protein and VCP protein can be primely interpreted by this complex structure.Chapter 2 deals with the investigation of interaction of dendrimer and bioactive surfactants and dendrimer-basic high-throughput screening (HTS). Firstly, the NMR techniques which were frequently employed to study the interactions between dendrimers and chemical molecules are introduced. These spectra can offer much useful information about the interaction detail of dendrimers and chemical components, binding sites, binding strength, the type of binding force, space orientation and so on. Then, we use NMR to investigate the complex structure of dendrimer and deoxycholic acid. The results suggest that deoxycholic acid locates in the hydrophobic pockets of dendrimers instead of binding in the surface. It indicates that the aggregation formation between dendrimers and deoxycholic acid is driven by hydrophobic interaction and hydrogen-bond interaction. From the 2D-NOESY, we obtain the orientation of deoxycholic acid in the interior pockets of dendrimer. And this hydrophobic encapsulation of deoxycholic acid by dendrimers is guest size-dependent and hydrophobic property dependent. It is proved by the similar research results of dendrimers and CHAPS, which possess the same steroidal core but the size of CHAPS (614 Da) is much larger than that of deoxycholate (392 Da). CHAPS molecule may exceed the size encapsulation limit of a G5 dendrimer. On the basis of the investigation of dendrimer-deoxycholic acid/CHAPS host-guest system, we develop NMR methodology on dendrimer-based drug high-throughput screening. Regular and new NMR experiments are applied for the fast screening of dendrimer-binding drugs. We design two screening strategies for insoluble and soluble drugs. To further reduce the experimental time in the HTS experiment in the 2D trNOE spectrum, we use the Hadamard-encoded cross-relaxation spectroscopy to speed up the HTS of dendrimer-binding guest molecules. The Hadamard-assisted NOE spectrum provides the same information with reduced experimental time from whole days to several minutes, which is essential in the HTS of dendrimer-binding guests. Based on this point, we can easily get higher signal to noise ratio (S/N) in the Hadamard-encoded NOE spectrum by increasing experimental time. It offers several features for the design of dendrimer-based drug formulations. The findings obtained in this study may help with the generation of a large family of guest molecules which may benefit from the dendrimer inclusion or binding technique. Also, the methodology developed for the dendrimer system is essential for fast discovery of new host-guest system and molecular recognition process.