Application Of Residual Dipolar Couplings In Biological Macromolecules In The Solution Structure Of Human Mical-1 Protein The Calponin Homolgy Domain And The Domain Structure And Function Of Brd7 Protein Bromodomain Study

Our work focuses on the structural and functional studies of proteins (domains) and correlative NMR methodology. This thesis is divided into three portions. At first, we set up a new method on applying residual dipolar couplings (RDC) to study protein structure, and use it to calculate the structure of SH3BGRL3 and MICAL-1 Calponin Homology(CH) domain. We also study the structure of human MICAL-1 CH domain by NMR, and speculate its potential function. Finally, we determine the solution structure of BRD7 bromodomain by NMR spectroscopy, and study its binding specificity by NMR titration with several acetylated histone peptides. Further, site-directed mutation of BRD7 bromodomain broadens our comprehension on molecular recognition of bromodomain and ligand.We describe application of RDC to study biomolecular structure in Chapter 1. We first provide a brief review of RDC, including its theory, alignment media, pulse program, data processing and structure calculating. We select several alignment media for measuring molecular orientation. Then we set up a series of method to apply the method of RDC. To identify this method, we study SH3BGRL3 protein using IP-AP [¹⁵N,¹H]-HSQC in Pf1 phage. We incorporate RDC into calculate the structure of SH3BGRL3, and compare the calculated structure with and without Pfl phage. Our result shows that application of RDC can impr6ve structural quality, which is mainly revealed by reducing RMSD and increasing residues in most-favoured region of Ramachandran.In chapter 2, we report the research result on the structure and function of human MICAL-1 CH domain. At first, we give a review of MICAL and CH domain. MICAL was first identified as CasL interacting protein. MICALs can interact with plexin and be involved in repulsive axon guidance. Recently, MICALs were also identified as rab1 interacting proteins, the cellular localization of which depends on microtubules. MICAL is composed of several domains, however, the function of CH domain is not clear. CH domain is an actin-binding domain. It only binds to filament actin. The structure of CH domain is made up of helices. Besides the binding to filament actin, some of CH domains play an important role in signal transduction and cytoskeleton regulation. Recombinant MICAL-1 CH was cloned, expressed in E. coli and purified by Ni-chelating column. The three-dimensional structure of the MICAL-1 CH was determined by NMR method based on NOEs and residual dipolar couplings experiments. The three-dimensional structure of the MICAL-1 CH is made up of 67% helices, six a helices and a 3₁₀ helix. Many hydrophobic residues form two main hydrophobic cores between these helices to maintain stability of whole domain. By chemical shift perturbation experiment we found MICAL-1 CH can't interact with F-actin alone and the reasons were explained based on sequence alignment, structural comparison and hydrophobic surface analysis. Finally, potential functions of MICAL-1 CH were discussed. It might interact with microtubule proteins, which needs further confirmation.Chapter 3 introduces the work on solution structure of BRD7 bromodomain and its interaction with acetylated peptides from Histone H3 and H4. Firstly, we review the BRD7 protein and bromodomain. Bromodomain is an evolutionarily conserved protein module of about 110 amino acids and associated with chromatin proteins. Bromodomain can specifically bind to acetyllysine of N-terminal of histone, and deliver the signal of acetylated of histone involved in chromatin remodeling and transcriptional activation. BRD7 is an important human bromodomain-containing protein tightly associated with Nasopharyngeal carcinoma (NPC). Previous studies have suggested that BRD7 is downexpressed in NPC biopsies and cell lines[1]. Moreover, overexpression of BRD7 inhibits cell growth and cell cycle progression from G1 to S phase of NPC cell by transcriptionally regulating the cell cycle related genes. BRD7 was also found to form a triple complex with adenovirus nuclear protein E1B-AP5 and histones, which represses the transcription activity of E1B-AP5. BRD7 has been reported to interact with the transcription factor, interferon regulatory factor 2. Recombinant BRD7 bromodomain was cloned, expressed in E. coli and purified by Ni-chelating column. We determine the solution structure of BRD7 bromodomain by NMR. The solution structure of BRD7 bromodomain is made up of five a helices and a 3₁₀ helix, noβ-sheet. BRD7 bromodomain contains the typical left-handed four-helix bundle topology, which is highly conserved in the bromodomain family. The four antiparallel a-helices are sequentially named αZ, αA, αB, and αC, respectively, from the N-terminal. We find that BRD7 bromodomain can bind to peptides from histone H4 with K8, K12 or K16 acetylated with weak affinity, as well as peptides from histone H3 with K9 or K14 acetylated. Moreover, we obtain dissociation constants of the complexes of BRD7 bromodomain with these peptides by fitting the titration curves. In our mutagensis study, the interaction between peptide residues near the acetylated-lysine and the mutated residues of BRD7 bromodomain are not as strong as in the case of other bromodomain. The broad range of acetylated histone ligands and low binding affinity imply that BRD7 bromodomain is significantly differed from other bromodomain.