The Man Coactosin Like Protein And Mical <sub>, 1 </ Sub> The Calponin Homolgy Domain Structure And Function

Our work focuses on the structural and functional studies of two important proteins (domains). The first one is human coactosin like protein (hCLP). The solution structure of hCLP was determined by NMR spectroscopy. Guided by the structure, a series of site-directed mutants were generated and their F-actin binding activities were measured by high-speed co-sedimentation assays. Furthermore, the structural model of the hCLP-F-actin complex was proposed using computational docking with the docking results filtered by the mutation data. The extended region of β4- β5 of hCLP (residue 66-75) was found to be very flexible and the flexibility of this region is very important for F-actin binding. Another one is calponin homology (CH) domain of human MICAL₁. We determined the three-dimensional structure of MICAL1 CH by NMR. Based on sequence alignment and hydrophobic surface analysis, potential functions of CH domain of human MICAL-1 were discussed.Chapter 1 provides a brief review of actin depolymerization factor homology (ADF-H) domain-containing protein family, which is very important in regulating actin dynamics, especially in the turnover rate of actin. ADF-H domain-containing protein can be subdivided into four classes. The structure and functions of the four classes of ADF-H domains were reviewed.In chapter 2, NMR, site-directed mutation, high speed co-sedimentation and computational docking were used to study the solution structure of hCLP and its binding model with F-actin. hCLP can bind to actin filaments but not globular actin and belongs to the fourth class of ADF-H-domain-containing proteins. Human CLP can also bind to 5-lipoxygenase (5LO), which plays an important role in cellular leukotriene synthesis. Recombinant hCLP was cloned, expressed in E. coli and purified by Ni-chelating column. Then the three-dimensional structure and backbone dynamics of hCLP were studied using multidimensional NMR spectroscopy. Guided by the structure of the protein in solution, a series of site-directed mutants were generated and their F-actin binding activities were measured by high-speed co-sedimentation assays. Several previously untested residues in hCLP are found...