指導教授：鄭貽生臺灣大學：植物科學研究所吳欣倚Wu, Hsin-YiHsin-YiWu2014-11-282018-07-062014-11-282018-07-062014http://ntur.lib.ntu.edu.tw//handle/246246/262831AtTLP18.3蛋白位於阿拉伯芥類囊體內腔，具有285個胺基酸。依序列分析可分為三段: N端為導引訊息，C端為穿膜的疏水性胺基酸序列以及一段功能未知的區域 (DUF477 domain)。之前研究認為AtTLP18.3是參與光系統II修復機制的輔助性蛋白之一，但其分子機制未明。為了進一步瞭解它的分子功能，本研究利用X光晶體繞射實驗決定AtTLP18.3的立體結構。藉由大腸桿菌系統大量表現已經去除N端導引訊息及C端穿膜區的AtTLP18.3，因分析此序列發現在這段僅帶有功能未知區域的截斷蛋白上並無甲硫胺酸，因此將第128,159位置的白胺酸及異白胺酸以定點突變方法置換成甲硫胺酸，並純化結晶得含重原子硒的甲硫胺酸衍生物晶體，以利用異常色散法取得相角，經過X-光繞射分析，野生型及突變型AtTLP18.3皆屬於正交晶系，所得之晶胞參數皆為a = 46.9, b = 49.8, c =76.7 Å, α=β=γ=90° 所屬空間群為P212121。以單波長異常色散法計算其相角，並進一步修正並取得其蛋白質結構，將此結構以MATRAS、DALI、CATH等蛋白質資料庫進行結構比對，發現其功能可能具有無機焦磷酸水解酶活性之可能，經以酵素活性分析證實其具有酸性磷酸酶之活性。AtTLP18.3 is a thylakoid lumen protein with 285 amino acids. The protein can be divided into three regions based on sequence analysis: a chloroplast transit peptide, a domain of unknown function (DUF477) and a transmenebrane α-helix (TMH). Previous studies indicated that the AtTLP18.3 protein is an auxiliary protein of photosystem II (PSII) repair cycle. In order to clarify the possible molecular function of the AtTLP18.3 protein, the crystal structures of the truncated AtTLP18.3 without targeting signal and TMH were resolved. Since there is no any methionine residue in the truncated AtTLP18.3 protein, we combined the prediction of secondary structure and solvent accessibility and selected leucine (L128M) and isolecine (I159M) residues for methionine substitution. The crystals of native and double mutated AtTLP18.3 shows isomorphous in space group P212121 with unit-cell parameters a = 46.9, b = 49.8, c =76.7 Å, α=β=γ=90°. Finally, the structure of mutant was resolved at a resolution 2.6 Å using single-wavelength anomalous dispersion method, and the native structure was resolved at 1.6 Å resolution. For further structural comparison, the native structure of truncated AtTLP18.3 was submitted to the CATH, DALI and MATRAS database to search similar folding of protein with known function. The results showed that the structure of AtTLP18.3 resembled to various inorganic pyrophosphatase. The enzymatic activity of AtTLP18.3 was further identified by alkaline/acid phosphatase assay. Therefore, we proposed that the function of AtTLP18.3 will act as phosphatase to remove the phosphate group from damage protein for repair cycle.中文摘要 i Abstract ii Table of Contents iii List of figures vii List of tables ix Abbreviations x Chapter 1 Introduction 13 1.1 Photosynthesis 13 1.2 Thylakoid membranes in chloroplast 14 1.3 Photoinhibition 14 1.4 Photodamage and the repair cycle of PSII 15 1.5 Auxiliary proteins in the PSII repair cycle 16 1.6 Chloroplast thylakoid lumen protein—AtTLP18.3 16 1.7 Deducing protein function from structure 17 1.8 Anomalous dispersion for phase problem 18 1.9 Goals for the thesis project 18 Chapter 2 Materials and Methods 20 2.1 Materials 20 2.1.1 Chemicals 20 2.1.2 Kits 20 2.1.3 Enzymes 20 2.1.4 Bacterial strains 21 2.1.5 Plasmids 21 2.1.6 Oligonucleotides 21 2.1.7 Synthetic phosphorylated oligopeptides 22 2.1.8 General buffers, media and solution 22 2.1.9 Bioinformatics analysis 26 2.2 Methods 28 2.2.1 Plant materials and growth conditions 28 2.2.2 cDNA cloning of AtTLP18.3 28 2.2.3 Culturing and storage of E. coli strains 28 2.2.4 Isolation and purification of plasmid DNA from E. coli 29 2.2.5 Determination of the DNA concentration 29 2.2.6 Agarose gel electrophoresis 30 2.2.7 Isolation of DNA fragments from agarose gels 30 2.2.8 Preparation of chemically competent E. coli cells 30 2.2.9 Chemical transformation of E. coli 31 2.2.10 Amplification of DNA fragments by PCR 31 2.2.11 Site directed mutagenesis by PCR 32 2.2.12 Screening Colonies by PCR 33 2.2.13 DNA sequencing 33 2.2.14 Small-scale protein expression test 33 2.2.15 Large-scale protein expression 34 2.2.16 Preparation of selenomethionine-labeled protein 34 2.2.17 Protein purification 35 2.2.18 Determination of protein concentration 36 2.2.19 Storage of purified proteins 36 2.2.20 Sodium dodecyl sulphate-polyacrylamide gel electrophoresis 36 2.2.21 Crystallization 37 2.2.22 Data collection, and structure determination 38 2.2.23 Phosphatase activity assay 39 Chapter 3 Results 40 3.1 Bioinformatics analysis 40 3.1.1 Multiple sequence alignment of AtTLP18.3 40 3.1.2 Sequence analysis of AtTLP18.3 40 3.1.3 In silico characterization of physicochemical parameters 41 3.1.4 Secondary structure and solvent accessibility prediction 42 3.2 Protein expression and purification 42 3.2.1 Optimization of Expression 42 3.2.2 Large-scale protein purification using Äkta prime plus 43 3.2.3 Enzymatic cleavage to remove the GST affinity tag 43 3.3 Residue selection for methionine substitution 43 3.4 Crystallization 45 3.5 Protein structure of AtTLP18.3 45 3.5.1 Structure determination 45 3.5.2 Overall structure 46 3.6 Structure comparison with known function protein 47 3.6.1 CATH classfication 47 3.6.2 MATRAS and DALI servers 47 3.7 Phosphatase activity assay 48 3.7.1 Phosphatase activity Assay using pNPP as substrate 48 3.7.2 Enzymatic kinetics with different substrates 49 3.8 Comparison of Wild-type and mutant 50 3.8.1 Structure comparison 50 3.8.2 Enzyme activity of mutant AtTLP18.3 51 3.8.3 Overall comparison of wild-type and mutants 51 3.9 Structure of the AtTLP18.3-pSer Complex 52 Chapter 4 Discussions 53 4.1 Combining secondary structure and protein solvent accessibility prediction in methionine substitution 53 4.2 AtTLP18.3 is an acid phosphatase in thylakoid lumen 53 4.3 Is AtTLP18.3 involved in the PSII repair cycle? 54 4.4 DUF477 is a novel acid phosphatase domain 54 Chapter 5 Summary 56 References 58 Figures 67 Tables 93 Publications 1075274388 bytesapplication/pdf論文公開時間：2014/07/22論文使用權限：同意有償授權(權利金給回饋本人)新穎酸性磷解酶類囊體內腔蛋白thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/262831/1/ntu-103-F92b42016-1.pdf