阿拉伯芥類囊體內腔新穎酸性磷解酶之晶體結構及功能分析

AtTLP18.3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。

關鍵字

新穎酸性磷解酶；類囊體內腔蛋白

並列摘要

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.

並列關鍵字

novel acid phosphatase ； thylakoid lumen protein

參考文獻

Adamczak, R., Porollo, A., and Meller, J. (2005). Combining prediction of secondary structure and solvent accessibility in proteins. Proteins: Structure, Function, and Bioinformatics 59, 467-475.

Altschul, S.F., Madden, T.L., Schäffer, A.A., Zhang, J., Zhang, Z., Miller, W., and Lipman, D.J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic acids research 25, 3389-3402.

Aro, E.-M., Virgin, I., and Andersson, B. (1993). Photoinhibition of photosystem II. Inactivation, protein damage and turnover. Biochimica et Biophysica Acta (BBA)-Bioenergetics 1143, 113-134.

Asada, K. (1999). The water-water cycle in chloroplasts: scavenging of active oxygens and dissipation of excess photons. Annual review of plant biology 50, 601-639.

Baena-González, E., Barbato, R., and Aro, E.-M. (1999). Role of phosphorylation in the repair cycle and oligomeric structure of photosystem II. Planta 208, 196-204.

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阿拉伯芥類囊體內腔新穎酸性磷解酶之晶體結構及功能分析

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