稻米Bowman-Birk蛋白酶抑制素與牛胰蛋白酶複合物晶體結構與功能分析之研究

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研究生：	李欣黛 Hsin-Tai Li
論文名稱：	稻米Bowman-Birk蛋白酶抑制素與牛胰蛋白酶複合物晶體結構與功能分析之研究 Crystal Structure and Functional Study of the Bowman-Birk Inhibitor from Rice Bran in Ternary Complex with Bovine Trypsin
指導教授：	吳文桂 Wen-Guey Wu 陳俊榮 Chun-Jung Chen
口試委員:
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 生物資訊與結構生物研究所 Institute of Bioinformatics and Structural Biology
論文出版年：	2007
畢業學年度：	95
語文別：	英文
論文頁數：	81
中文關鍵詞：	單子葉植物、蛋白酶抑制素、植物與病蟲害系統、稻米BBI
外文關鍵詞：	Bowman-Birk Inhibitor, monocotyledonous plant, reactive-site loop, protease-inhibitor, plant-pest systems, RBTI
相關次數：	點閱：3 下載：0
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從米糠純化得到的稻米胰蛋白酶抑制素（簡稱RBTI）是屬於Bowman-Birk抑制素（BBI）家族的一員，其分子量為15kDa。本研究中，一個RBTI分子與一個牛胰蛋白酶分子結合之複合物晶體結構是利用分子置換與電子密度修飾兩種方法而決定，在解析度至3 Å的繞射數據裡得到剩餘值為26.2%與自由剩餘值為31.6%。RBTI由七個平行折板與環線組成，不具有螺旋之二級結構，並且折疊成兩個分子量相當、較小的區域（稱為N-或C-區域），這兩個區域分別與大麥BBI具有相似的三級結構。但是RBTI兩個區域之間的方向連結與大麥BBI兩個區域之間的方向連結差異極大，這使得RBTI位於兩區域內的兩個與胰蛋白酶反應殘基相距只有23 Å，然而在大麥的例子裡，此距離為40 Å。這個較近的距離提供了證據支持活性測試的結果，RBTI兩區域裡的兩個分別的反應殘基具有不同的能力抑制胰蛋白酶，而位於RBTI C-區域P1’位置之向外指出的Met殘基是破壞反應區域的典型標準構造的主要原因。因此，RBTI主要與胰蛋白酶作用的區域為N-區域，但是C-區域也扮演了輔助的角色。

The 15-kDa trypsin inhibitors from rice bran (RBTI) are members of the Bowman-Birk protease inhibitor (BBI) family. The crystal structure of a 1:1 complex between RBTI and bovine pancreatic trypsin (BPT) was determined by combination of molecular replacement and electron density modification methods. This complex model has been refined to a crystallographic R-factor of 26.2% and free R-factor of 31.6% at 3.0 Å resolution. The RBTI structure consists of seven beta-strands and loops without alpha-helices structure and folds into two compact domains (N- and C-domain) which are similar to each domain from barley BBI. However, orientation between two domains is quite different to barley BBI, this makes the distance between two P1 residues (17Lys and 83Lys) in RBTI is only 23 Å apart rather than distance of 40 Å in barley BBI. The closer distance provides evidence to support result from activity assay that two domains show different abilities to inhibit trypsin. RBTI C-domain with protruding 84Met at P1’ position, moreover, mainly leads into breakdown of the classically canonical conformation of reactive site loop. Major interaction with BPT is achieved by RBTI N-domain but C-domain plays an auxiliary role to block trypsin molecule.

Chapter 1  Introduction                                  1
1.1    Inhibitor-proteases in plant-pest systems       1
1.2    Bowman-Birk Inhibitors in plants                2
1.2.1    BBIs from dicotyledonous plants                2
1.2.2    BBIs from monocotyledonous plants                4
1.3    Rice Bran Trypsin Inhibitor                         5
1.4    Trypsin                                           8
1.5    Aim of this study                                 10

Chapter 2 Materials and Methods                        11
2.1    Protein Purification and identification      11
2.2    The method of inhibitory activity assays      12
2.3    Protein Crystallization                        14
2.3.1    Methods of protein crystallization               15
2.3.2    Crystallization of RBTI-BPT complex crystals      16
2.4    X-ray data collection and processing               18
2.4.1    Crystal selection and mounting               18
2.4.2    Data collection                                 20
2.4.3    Data processing                                 20
2.5    Phase problem and different methods for phase
         problem                                          22
2.6    Density modification and phase improvement      23
2.7    Structure modeling, refinement and validation      24
2.7.1    Model building                                 24
2.7.2    Refinement                                 24
2.7.3    Validation                                 25
2.8    Solving the phase problem by molecular
         replacement                                 26
2.9    RBTI-BPT complex structure determination by
         combination of MR and DM methods               27
2.9.1    Finding the position of BPT in complex
         structure                                          27
2.9.2    Slotting N-domain of barley BBI model into
         the electron density map as the initial phase
         for N-domain of RBTI                        28
2.9.3    Placing C-domain of barley BBI model into the
         electron density map as the initial phase for
         C-domain of RBTI                                 30

Chapter 3 Results and discussion                        31
3.1    Inhibitory activity assay                        31
3.2    RBTI-BPT complex model quality               31
3.3    Overall structure of RBTI                        33
3.4    Interaction between RBTI and BPT               36
3.5    Comparison of the RBTI and other BBIs
         structure                                          38
3.5.1    Sequence comparison between monocotyledonous
         and dicotyledonous BBIs                        38
3.5.2    Comparison of domains between RBTI and barley
         BBI or soybean BBI                                 39
3.5.3    Comparison of overall structures between RBTI
         and barley BBI                                 41
3.6    Inhibitory loops of RBTI and conformation and
         comparison with other inhibitors               43
3.6.1    Reactive site loop of RBTI N-domain               43
3.6.2    Reactive site loop of RBTI C-domain               45

Chapter 4 Conclusion                                 47

References                                          49
Tables and Figures                                          53

                                

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