利用核磁共振技術研究人類鈣離子結合 S100A12 蛋白質水溶液結構以及其與 Siah-1 作用蛋白質(SIP)中序列189-219 之間交互作用的探討

NMR Structure of Calcium-bound Human S100A12 Protein and its Interaction with Sequence 189-219 of Siah-1 interacting Protein(SIP)

許展嘉

指導教授 ： 余靖

國立清華大學/理學院/化學系/碩士(2013年)

摘要

在生物系統中的許多蛋白質有其獨特的作用區域，可與特定的分子結合，決定蛋白質的活性與功能。這些作用的分子(統稱為ligands)包含蛋白、胜肽、金屬離子及藥物等等…。本篇論文進行的研究案例，即是蛋白質與胜肽之交互作用。
人類S100A12蛋白是S100蛋白質家族之一份子，在水溶液狀態下為一同質雙聚體蛋白，可藉由與鈣離子結合改變其構形，進一步與目標蛋白交互作用；SIP為一種Siah-1作用蛋白，與配體結合後，可以調控細胞訊息傳遞，以進行泛素化水解的工作。在本篇論文中，藉由兩種蛋白質間的交互反應，探討其形成複合物之後的結構特性。
我們利用二維1H-15N HSQC滴定實驗找出S100A12與SIP可能的結合位置，並且藉由螢光實驗以及圓二色光譜儀，探討兩者間的交互作用。搭配三維核磁共振實驗HNCA, HN(CO)CA, HCCH-TOCSY以及HCCH-COSY等來完成蛋白質的化學位移判定，並利用實驗所得的距離、雙面角、氫鍵限制條件經由軟體計算，解出人類鈣離子結合S100A12水溶液結構，以及S100A12與SIP作用後的複合物水溶液結構。從實驗結果歸納出，S100A12與SIP以1：1之比例結合形成複合物，此研究幫助我們更了解S100家族與SIP反應情形。然而，後續仍需更進一步研究，以探討此反應是否與實際生理情形相關。

關鍵字

核磁共振 ； 結構計算 ； 蛋白質水溶液結構 ； S100A12蛋白質

並列摘要

Many proteins in biological systems have specific binding sites for certain molecules, and these binding events are critical to the protein’s activity and function. These molecules, often called "ligands" include proteins, peptides, metals and drugs and so on. In this thesis, the case which we study is protein-peptide interactions.
Human S100A12 protein belongs to S100 protein family which shows a homodimer in solution. After binding with calcium, S100A12 undergoes a calcium-induced conformational change and interacts with target protein. SIP is one kind of Siah-1 interacting protein. It controls cellular signal transmitting and ubiquitination via interacting with several ligands. We study the interaction between S100A12 and SIP, and we’d like to realize the complex’s characteristics.
According to 1H-15N HSQC titration, the binding sites between S100A12 and SIP can be found. Furthermore, through fluorescence and circular dichroism spectrums to study the protein-protein interaction, and chemical shift assignments were made from HNCA, HN(CO)CA, HCCH-TOCST and HCCH-COSY, etc. The solution structure of human calcium bound S100A12 and complex-form S100A12 were determined by ARIA/CNS software, using the experimental restraints such as distance, dihedral angle, hydrogen bond. We make a conclusion that binding ratio between S100A12 and SIP is 1:1 to form the complex. However, Further studies would be necessary to characterize this interaction in more detail and to investigate if this interaction is physiologically relevant.

參考文獻

[8] Donato, Rosario. Microscopy research and technique 2003, 60, 540-551.

[11] K. Yamashita, Y. Oyama, T. Shishibori, O. Matsushita, A. Okabe, R. Kobayashi,

Protein Expression Purif. 1999, 16, 47–52

[16] Moroz, O.V., Blagova, E.V., Wilkinson, A.J., Wilson, K.S., Bronstein, I.B., Journal of Biological Chemistry 2009, 391, 536-551.

[20] Gabriela Schneider, Anna Filipek, Amino Acids 2011, 41, 773–780.