Dynamics of Calmodulin/MARCKS Complex Studied by NMR Spectral Density Analysis

利用核磁共振頻譜密度分析調鈣素和肉豆蔻酸化之富含丙氨酸蛋白質激酶C受質的複合物的動力學研究

陳姵君(Pei-Chun Chen)

指導教授 ： 張壽麟

國立清華大學/生命科學院/生物資訊與結構生物研究所/碩士(2008年)

摘要

Calmodulin (CaM) is a ubiquitous Ca2+ binding protein that plays a key role in numerous cellular Ca2+-dependent signaling pathways and exists broadly in eukaryotes. CaM folds into two separate globular domains in solution, namely the N- and C-terminal domain. Each domain consists of two helix-loop-helix motifs (EF-hand). The central helix connecting the two domains in CaM is flexible, allowing the N-domain and C-domain to adopt various relative orientations and separation in solutions. Recently, many natively unfolded proteins have been reported. These proteins are highly flexible with no average structure in solution. Myristoylated alanine-rich C kinase substrate (MARCKS) is one of such natively unfolded proteins and a prominent substrate for protein kinase C (PKC) and CaM. MARCKS can mediate crosstalk between the PKC and CaM signal transduction pathways. The MARCKS and Calmodulin interaction is differed from other globular-type target-CaM complex. In this study, the dynamics of CaM upon binding of the natively unfolded substrate, MARCKS, is characterized by NMR spectral density analysis of 15N NMR relaxation data. The dynamics of CaM in bound and unbound forms is compared. The results show that the N- and C-domain, and central helix of free CaM exhibit distinct dynamics. However, such distinction in dynamics is diminished in the presence of MARCKS. Moreover, the backbone flexibility on the fast time scale is in general reduced upon substrate binding with the exception of the N-domain residues.

關鍵字

核磁共振 ； 調鈣素 ； 動力學 ； MARCKS ； HASH(0x1c73c880)

並列摘要

調鈣素〈Calmodulin〉是一個可以和鈣原子鍵結的蛋白質，其常常廣泛的出現在和鈣有關的訊號傳遞路徑當中。調鈣素在溶液中可以折疊成兩個球狀區域─Ｎ端和Ｃ端區域。而每個區域存在著兩個螺旋─環─螺旋的二級結構（EF-hand）。這兩個區域中間是利用一個中心螺旋來銜接，而中心螺旋也是調鈣素中有彈性的區域，它允許Ｎ端區域跟Ｃ端區域有各式各樣的位向。最近，一個新的無法摺疊的蛋白質已經被發現，這類的蛋白質特性是在溶液中沒有一個平均的結構。而這個蛋白質稱作肉豆蔻酸化之富含丙氨酸的蛋白質激酶Ｃ受質Myristoylated alanine-rich C kinase substrate 〈簡稱MARCKS〉。MARCKS可以鍵結蛋白質激酶C〈protein kinase C〉和調鈣素，並藉此調控與這兩個蛋白質的訊號傳遞路徑。MARCKS和調鈣素所形成的結構和一般球型的目標蛋白質和調鈣素的複合物不大相同。在這篇論文當中，我們將要探討MARCKS和調鈣素所形成的複合物的動力學，而我們利用空間密度來分析氮十五核磁共振弛緩速度，並對於調鈣素在有鍵結MARCKS下跟沒有鍵結MARCKS下的動力學做比較。結果我們發現調鈣素在沒有鍵結下其Ｎ端Ｃ端跟中心螺旋存在著不同的動力學，而在鍵結MARCKS蛋白質以後其動力學就變的相似。更進一步的發現調鈣素除Ｎ區域外，其餘骨架的彈性也會因為和MARCKS鍵結而有所不同。

並列關鍵字

NMR relaxation ； calmodulin ； CaM ； protein dynamics ； MARCKS

參考文獻

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