行政院國家科學委員會補助專題研究計畫成果報告
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※ 蛋白質磷酸化之結構探討(I) ※
※ (Structural studies on protein phosphorylation (I) ※
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計畫類別：■個別型計畫 □整合型計畫
計畫編號：NSC89-2113-M-006-022－
執行期間：89 年 8 月 1 日至 90 年 10 月 31 日
計畫主持人：鄭 梅 芬
共同主持人：
執行單位：國立成功大學生物科技研究所
中 華 民 國 91 年 1 月 31 日
中文摘要
關鍵詞：BICKS、磷酸化、結構、核磁共振
本計畫之重點在於比較磷酸化及未磷酸化蛋白結構之差異，從結構上之差異我們 可推測磷酸化過程所需要的結構因子和構形變化、磷酸化反應所調控的各種機制及接合 作用。在研究結構之前,首先需先得到足夠量之蛋白質以供做物性、功能及結構之研究。
本年度之研究重點在於以重組 DNA 技術、基因轉殖及蛋白質表現系統來生產重組 蛋白，並改變環境因子(包括 Ca+2,, DTT, Calmodulin (CaM)等)以 circular dichroism 及核磁 共振光譜來研究蛋白質之構形變化。我們已成功地將 Bicks 基因接進 pET-21a vector 中, 並轉殖入 E. Coli BL21(DE3)菌中,以 IPTG 誘導表現。Circular dichroism (CD)光譜實驗結 果顯示氧化(具雙硫鍵)或還原態,未磷酸化及磷酸化,加入含 Ca+2溶液之 Bicks 其 CD 光譜 幾少變化,且僅顯示含少許之α-helix 構形。但加入 CaM 之 Bicks, 其α-helical 構形稍許 增加。
NMR 光譜亦顯示 Bicks 大部份之構形是 random coils。
英文摘要
Keywords: Bicks. Phosphorylation. Structure, NMR
Many proteins’ function and interactions are mediated by phosphorylation. Studies of the difference of the structures of both phosphorylated and unphosphorylated forms of proteins and of the interaction with other biomolecules from both forms, will provide a great opportunity to understand this event better. The phosphorylated and unphosphorylated forms of BICKS protein are used as model compounds. Their structures, stability and dynamics are going to be studied. Before structure determination, a large amount of protein must be obtained.
BICKS is a forebrain-enriched, neuron-specific, neuromodulin-immunoreactive, postsynaptic, thyroid hormone-dependent, cytosolic, and protein kinase C (PKC)-substrate protein, containing 78 amino acids with a conserved 19-amino acid sequence region (IQ motif) for calmodulin (CaM) binding and protein kinase C phosphorylation site. It is involved in several important biological events such as signal transduction, long-term potentiation, synaptogenesis, neural plasticity, and local calcium homeostasis. Its functions are mediated by phosphorylation and calmodulin-binding. BICKS is highly conserved during mammalian evolution. Its c-DNA has been cloned into pET-21a vector and can be expressed in Escherichia coli (BL21(DE3) expression system by IPTG induction. The recombinant BICKS retained native BICKS functions.
The CD spectra generated by Bicks have shown marginal negative ellipticities at 222 nm. Studies of the secondary structure by circular dichroism spectra have shown that modification of Bicks by phosphorylation, oxidation forming intramolecular disulfides, did not affect the α-helical content of this protein. Addition of Ca+2 ion did not increase α-helical content of both phorphorylated and unphosphorylated or oxidative and reductive
forms of Bicks either. Interaction of CaM with Bicks with the reduced and
unphosphorylated in the Ca+2 ion free solution have resulted in an increase in the α-helical structure, but there is little change in the forms of the oxidized or /and phosphorylated of Bicks. NMR data were unable to detect any α-helix structure existence.
計畫緣由及目的
生物體的生命現象是由不同層次的生物分子進行一連串複雜的傳導及反應所產 生的，這些生命表象均由生物分子間之交互作用所產生。為了進一步了解這些重要之 生命現象，在原子的層面上對生物分子的結構、功能、及交互作用做深入詳盡的瞭解 是必需的，而研究這些生物分子及其複合物的三度空間結構為首要步驟。充份洞察蛋 白質結構有助於了解其接合、催化和調節等功能。
很多蛋白質的功能及交互作用受磷酸化反應所調控，為了進一步了解磷酸化反應 所調控的機制及接合作用，研究磷酸化及未磷酸化蛋白的結構及動力學上的差異是必 需的。因此，我們將以磷酸化及未磷酸化的 BICKS 蛋白當做模型化合物，用核磁共 振儀、分子生物學、分子動能學、及電腦模擬等方法對其結構、穩定度、及動力學等 特性做深入的研究，期能在原子的層面上來探討磷酸化反應及與磷酸化有關之分子間 的辨識作用及各種現象。
BICKS 常見於前腦，具神經元特異性及 neuromodulin 免疫反應性，在訊號傳遞、
long-tern potentiation、synaptogenesis、神經成形性、及區域性之鈣的等穩性中扮演 重要的角色。哺乳動物之 BICKS 蛋白在演化過程中少有變化。它由 78 個氨基酸所 組成，可溶於水，具有一段由 19 個氨基酸組成的 IQ motif 區域，IQ motif 包含與 calmodulin 接合及受 protein kinase C (PKC) 磷酸化的位置。 它的功能經由磷酸化反 應及與 calmodulin 接合來調節。它的 cDNA 已被合成及轉殖，並可在 Escherichia coli 中表達。重組 BICKS 具有與自然界存在的 BICKS 相同之特性及功能。
研究磷酸化及未磷酸化蛋白結構之差異及其與它種分子間交互作用的不同，不 但可幫助我們從結構的觀點，來了解磷酸化過程所需要的結構因子和構形變化、磷 酸化反應所調控的各種機制及接合作用、及這些蛋白所參與的各種反應，並可進一 步做為研究分子間之辨識及結構-功能關係之基本模型。
結果與討論
(1) Bicks coding sequence was polymerase chain-reaction-amplified from human brain cDNA library with the addition of NdeI and EcoRI restriction sites using the Bicks’ sense and antisense sequence as primers. The polymerase chain reaction product was then cloned into a TA vector (Promega) and then digested with NdeI and EcoRI. The gel-isolated Bicks’ gene was ligated into pET-21a vector. Plasmid DNA was sequenced to ensure accuracy during cloning. Plasmid DNA was then transformed into BL21(DE3) expression strain by heat shock method. The protein was then overexpression by IPTG induction. The protein was purification as Mahoney’s method (1996).
(2) the CD spectrum by Bicks alone showed marginal negative ellipicity at 222 nm suggesting there is little α-helical content in this protein, and most parts of this protein in the random coil conformation. The CD spectra ware not altered appreciably by the presence of NaCl salt, Calcium ion, the reducing agent DTT, or changes in the protein concentration.
Mixture of Bicks with calmodulin without presence of calcium ion, the α-helical content was increased comparing with the sum of the spectra of Bicks and calmodulin. The results were similar with the data obtained by Gerendasy et al. (1995) for bovine Bicks (neurogranin).
(3) Nuclear magnetic resonance spectroscopic data were unable to detect any α-helical conformation in Bicks either.
Gerendasy et al. (1995b) have concluded that CaM stabilizes a basic, amphiphilic α-helix within BICKS under physiological salt concentrations only when Ca2+ is absent.
This provides structural confirmation for two binding modes and suggests that CaM regulates the biological activities of BICKS through an allosteric, Ca2+-sensitive mechanism that can be uncoupled by protein kinase C-mediated phosphorylation.. A pair of strongly hydrophobic amino acids separated by 8 or 12 residues (Ile44, or Phe37, and lie 46) in BICKS may correspond to residues of Ca2+'/CaM-activated proteins that interact with one or both of the hydrophobic patches exposed by CaM in response to Ca2+.(Ikura et al, 1992; Meador et al., 1993)
Spectroscopy of Bicks alone indicates that its conformation is mostly unstructured and is not significantly affected by calcium. Interaction between CaM and BICKS lead to an increased α-helicity when Ca2+ is absent, i.e., an α-helix is stabilized when RC3 binds to calmodulin. This result indicates that an α-helix is stabilized within the CaM-binding site of RC3 upon binding to calmodulin when calcium is absent.
The stabilization of α helix within BICKS only by the Ca2+-free form of CaM suggests that CaM regulates their activities in a Ca2+-sensitive manner. CaM induces α helical conformation within certain BICKS only when Ca2+ is low or absent, implying that the Ca2+-free form of CaM may also be an active regulatory molecule (Gerendasy et al., 1995).
本實驗進度到目前為止大致與預計進度相符.
Reference
1. Gerendasy, D. D., Herron, S. R., Jennings, P. A., and Sutcliffe, J. G. (1995) "Calmodulin stabilizes an amphiphilic alpha-helix within BICKS/neurogranin and Gap-43/neuromodulin only when Ca2+is absent" J. Biol. Chem. 270, 6741-6750.
2. Gerendasy, D. D., Herron, S. R., Jennings, P. A., and Sutcliffe, J. G. (1995b) "Calmodulin stabilizes an amphiphilic alpha-helix within BICKS/neurogranin and
Gap-43/meuromodulin only when Ca2+ is absent" J. Biol. Chem. 270, 1-10.
3. Ikura, M., Clore, G. M., Gronenborn, A. M., Zhu, G., Klee, C. B., and Bax, A. (1992) Science 256, 632-638.
4. Mahoney, C. W., Pak, J. H., and Huang K.-P. (1996) "Nitric oxide modification of rat brain neurogranin" J. Biol. Chem. 271, 28798-28804.
5. Meador, W. E., Means, A. R., and Quiocho, F. A. (1993) Science 262, 1718-1721.
