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研究生:王興祥
研究生(外文):Hsing-Hsiang Wang
論文名稱:利用螢光光譜研究調鈣素和肉豆蔻酸化之富含丙氨酸的蛋白質激酶C受質的複合物
論文名稱(外文):Fluorescent study of Myristoylated Alanine-Rich C-Kinase substrate and Calmodulin complex
指導教授:張壽麟
指導教授(外文):Shou-Lin Chang
學位類別:碩士
校院名稱:國立清華大學
系所名稱:生物資訊與結構生物研究所
學門:生命科學學門
學類:生物訊息學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:英文
論文頁數:48
中文關鍵詞:調鈣素肉豆蔻酸化之富含丙氨酸的蛋白質激酶C受質螢光
外文關鍵詞:CalodulinMARCKSFluorescence
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摘要

調鈣素是真核細胞主要的鈣離子訊號傳遞蛋白。目前已知它可和一百種以上不同的受質作用,而且參與了包含了生長、肌肉收縮以及分化等生理現象。肉豆蔻酸化之富含丙氨酸的蛋白質激酶C受質(MARCKS)是調鈣素的受質之一,也是一個和膜能結合的蛋白。他在包含出生後的存活、細胞的移動和腦部發育之不同的細胞功能上扮演重要的角色。效應器功能區塊(effector domain)是MARCKS和調鈣素的結合部位,並且也是MARCKS主要和受質有作用的功能區域。MARCKS的效應器功能區塊和調鈣素的結晶結構以及兩者間的解離常數先前已被發表(Kd = 8.8 ± 1.6 nM)。調鈣素對於MARCKS有很高的親合力。在此我們可利用吸收光譜以及螢光光譜來研究調鈣素與MARCKS間之交互作用。利用光譜訊號的變化來監控二者於滴定實驗過程中的結合並且得到了解離常數(Kd = 357.41 ± 26.91 nM)。在本實驗中螢光給予者和螢光接受者之間並沒觀察到FRET的發生。我們推測這可能是因為螢光給予者和螢光接受者之間的距離太遠。根據以上的結論我們可以提出符合FRET發生的螢光給予者和螢光接受者之間的距離。這些研究成果將作為日後將進行單分子螢光相關光譜研究之前導實驗。
Abstract

Calmodulin (CaM) is the major calcium sensor protein in eukaryotic cells. It has been known to interact with more than one hundred substrates and control the physiological events including development, muscle contraction, and differentiation. Myristoylated alanin-rich C kinase substrate (MARCKS) is the substrate of CaM, and is a membrane associated protein. It plays important roles in various cellular functions including postnatal survival, cellular migration, and brain development. The effetor domain (ED) is its binding domain to CaM and is also the major functional region to interact with many substrates. The crystal structure of MARCKS effector domain-CaM complex, and the affinity between MARCKS effector domain and CaM have been reported. The CaM shows high affinity to the MARCKS effector domain. In this study, we apply fluorescence and absorption spectroscopy to investigate the interaction of the two proteins. The binding process was monitored by the change of fluorescence and absorption spectra in the titration experiment. The dissociation constant Kd was determined to be about 357 nM. In this experiment, the FRET between donor and acceptor was not observed. We suggested that this is due to the long distance between donor and acceptor. This is our pilot experiment for the future fluorescence correlation spectroscopy studies.
CONTENT

Abstract (in Chinese)..………………………………………………………………..i
Abstract (in English)…………………………………………………………………ii
List of Figures………………………………………………………………………..iv
List of Tables………………………………………………………………………….v
Abbreviations………….………………………………………………………….....vii
Introduction…………………………………………………………………………..1
Signal transduction of Ca2+…………………………………………………….2
Calmodulin…………………………………………...…………………………2
The interaction between CaM and protein targets……….…………………..3
The unique property of the central linker……………………………………..5
MARCKS………………………………………………………………………..5
Interaction between MARCKS peptide and Cam………………………….....7
Materials and methods………………………………………………………...16
Preparation of CaM single mutant…………………………………………...16
Expression and purification…………………………………………………..18
Labeling procedures…………………………………………………………..20
Procedures of using HPLC……………………………………………………21
FRET…………………………………………………………………………...21
Fluorescence quenching……………………………………………………….25
Absorption spectroscopy and emission spectroscopy………………………..25
Result and Discussion……………………………………………………….………34
Conclusion……………………..…………………………………………………….43 References…………………………………………………………………………...44

FIGURES LIST

Figure 1. Schematics of the domain organization of CaM…………………...…..10 Figure 2. EF-hand is a helix-loop-helix motif where calcium is hided………......10
Figure 3. Ribbon representation of apo-CaM…...………………………………...11 Figure 4. Ribbon representation of Ca2+-CaM…...………………………….........11
Figure 5. Surface respresentation of CaM, skMLCK-CaM complex, and C20W-CaM complex………...……………………………………………………...12
Figure 6. Ribbon representation of CaMBD-CaM complex…...…………….......12 Figure 7. The overall structure of MARCKS……………………...………….…..13
Figure 8. The primary structure of MARCKS showing, effector domain is consisted of 25 amino acids…...………………………………………………….....13
Figure 9. MARCKS plays the cross-talk role between PKC and CaM……….…13
Figure 10-a. Ribbon representation of CaMKII -CaM complex…...…………....14
Figure 10-b. Ribbon representation of MARCKS peptide-CaM complex…........14
Figure 11. Ribbon representation of the MARCRKS peptide-CaM comple…....15
Figure 12. The primers for making the mutant………...…………………………27
Figure 13. The amino acid sequence of wild type CaM and T34C-CaM………..28
Figure 14. Map of pET-28a(+)-T34C-CaM gene construction……...…………....28
Figure 15. The mutant site of T34C-CaM……………...………….………………29
Figure 16. The T34C-CaM was purified with DEAE column…...…………….…29
Figure 17. The fractions from DEAE column were further purified by applying a phenyl column……..……………………………………………………….………..30
Figure 18. Elution profile from HPLC column…………...………..……………...31
Figure 19. Jablonski diagrams for FRET…...…………………………………..…31
Figure 20. Diagram of absorbance and emission band for fluorophores that exhibit FRET, and illustrate the emission FRET procedure………..…………....32
Figure 21. Absorption spectra of Alexa Fluor 488 and CaM-AF..…..……..…….37
Figure 22. Absorption spectra of Texas Red and MARCKS-TR.…………..……37
Figure 23. Emission spectra of Alexa Fluor 488 and different concentration of CaM-AF……..…………………………………………………………..…………...38
Figure 24. Emission spectra of Texas Red and different concentration of MARCKS-TR.………..……………………………………………..………………38
Figure 25. Absorption spectra of CaM-AF titrated with MARCKS………….....39
Figure 26. Emission spectra of CaM-AF titrated with MARCKS…………….....39
Figure 27. Emission spectra CaM-AF titrated with MARCKS-TA………….......40
Figure 28. The distance between C34 and K191………………….…...………......40
Figure 29. Titration of CaM-AF with MARCKS-TR or non-labeled MARCKS, and MARCKS-TR with CaM, respectively….....…………………………………41
Figure 30. Binding constant Kd and nunber of binding sites n of MARCKS-TR/CaM-AF, MARCKS/CaM-AF, and MARCKS-TR/CaM complex………………………………………………………………………………41
Figure 31. Emission spectra of MARCKS-TR titrated with CaM………….........42

TABLES LEST

Table 1. The DNA and amino acid sequence, molecular weight, number of amino aicds, and the pI value of CaM…………….…………………………………...….27
Table 2. The gradient of the buffer applied with HPLC using process…..……...30
Table 3. The conditions of different emission spectra of Alexa Fluor 488, Texas Red, CaM-AF, and MARCKS-TR………………………………….……………...33
Table 4. The conditions of different emission and absorption spectra of CaM/MARCKS, CaM-AF/MARCKS-TR, and MARCKS-TR/CaM complexes……………………………………………………………………….…...33
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