耐熱性蛋白質Sac7d和DNA錯合物之晶體結構解析以及其結合機制之探討

陳青諭

doi:10.6342/NTU.2006.02535

3.239.51.78，Hello！

Close
Search Quick Search Advanced Search Publication Search	Personal Service Member Login Searching History Use Bonus Card SYMSKAN
Browse Browse by University of Graduate Thesis Browse by Pinyin of Graduate Thesis Browse by Conference Proceedings Topic Browse by Conference Proceedings Publisher Browse by Pinyin of Conference Proceedings Browse by eBook Category Browse by eBook Topic Browse by eBook Publisher
Customer Services Authorize Airiti Recommend a Journal

Search Symbol (Half-width)	Description of Search Symbols
Space	"AND" indicates the intertwining of key terms used in a search
Double Quotation Marks ("") ( " " )	Double quotation marks indicate the beginning and end of a phrase, and the search will only include terms that appear in the same order of those within the quotations. Example: "image process" ： " image process "
?	Indicates a variable letter. Entering two ? will indicate two variable letters, and so on. Example: "Appl?", search results will yield apple, apply… e , appl y … ( (often used to English word searches) )
*	Indicates an unlimited number of variable letters to follow, from 1~n. Example: Enter "appl*", search results will yield apple, apples, apply, applied, application…(often used in English word searches) e , appl es , appl y , appl ied , appl ication … ( (often used to English word searches) )
AND、OR、NOT	Boolean logic combinations of key words is a skill used to expand or refine search parameters. (1) AND (1) AND: Refines search parameters (2) OR (2) OR: Expands search parameters (3) NOT: Excludes irrelevant parameters

DOI stands for Digital Object Identifier （ D igital O bject I dentifier ） ,
and is the unique identifier for objects on the internet. It can be used to create persistent link and to cite articles.

Using DOI as a persistent link

To create a persistent link, add「http://dx.doi.org/」「 http://dx.doi.org/ 」 before a DOI.
For instance, if the DOI of an article is 10.5297/ser.1201.002 , you can link persistently to the article by entering the following link in your browser: http://dx.doi.org/ 10.5297/ser.1201.002 。
The DOI link will always direct you to the most updated article page no matter how the publisher changes the document's position, avoiding errors when engaging in important research.

Cite a document with DOI

When citing references, you should also cite the DOI if the article has one. If your citation guideline does not include DOIs, you may cite the DOI link.

DOIs allow accurate citations, improve academic contents connections, and allow users to gain better experience across different platforms. Currently, there are more than 70 million DOIs registered for academic contents. If you want to understand more about DOI, please visit airiti DOI Registration （ doi.airiti.com ）。

Data Source

Basic & Applied Sciences > Chemistry
College of Science > Graduate Institute of Chemistry

Bibliography Management Tool

Related Links

Report an Issue

Purchase Single Articles

Download PDF

耐熱性蛋白質Sac7d和DNA錯合物之晶體結構解析以及其結合機制之探討

Structural basis of DNA binding mechanism of the hyperthermophilic chromosomal protein Sac7d from Sulfolobus acidocaldarius

陳青諭 , Ph.D　　Advisor：王惠鈞　　Co-advisor ：陳長謙

英文 DOI： 10.6342/NTU.2006.02535

耐熱嗜酸性蛋白質；蛋白質-去氧核糖核酸之錯合物；去氧核糖核酸結構的彎曲； X-光繞射技術； Sac7d ； protein-DNA complex ； DNA bending ； DNA kink ； X-ray crystallolography ； hyperthermophilic ； sequence-general DNA binding protein

Abstract │ Reference (165) │ Altmetrics

Abstract 〈TOP〉

Sulfolobus是一種生活於硫磺溫泉中，嗜熱耐酸的古生菌。而Sac7d是存在Sulfolobus菌體內，含量最為豐富的一種非特異性的DNA結合蛋白質。Sac7d是分子量7.6 kDa的小蛋白質，對熱、對酸以及化學試劑都有很高的穩定性。由於古生菌體內缺乏組織蛋白質histone來幫助DＮＡ纏繞，因此一般相信Sac7d蛋白質是扮演類似histone 的角色，並且在高溫下，有助於穩定DNA維持雙股螺旋的二級結構。
當Sac7d與DNA結合形成錯合物時，會利用Val26以及Met29兩個疏水性的支鏈，嵌合插入DNA的鹼基對之中。此蛋白質特殊的結合模式，會於DNA局部的構造，產生極大的偏折，進而形成彎曲的結構。然而DNA結構的彎曲，在許多生理過程中，扮演極為重要的角色。例如，調控基因的轉錄（gene transcription），啟動DNA的複製(DNA replication)，DNA的修復（DNA repair），以及蛋白質對DNA的辨識(protein-DNA recognition)等等。因此，我們利用Sac7d蛋白質為模版，利用點突變的方式，將Val26以及Met29兩個胺基酸，分別置換成支鏈小的Ala以及支鏈大的Phe，並以X-光繞射的技術，將Sac7d 突變蛋白質與DNA結合的八個錯合物的晶體結構解出，來研究不同的Sac7d突變蛋白質如何與DNA作用。實驗結果證實，擁有不同疏水性支鏈大小組合的突變蛋白質，確實可以令DNA分子的結構以不同的角度和方式產生偏折和彎曲。我們成功地利用簡單的實驗技術，達到調控DNA分子曲度的目的。此外，這些protein-DNA錯合物的晶體結構，可以提供準確的結構資訊，讓我們對於蛋白質如何辨識DNA，以及如何與DNA產生作用，有更深一層的認識。對於改良或設計新的具選擇性的基因調控藥物，或是抗癌藥物有所幫助。
除此之外，兩個新的wt-Sac7d-decamer錯合物的晶體結構已被成功地解析。這些高解析度的晶體結構提供一個良好的機會，用以比較同一種非特異性的DNA結合蛋白質Sac7d，如何與不同序列的DNA作用，並歸納出Sac7d對於特定的DNA序列，有一定程度的結合偏好。另一方面，我們利用紫外光以及螢光光譜分析法，來研究各種Sac7d突變蛋白質（Trp24/Val26/Met29 mutants）之熱穩定性以及DNA結合力，並與野生型的Sac7d做比較。實驗結果顯示，幾乎所有的Sac7d突變蛋白質其耐熱性與DNA結合力皆小於野生型。而residue 26 和29的支鏈與DNA 分子間的疏水性作用力以及Trp24與DNA鹼基之間的氫鍵作用力，對於Sac7d與DNA之間結合力的強弱有很大的影響。而利用Raman光譜的研究，我們證實了當DNA與Sac7d形成錯合物，對於DNA被結合的部位，其骨架結構會由B-form轉換成A-form。這個結果與我們在Sac7d-DNA錯合物的晶體結構中，所觀察到的結構資訊是一致的，同時也間接證明了，這個錯合物不論在晶體中或水溶液中，其結構是相似的，因此我們由X-光繞射所獲得的結構資料是可信的，而非由於晶體堆疊所造成。
另一部份，我們與日本的Prof. Hiroshi Sugiyama 合作，進行Sac7d-DNA錯合物的光化學研究。實驗證明，於紫外光(302 nm)的激發下，在Sac7d與DNA形成的錯合物中，電子會從Trp24這個胺基酸，轉移至含有BrU(bromouracil)的DNA分子上，而Trp24會進一步與氧氣作用氧化形成N’-formyl kynurenine。這個光誘發導致電子由蛋白質轉移至DNA的過程，對於 photolyase這個修補酵素而言，是一個良好的模型，用以解釋這個酵素如何在光照的環境下，修補並移除DNA分子上T-T雙聚體的結構。
最後，我們探討存在於各式各樣的蛋白質與DNA錯合物介面中的水分子所扮演的角色。結果十分有趣，不同功能的DNA結合蛋白質對於水分子的作用方式也迥異，但水分子對生物體的重要性是無庸置疑的。在非特異性的DNA結合蛋白質（例如Sac7d、HMG和nucleosome）與DNA結合的介面裡，水分子通常扮演調控者（modulator）的角色，讓同一種蛋白質可以跟不同序列的DNA做適當的結合。若具有序列專一性的蛋白質與非目標的DNA序列結合，常會形成結構鬆散的錯合物，且其介面常常會留存許多水分子當作潤滑劑，令蛋白質可以在DNA分子上滑動，來尋找其特異性的結合序列。而有一些蛋白質，藉由以水分子媒介的氫鍵取代胺基酸支鏈與DNA鹼基之間的直接氫鍵，來轉換其特異性或DNA結合力的強弱。在特異性的蛋白質和DNA形成的錯合物中，水分子對於蛋白質對特定DNA序列的辨識力，以及錯合物的穩定度，都有很大的貢獻。此外，由於不同的DNA序列會產生不同的水合模式，因此有一些蛋白質是藉由辨識DNA的水合模式與其結合，而非辨認DNA的序列。

Parallel Abstract 〈TOP〉

Sac7d is a small (~7600 Da.), but abundant, chromosomal proteins from the hyperthermophilic archaeon Sulfolobus acidocaldarius. The protein is extremely stable to heat, acid and chemical agents. Sac7d binds to DNA as monomer non-cooperatively with micro-molar affinity, without marked sequence preference and increases the Tm of DNA by ~ 40°C. Previously, two crystal structures of Sac7d-octamer complexes have been solved at high resolution. These structures reveal that Sac7d binds in the minor groove of DNA and causes a single-step sharp kink in DNA (~60°) via the intercalation of both Val26 and Met29. These two amino acids were systematically changed in size to probe their effects on DNA kinking. DNA bending has long been recognized as an important component of biological activity. Eight crystal structures of five Sac7d mutant-DNA complexes have been analyzed. The DNA binding pattern of the V26A and M29A single mutants is similar to that of the wild type, whereas the V26A/M29A protein binds DNA without side chain intercalation, resulting in a smaller overall bending (~50°). The M29F mutant inserts the Phe29 side chain orthogonally to the C2pG3 step without stacking with base pairs, inducing a sharp kink (~80°). In the V26F/M29F-GCGATCGC complex, Phe26 intercalates deeply into DNA bases by stacking with the G3 base, whereas Phe29 is stacked on the G15 deoxyribose, in a way similar to those used by the TATA-box binding proteins. All mutants have reduced DNA-stabilizing ability, as indicated by their lower Tm values. The DNA kink patterns caused by different combinations of hydrophobic side chains may be relevant in understanding the manner by which other minor groove binding proteins interact with DNA.
Two new crystal forms of Sac7d in complex with the DNA decamers CCTATATCGG and CCTACGTACC were obtained and their structures were determined by molecular replacement. The protein structures are similar to the previously determined structure of Sac7d-GCGATCGC, but the DNA molecules are more bent overall, by 14-20°. Analysis the interactions of the same protein bound to different DNA sequences showed weak DNA binding sequence preferences of Sac7d, a sequence-general DNA binding protein. The preferred intercalation sites in DNA were found at either the CpG, TpT(=ApA) or TpA steps, likely due to their weak stacking forces. The base at the 3’ end of the intercalating site is always a purine (G or A), with Trp24 NE1 forming a hydrogen bond to its N3 atom. The second or third base at the 3’-side of the intercalation site is a thymine that forms hydrogen bond(s) with its O2 atom to Arg42.
The spectroscopic methods such as UV-Vis, fluorescence and Raman spectroscopy have been used to study variety properties of Sac7d in solution. A Raman spectroscopic analysis of Sac7d binding to decamer GAGGCGCCTC reveals that large changes in the DNA backbone and partial B- to A-form DNA transitions in the DNA structure upon complex formation. A hydrophobic cluster on the surface of Sac7d is composed of Trp24, Val26, and Met29 residues which play a key role in defining thermal stability and DNA binding affinity of Sac7d. All of the Sac7d W24/V26/M29 mutations resulted in a decrease in protein thermal stability and DNA binding affinity. In addition, the photochemical study show evidence for the photoinduced specific electron transfer process from Trp24 to bromo-uracil containing DNA in the Sac7d-DNA complex.
Recent surveys of high-resolution protein-DNA crystal structures have noted that solvent molecules are commonly present within the protein-DNA interfaces. Putting these results together has revealed that protein-DNA complexes are quite diverse in their use of water. In the non-sequence specific DNA binding proteins such as Sac7d, interfacial water molecules may act as “modulators” for their binding to DNA of varying sequence without adding specificities. When sequence specific DNA binding proteins bound to non-cognate DNA, more waters remained at the interface of the complexes. These waters may behave as a kind of molecular glue allowing the protein to slide along the DNA for their target sites. Some proteins switch their specificity, i.e., transformation of the high affinity complex to a low affinity complex revealed that direct hydrogen bonds at the interface of protein and DNA are often replaced by water-mediated hydrogen bonds. Water molecules could act as major contributions to stability and specificity in some specific protein-DNA complexes. Since DNA hydration patterns are sequence dependent, proteins recognize the DNA hydration structures rather than DNA sequence upon forming the complexes.

Reference ( 165 ) 〈TOP〉

1. Jaenicke, R. and Zavodszky, P. (1990) Proteins under extreme physical conditions. FEBS Lett. 268, 344-349.
連結：
2. Woese, C., Kandler, O. and Wheelis, M. (1990) Towards a natural system of organisms: proposal for the domains archaea, bacteria, and eucarya. Proc. Natl. Acad. Sci. U.S.A. 87, 4576-4579.
連結：
3. Stetter, K. O., Fiala, G., Huber, G., Huber, R. and Segerer, A. (1990) Hyperthermophilic microorganisms. FEMS Microbiol. Rev. 75, 117-124.
連結：
4. Kimura, M., Kumura, J., Phillips, D., Reinhardt, R. and Dijk, J. (1984) The amino acid sequence of a small DNA binding protein from the archaebacterium Sulfolobus solfataricus. FEBS Lett. 176, 176-178.
連結：
5. Grote, M., Dijk, J. and Reinhardt, R. (1986) Ribosomal and DNA-binding proteins of the thermoacidophilic archaebacterium Sulfolobus acidocaldarius. Biochim. Biophys. Acta 873, 405-413.
連結：

Altmetrics

〈TOP〉

E-mail ：

When an article is available to download, a notice will be sent to your mailbox address.

E-mail ：