古生菌硫磺礦硫化葉菌中的蛋白質Sso7c4 在溶液中以雙聚體的形式存在，一
般相信其為染色體蛋白質，功能是參與去氧核醣核酸組裝或基因轉錄調控。我們
利用X 光單晶繞射技術解析出Sso7c4 蛋白質的晶體結構，解析度為1.63 埃。而
在這個蛋白質晶體的繞射數據中，蛋白質的C 端完全沒有電子密度，顯示C 端是
相當動態的。利用螢光極化光譜量測Sso7c4 突變蛋白質(R11A,R22A,R11A/R22A)
與DNA 的結合常數並與野生型的Sso7c4 比較。實驗結果顯示Sso7c4 突變蛋白質
(R11A,R22A,R11A/R22A)與DNA 分子的結合力皆小於野生型，而其中蛋白質
R11A/R22A 幾乎不與DNA 作用。這證明了位於Sso7c4 上方表面的R11 與R22 兩
個帶正電的胺基酸，有參與DNA 的結合。由電子顯微影像的分析，證實野生型
Sso7c4 主要是以架橋與彎曲兩種方式與DNA 作用。相反地C 端最末六個胺基酸
被刪除的Sso7c4 蛋白質，在高濃度下，會將環狀的DNA 分子撐開，且沒有任何
讓DNA 更緊密結實的現象。而綜合螢光極化光譜、電子顯微影像以及螢光共振能
量轉移技術的數據，我們證明了Sso7c4 蛋白質動態且帶正電的C 端，不僅參與
DNA 分子結合，也會使DNA 分子彎曲。
在前人的研究中，依照胺基酸序列的比對，將Sso7c4 歸類於類轉錄抑制蛋白
質。綜合以上所有數據資料，我們利用代謝產物活化蛋白與DNA 複合體中，彎曲
的DNA 分子結構來建構Sso7c4 與DNA 複合體的模型。更進一步利用螢光共振能
量轉移實驗量測出與Sso7c4 結合的DNA 分子兩端的距離與模型中的距離吻合，
驗證了Sso7c4-DNA 模型的準確度。螢光共振能量轉移數據同時也驗證了電顯影
像分析的結果，野生型Sso7c4 會使得DNA 分子長度縮短(表示DNA 分子被蛋白質
彎曲)，但是C 端刪除的蛋白質無法使DNA 分子長度改變。綜合以上結果，我們
證明Sso7c4 蛋白質建構或組裝染色體DNA 或是參與基因轉錄的調控，主要是藉
由架橋與彎曲兩種方式來與DNA 分子作用。

The Sso7c4 from Sulfolobus solfataricus forms a dimer, which is believed to
function as a chromosomal protein involved in genomic DNA compaction and gene
regulation. Here, we present the crystal structure of wild-type Sso7c4 at a high
resolution of 1.63 Å, showing that the two basic C-termini are disordered. Based on the
fluorescence polarization (FP) binding assay, two arginine pairs, R11/R22¢ and
R11¢/R22, on the top surface participate in binding DNA. As shown in electron
microscopy (EM) images, wild-type Sso7c4 compacts DNA through bridging and
bending interactions, whereas the binding of C-terminally truncated proteins rigidifies
and opens DNA molecules, and no compaction of the DNA occurs. Moreover, the FP,
EM and fluorescence resonance energy transfer (FRET) data indicated that the two
basic and flexible C-terminal arms of the Sso7c4 dimer play a crucial role in binding
and bending DNA. Sso7c4 has been classified as a repressor-like protein because of its
similarity to Escherichia coli Ecrep 6.8 and Ecrep 7.3 as well as Agrobacterium
tumefaciens ACCR in amino acid sequence. Based on these data, we proposed a model
of the Sso7c4-DNA complex using a curved DNA molecule in the catabolite activator
protein-DNA complex. The DNA end-to-end distance measured with FRET upon wildtype
Sso7c4 binding is almost equal to the distance measured in the model, which
supports the fidelity of the proposed model. The FRET data also confirm the EM
observation showing that the binding of wild-type Sso7c4 reduces the DNA length
while the C-terminal truncation does not. A functional role for Sso7c4 in the
organization of chromosomal DNA and/or the regulation of gene expression through
bridging and bending interactions is suggested.

Table of Contents
Chapter 1 Introduction .............................................................................................................. 6
Architectural proteins across three kingdoms of life ............................................................. 6
Architectural proteins from Sulfolobus species ................................................................... 10
Architectural protein Sso7c4 ............................................................................................... 11
3D complex structures among three domains of life ........................................................... 12
AFM images of Sso10-DNA complexes ............................................................................... 14
Specific aim ......................................................................................................................... 15
Chapter 2 Materials & Methods .............................................................................................. 16
Gene cloning and protein overexpression and purification ................................................ 16
Crystallization and X-ray diffraction data collection.......................................................... 19
Structure determination and refinement .............................................................................. 20
Oligonucleotides used in the FP assays .............................................................................. 23
Tryptophan fluorescence quenching assay .......................................................................... 23
Fluorescence polarization ................................................................................................... 24
Electron microscopy ............................................................................................................ 25
Molecular docking model of the Sso7c4-DNA complex ...................................................... 26
Oligonucleotides used in the bulk FRET study ................................................................... 27
Bulk FRET measurement ..................................................................................................... 28
FRET efficiency calculations ............................................................................................... 29
Chapter 3 Results & Discussion ........................................................................................... 31
Overall structures of wild-type and C-terminally truncated Sso7c4 ................................... 31
Sulfates mediate protein-protein interactions in the wild-type crystal ................................ 38
Arginine pairs on the top surface of Sso7c4 are involved in binding DNA ......................... 39
The flexible C-termini of Sso7c4 are crucial for interactions with DNA ............................ 45
Sso7c4 mediated DNA compaction, as visualized by EM ................................................... 50
Proposed Sso7c4-DNA model and validation using FRET ................................................. 59
Biological implications ........................................................................................................ 67
4
Chapter 4 Conclusion .............................................................................................................. 74
References ............................................................................................................................... 76
5
List of figures
FIG. 1 THE MODELS OF DNA BINDING MODES. ....................................................................................... 9
FIG. 2 AMINO ACID SEQUENCES OF WILD-TYPE AND C-TERMINALLY TRUNCATED SSO7C4. ................ 12
FIG. 3 REPRESENTATIVE CRYSTAL STRUCTURES OF DNA-BENDING PROTEINS IN COMPLEX WITH
DNA AMONG THE THREE KINGDOMS OF LIFE. ............................................................................ 13
FIG. 4 AFM IMAGING OF SSO10-DNA COMPLEX.................................................................................. 14
FIG. 5 COOMASSIE BLUE STAINED SDS-PAGE (15 %) ANALYSIS OF PURIFIED SSO7C4 PROTEINS. .... 18
FIG. 6 CRYSTAL STRUCTURE OF THE WILD-TYPE SSO7C4 PROTEIN. .................................................... 33
FIG. 7 CRYSTAL STRUCTURE OF C-TERMINALLY TRUNCATED SSO7C4. ............................................... 34
FIG. 8 INTERMOLECULAR INTERACTIONS OF THE SSO7C4 MONOMERS AT THE DIMERIZATION
INTERFACE. .................................................................................................................................. 35
FIG. 9 PROTEIN-PROTEIN INTERACTIONS IN THE WILD-TYPE SSO7C4 CRYSTAL. ................................ 37
FIG. 10 SULFATE BOUND TO THE TOP SURFACE OF SSO7C4. .................................................................. 37
FIG. 11 SSO7C4-DNA BINDING ASSAY USING FP. .................................................................................. 43
FIG. 12 THE FLUORESCENCE SPECTRA OF SSO7C4 AND ITS DNA-BINDING CURVE.............................. 49
FIG. 13 ELECTRON MICROGRAPHS OF THE PHIX174 PLASMID WITH OR WITHOUT THE WILD-TYPE/CTERMINALLY
TRUNCATED SSO7C4 PROTEINS. ............................................................................ 54
FIG. 14 RAW EM IMAGES OF NICKED CIRCULAR PHIX174 PLASMID WITH AND WITHOUT THE SSO7C4.
..................................................................................................................................................... 55
FIG. 15 MODEL OF THE INTERACTION OF SSO7C4 WITH DNA. ............................................................ 61
FIG. 16 IMAGES OF A 384-WELL MICROPLATE AFTER EACH OF THREE SCANS. ................................... 65
FIG. 17 ALIGNMENT OF THE SSO7C4 AND HISTONE H3 STRUCTURES AND STRUCTURAL COMPARISON
BETWEEN HISTONE H3 AND HMFA. ............................................................................................ 73
List of tables
TABLE 1. DIFFERENT CLASSES OF ARCHITECTURAL PROTEINS (BRIDGERS, BENDERS, WRAPPERS OR
STIFFENER) IN EUKARYOTES, BACTERIA AND ARCHAEA. .............................................................. 9
TABLE 2. DATA COLLECTION AND REFINEMENT STATISTICS FOR THE SSO7C4 CRYSTALS. ................ 22
TABLE 3. BINDING AFFINITIES FOR THE ARGININE MUTANT/WILD-TYPE/C-TERMINALLY
TRUNCATED/PROTEINS IN COMPLEX WITH THREE DSDNA FRAGMENTS OF DIFFERENT
LENGTHS. ..................................................................................................................................... 44
TABLE 4. STATISTICS OF NUCLEOPROTEIN COMPLEXES IN DIFFERENT FORMS. .................................. 53
TABLE 5. AVERAGE LENGTHS AND WIDTHS OF THE PLASMID AND NUCLEOPROTEIN COMPLEXES. EM
IMAGES WERE MEASURED USING IMAGEJ SOFTWARE. ............................................................... 57
TABLE 6. FRET EFFICIENCY AND DISTANCE OF THE DNA DUPLEX ALONE OR BOUND TO SSO7C4 IN
BULK FRET EXPERIMENTS. ......................................................................................................... 66

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