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探討 BCDX2 蛋白質複合體的生化特性與其與 RAD51 重組酵素的交互作用

為了解決Ogawa dx2的問題，作者江宜蓁 這樣論述：

同源重組 (homologous recombination) 主要透過修復去氧核醣核酸雙股斷裂 (DNA double-strand breaks) 來維持基因體的穩定。在同源重組中，演化上被高度保留的 RAD51 重組酵素 (RAD51 recombinase) 是執行同源重組反應來修復雙股斷裂的去氧核醣核酸所不可或缺的。除此之外，研究發現在細胞內除了 RAD51 之外，去氧核醣核酸的修復還需要另外五個 RAD51 的同源蛋白質 (paralog) 形成兩個主要的蛋白質複合體，分別是 BCDX2 以及 CX3。目前已知這些 RAD51 的同源蛋白質與 RAD51 約有二十至三十百分比的胺

基酸序列相似性，並且也具有結合與水解三磷酸腺苷 (ATP) 的能力。然而，由於過去純化 BCDX2 蛋白質複合體的困難，導致目前關於 BCDX2 複合體的生化特性以及與 RAD51 作用相關的詳細機制仍有許多未知。在我們的研究中，我們成功地建立了表達和純化 BCDX2 複合體重組蛋白質的系統，並且得到了 BCDX2 複合體重組蛋白質。我們的研究證明了重組蛋白質 BCDX2 複合體具有結合單股去氧核醣核酸 (single-strand DNA) 的能力也和 RAD51 有直接的蛋白質交互作用。 然而，出乎我們意料之外，純化的 BCDX2 蛋白質複合體，特別是 RAD51B 能夠在體外實驗被磷酸化

(phosphorylation)，並且我們初步的實驗證明 RAD51B 的磷酸化能夠影響 BCDX2 複合體與 RAD51 的交互作用。

大環變形對三價鐵卟啉電子組態的影響

為了解決Ogawa dx2的問題，作者李俞諼 這樣論述：

Metalloporphyrin compounds are well known to be a considerable of models in the active site of hemopoteins with respect to the interpretations of the biological activity and electronic configuration of central metal. Recently, a series of nonplanar heme were identified by crystal structures or mol

ecular dynamics calculations, which infer the universality of the porphyrin ring deformation extensively existed in nature. Octaethyltetraphenylporphyrin (OETPP) was the early noticed nonplanar porphyrin with saddle-shaped deformation. Interestingly, its corresponding FeIII(OETPP)Cl is quantum-mix

ed intermediate spin state, which is very common for cytochrome c’, an electron transfer protein, but different to the planar iron(III) porphyrin such as FeIII (TPP)Cl and FeIII (OEP)Cl with pure high spin state ( S=5/2 ).In this study, we report the synthesis and characterization of two five coordi

nate FeIII(OETPP)CN and FeIII(OiPTPP)Cl complexes. We employed spectroscopic techniques, including 1H NMR, 13C NMR, EPR, DFT theoretical calculations and the measurement of magnetic moment, to characterize and study the spin states with regard to porphyrin ring deformation. FeIII(OETPP)CN shows the

reduced 1H NMR extent of FeIII(OETPP)Cl, ranging from 0 ppm to 25 ppm; the 13C NMR chemical shift of axial CN- ligand in a characteristic position of 13037 ppm, and the rest carbon peaks of the OETPP spread from -20 ppm to 400 ppm. The 77K EPR spectra are incompatible between pure solids, inclined

to S = 3/2 and in the solvents, such as CH2Cl2 or CHCl3, with S = 1/2. This unusual conflict was proposed that the reassembly of axial CN- ligand and FeIII(OETPP) forms 6-coordinate FeIII(OETPP)(CN)2 in low temperature. The DFT calculations shows the diagnostic nuclear spin density which is propor

tional to the Fermi contact shift, the main contribution of paramagnetic NMR, with varied S = 3/2, 4A2 (dxy)2(dxz,yz)2(dz2)1, 4B1 (dxy)1(dxz2,yz1)(dz2)1,and 4B2 (dxy)1(dxz1,yz2)(dz2)1 and show the 4A2 most consistent to both of 1H NMR and 13C NMR data.Octaisopropyltetraphenylporphyrin ( OiPTPP ) is

designed for more saddle-shaped deformation. The 1H NMR spectrum of FeIII(OiPTPP)Cl shows the upfiledward shifts comparing to FeIII(OETPP)Cl or FeIII(OMTPP)Cl. 77K EPR shows an axial symmetry spectrum with g⊥= 4, g// =2. These signals show Fe(OiPTPP)Cl a pure intermediate-spin state (S=3/2). In th

e lack of single crystal structure of Fe(OiPTPP)Cl, we adopt structure optimization by ADF calculation. The energy of intermediate-spin state (S=3/2) is the lowest than high-spin (S=5/2) and low-spin (S=1/2) , showing S=3/2 is a ground state for Fe(OiPTPP)Cl. Additionally, the magnitude of saddle-sh

aped deformation of Fe(OiPTPP)Cl is larger than Fe(OETPP)Cl and Fe(OMTPP)Cl crystal structure. Molecular orbital analyses further present when porphyrin ring symmetry lower to C2v and consequently the dx2-y2 and dz2 have same symmetry (a1) to mix with porphyrin a2u orbital. This orbital interactio

n will not only raise the dx2-y2 orbital higher than planar porphyrin but also enlarge the gap between dx2-y2 and dxy, which stabilizes the S = 3/2 spin state accordingly.