蛋白質結晶學在結構學上是一種很有力的工具，但是很多蛋白因其結構不穩定或動態而難以結晶，Fusion partner是利用額外的融合蛋白提供蛋白質堆疊所需要的接觸面以提高結晶的機率，此實驗的目的是希望能開發出更多Fusion partner的同時還能一併解出未知的蛋白質結構。MrPd和PvPd因為已被證實可以很容易形成高解析度的晶體而被列入Fusion partner的研究，GNNVPd是因為能夠形成穩定的三聚體，PvSd是想利用它會形成空心球狀多聚體的特性將目標蛋白質包裹在內來形成晶體，GST則是已經被多次用來當作Fusion partner，而實驗室也已有建立好的表現質體，所以也一併進行測試。WWOX主要出現在對抗癌症的細胞自噬過程中並扮演著重要的訊息傳遞角色，其中WW domains是主要與其他蛋白質交互作用的區域，然而因其結構不穩定，目前還沒有利用結晶學解出來的結構，所以我們便想利用WW domains當作試金石檢驗我們挑選的Fusion partners。
我們總共利用了8種蛋白進行長晶，包括MrPd-WW、PvPd-WW、WW-GNNVPd、WW-EcoRI-PvSd和GST-WW，還有為了減少結構不穩定性所進行修飾的P256-WW、MrPd-WW1跟MrPd-WW2。目前為止我們成功獲得了MrPd-WW和GST-WW的晶體，然而在解出MrPd-WW的結構後，發現看不到WW domains的結構，於是懷疑WW domains是否發生了降解，經過基質輔助雷射脫附游離飛行時間質譜法驗證其蛋白質分子量後，證實MrPd-WW中WW domains只剩下1.4 kDa的氨基酸片段。另外在解出GST-WW的結構後，我們仍未看到WW domains的結構，於是利用膠體電泳確認晶體內的組成，發現蛋白是完整的GST-WW，推測可能是因為WW domains的區域因為結晶時沒有進行規律的排列，而無法觀察到其電子雲密度。
雖然無法解出完整的WW domains的結構，但是我們發現了新的P6122空間群組成方式，MrPd-WW能夠形成六邊形的空心柱體並將C端包裹在內，文獻中有類似的包裹形式來解出目標蛋白質結構的例子，我們相信MrPd也有機會利用這樣的包裹形式解出目標蛋白質結構。比較新舊的晶體排列，可以觀察到在Val52到Leu57和Thr78到Ala80的區域會因作用力的不同而改變其結構，這些區域在文獻上指出可能負責強化病毒和細胞間接觸的功能。因此我們認為這些區域在病毒和細胞接觸時會藉由改結構改變以增強病毒與細胞連結的能力。在Leu10之前的氨基酸負責連結P-domain和S-domain，具有高度的動態而無法呈現結構，而Val123後的氨基酸區域也因可能是接著的WW domains而影響到結構的穩定性，使得原本穩定的C端無法呈現出結構。另外在GST-WW的方面，若因WW domains不易形成規則性的排列而無法解出WW domains的結構，我們或許可以改變GST和WW domains的連接處或是加入能夠和WW domains交互作用的蛋白，來增強WW domains的穩定性，如此將有機會讓利用蛋白質結晶學解出其精確的結構。

X-ray crystallography is a powerful tool to solve protein structures. However, it is sometime difficult to obtain suitable protein crystals due to structural instability and flexibility. Previous studies suggested that fusion partners and protein complex formation can help protein crystallization. In this study, the protruding domains of nodavirus Macrobrachium rosenbergii (MrPd, residues 246-371), Penaeus vannamei (PvPd, 250-368) and Grouper nervous necrosis virus (GNNVPd, 214-338), the shell domains of nodavirus Penaeus vannamei (PvSd, 38-250) and glutathione-S-transferase (GST) are investigated as fusion partners to enhance protein crystallization.
WW domain-containing oxidoreductase (WWOX) is a regulatory and signaling protein that mediates specific protein-protein interactions in cell death pathway, such as p53 or Zfra induced apoptosis. The WW domains (residues 15-90) serve the important binding site of WWOX for protein-protein interactions. Nevertheless, the structure of the human WW domains has not been determined. To determine the structure of the WW domains and verify the usage of designed fusion partners, the expression plasmids of SUMO-MrPd-WW, SUMO-PvPd-WW, SUMO-WW-GNNVPd, SUMO-WW-EcoRI-PvSd and GST-WW were constructed. To further reduce the flexibility of fusion proteins, SUMO-P256-WW (PvPd, residues 256-368), SUMO-MrPd-WW1 (WW domains, residues 15-52) and SUMO-MrPd-WW2 (WW domains, residues 53-90) were constructed. All the plasmids were transformed to BL21(DE3) for over-expression of the fusion proteins. All SUMO His-tag recombinant proteins were purified with nickel affinity chromatography. The SUMO His-tag was subsequently removed and the recombinant proteins of high purity were collected for crystallization.
Among these recombinant proteins, MrPd-WW and GST-WW protein proteins could be crystalized at the resolutions 1.3 Å and 2.1 Å, respectively, with synchrotron X-ray diffraction. The refined MrPd-WW structure showed only MrPd molecules packed in a new space group (P6122). To analyze this result, we used mass spectrometry to measure its molecular weight as 31.1 kDa in solution, which showed a degradation of WW domains from MrPd-WW. The C terminus of MrPd is followed by 1.4 kDa amino-acid fragment of the WW domains. The new crystal packing leads to structural variations of MrPd at the Val52 to Leu57 and Thr78 to Ala80 loop regions compared to our previously published structures, suggesting that these regions might be involved in the virus attachment with host cells. As for GST-WW crystals, only the model of GST could be built based on interpretable electron density. After verifying the molecular weight of GST-WW crystals with SDS-PAGE by dissolving crystals, the complete GST-WW remained intact within crystals. We surmise the reason why no WW domains could be observed was because of the high flexibility of the WW domains. Our MrPd-WW and GST-WW demonstrated that the proteins could be crystallized with a new space group and with the protein contacts provided by fusion partners. Therefore, the usage of fusion partners in this study might be a potential approach to enhance protein crystallization after resolving the degradation and flexibility issue.

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