(第一部份)
熱休克蛋白質Hsp70是一種存在於大多數生物體內有高度保留性的分子伴護子，其主要的功能是維持細胞內其他蛋白質的正常摺疊，以幫助細胞對抗外來的不正常環境(如高溫、低溫、壓力等…)。該蛋白質由兩個主要的區段組合而成，分別是氨基末端的核酸結合區段以及羧基末端的受質結合區段，而區段之間的交互作用則主導了該伴護子的功能。本報告中為了提供該種蛋白質之詳細結構資訊以利進一步之研究，共解出了嗜熱菌Geobacillus kaustophilus HTA426內之Hsp70同源蛋白質DnaK，以及褐鼠Rattus norvegicus內之Hsp70同源蛋白質Hsc70的三度空間立體結晶結構。此二結構皆為與雙磷酸腺苷－無機磷之結合狀態，並且呈現出獨特的區段分離構型。而為了進一步偵測此區段分離動作的成因，一個色氨酸更被建構於兩個主要區段的交互作用界面上，藉由測量其螢光強度的變化，我們成功得知水溶液中之熱休克蛋白質Hsp70在與雙磷酸腺苷以及與受質結合時，確實會促進其兩主要區段的分離。

(第二部份)
尾端固定膜蛋白是一類利用其梭基末端固定於細胞或胞器之磷脂質雙層膜上的蛋白質。在酵母菌體內，將此類膜蛋白插入內質網上的工作已知是由蛋白質Get1、Get2、Get3、Get4和Get5所負責，為了進一步研究其間的詳細分子機轉，此報告中解出了酵母菌Saccharomyces cerevisiae內之Get4全長與Get5氨基末端的蛋白質複合體晶體結構，該結構顯示出Get4與Get5之間的結合力量主要來自於疏水性作用力，在充滿水溶液的生理環境中該兩蛋白質的結合非常穩定。此外，酵母菌雙雜交實驗結果顯示出Get3與Get5之間的交互作用需要有Get4的存在才能達成，而在進一步利用Get4的不同片段進行酵母菌雙雜交實驗後，更確認了Get4是分別利用其氨基半部以及其梭基半部與Get3以及Get5進行交互作用。

(Part1)
The 70-kDa heat shock proteins (Hsp70s) are highly conserved ATP-dependent molecular chaperones composed of an N-terminal nucleotide binding domain (NBD) and a C-terminal protein substrate binding domain (SBD) in a bilobate structure. Interdomain communication and nucleotide-dependent structural motions are critical for Hsp70 chaperone functions. Our understanding of these functions remains elusive due to insufficient structural information of functionally intact Hsp70s in different chaperone cycle states. We report here the crystal structures of DnaK from Geobacillus kaustophilus HTA426 bound with ADP-Mg2+-Pi at 2.37 Å and 70-kDa heat shock cognate protein from Rattus norvegicus bound with ADP-Pi at 3.5 Å. The NBD and SBD in these structures are significantly separated from each other and they may be corresponding to the ADP-bound conformation. Moreover, a Trp reporter was introduced at the potential interface region between NBD and interdomain linker of GkDnaK to probe the environmental changes. The result of fluorescence measurement further supports that the substrate binding enhanced domain disjoining behavior for Hsp70 chaperone family.

(Part2)
The tail-anchored (TA) proteins are a typical class of membrane proteins, which present a single transmembrane domain (TMD) located near their C-termini. By anchoring the single TMD into the phospholipid bilayer surrounding cellular organelles, the N-terminal cytosolic portion of TA proteins can be properly arranged to cytosol for particular functional purposes. Get3, Get4 and Get5 in Saccharomyces cerevisiae participate in the insertion of tail-anchored proteins into the endoplasmic reticulum membrane. We elucidated the interaction between Get4 and Get5 and investigated their interaction with Get3. Based on crystallographic studies, Get4 and Get5 formed a tight complex, suggesting that they constitute subunits of a larger complex. The Get4 structure shows an overall oblong shape and uses its C-terminal part to interact with Get5. Yeast two-hybrid experiment revealed that Get4 mediates the communication between Get3 and Get5 by interact with these two proteins through its different bisections.

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