整合蛋白（integrins）是一群位於細胞表面的alpha、beta異質雙體，其功能與細胞的黏著、移動、和訊息傳遞有關。很多生理及非生理方面的整合蛋白受質是利用RGD、LDV、或相關序列做為它們與整合蛋白黏結的基本結構。Rhodostomin（簡稱Rho）是個含有68個胺基酸及6對雙硫鍵的蛋白，其中含有一段48PRGDMP53序列會高親和力競爭纖維蛋白原（fibrinogen）在血小板上的整合蛋白alphaIIbbeta3之黏結部位，而抑制了血小板的凝集。最近的研究也證實了與整合蛋白黏結的RGD序列附近胺基酸會對整合蛋白alphaIIbbeta3、alphaVbeta3、及alpha5beta1產生特異性。為了研究整合蛋白alphaIIbbeta3、alphaVbeta3、及alpha5beta1可辨識的特異性序列，我們將Rho的RGD loop或C端附近區域的胺基酸作突變，並表現在可正確表現Rho的Pichia pastoris系統中，利用血小板凝集抑制試驗及細胞黏著抑制實驗作為這些序列的功能性分析。我們發現在RGD loop中含有AKGDWN以及RLD和RGDD序列可以分別特異性地抑制表現整合蛋白alphaIIbbeta3以及alphaVbeta3細胞的黏結，而含有ARGDGW及ARGDXX序列的突變蛋白對整合蛋白alpha5beta1有較好的特異性或活性。相對地，在Rho的C端附近區域對這3種整合蛋白的黏結只有些微的影響。為了了解這些對整合蛋白有特異性的去整合蛋白之結構與功能的關係，利用NMR分析它們的3D結構發現在RGD motif的殘基R49和D51所在的方位及動態在整合蛋白的辨識上扮演著重要角色。另外，為了在P. pastoris系統中成功表現出胺基酸選擇性（amino-acid-type selective；AATS）同位素標示蛋白，利用Rho去嘗試AATS同位素標示蛋白的培養條件且成功地應用生產出AATS同位素標示Rho，這些[alpha-15N]-Cys、-Leu、-Lys、和-Met Rho都有大於50%的同位素胺基酸嵌入程度。此外，同樣的培養條件也可成功地應用在P. pastoris系統中表現出含有4對雙硫鍵AATS同位素標示的蛋白dendroaspin。這是第一個在P. pastoris系統中表現出AATS同位素標示蛋白的研究。將來可作為我們探索整合蛋白與去整合蛋白複合體間的結構與活性關係的基礎，也可以幫助整合蛋白相關的藥物設計。

　Integrins are alpha/betaheterodimers on cellular membrane and involved in cell adhesion, mobility, and signal transduction. Many physiological and non-physiological integrin ligands utilize RGD, LDV, or a related sequence as a key structural component of their integrin-binding site. Rhodostomin (Rho) is a potent integrin antagonist, which consists of 68 amino acids including six disulfide bonds and a PRGDMP sequence at the positions of 48-53. Rho competes against fibrinogen in the binding of integrin alphaIIbbeta3 on platelet and results in inhibiting platelet aggregation. Many studies have also demonstrated that the amino acid residues flanking the RGD sequence of integrin ligands modulate their specificity of interaction with integrin alphaIIbbeta3,alphaVbeta3, and alpha5beta1 complexes. In order to identify the amino acid residues required for selective recognition of integrins alphaIIbbeta3, alphaVbeta3, andalpha5beta1, we mutated the residues in the RGD loop or C-terminal region of Rho, expressed Rho mutants in P. pastoris, and used the platelet aggregation and cell adhesion assays for functional studies. We found that Rho mutants containing the sequences AKGDWN, as well as RLD and RGDD can selectively inhibit integrins alphaIIbbeta3 and alphaVbeta3, respectively. In addition, the mutants containing the sequences ARGDGW and ARGDXX have better selectivity or activity in inhibiting integrin alpha5beta1. 3D structures of integrin-specific Rho mutants were determined by NMR spectroscopy. Our results showed that the orientations of R49 and D51 residues and dynamic properties of the RGD motif in Rho play important roles in integrin recognition. To develop the protocol to produce amino-acid-type selective (AATS) isotope labeling of protein expressed in P. pastoris, Rho was used to optimize the culture condition. The optimized protocol was successfully applied to produce AATS isotope-labeled Rho and other protein, dendroaspin. The labeling of [alpha-15N]-Cys, -Leu, -Lys, and -Met amino acids has an incorporation rate greater than 50%. In addition, the culture condition was successfully applied to label dendroaspin, a four disulfide-bonded protein expressed in P. pastoris. This is the first study to present a protocol for AATS isotope labeling of protein expressed in P. pastoris. This study will serve as the basis for exploring structure-activity relationships of the integrin/disintegrin complexes as well as rational drug design on integrins.

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