SYNCHROTRON CRYSTALLOGRAPHY STRUCTURAL ANALYSIS OF PROFILIN

Profilin是一种普遍存在的15kD蛋白质，是正常 丝状肌动蛋白结构在体内的分布。Profilin结合 PIP2，提示可能与信号转导途径有关。 来自不同生物体的Profilin序列同源性有限，但 高度同源的3D结构，表明进化压力 以维持蛋白质折叠。这一直是我们实验室的目标 将结晶学和活体研究相结合，以检查 细胞内的Profilin。在X9B收集了两个Profilin数据集。 P2蛋白晶体(棘阿米巴的一种蛋白亚型 Castellani)，它使用旋转阳极仅衍射到4.0A，并且 在X9B处衍射到2.0A，具有F432空间群和单胞 参数a=b=c=241.28，a=b=g=90o。P2的原子结构 Profilin将有助于创建突变体，用于研究 该蛋白具有特定的脂结合活性。晶体： 酿酒酵母Profilin的弱衍射性 旋转阳极，在X9B衍射率为3.0A。酿酒酵母 晶体属于P432空间群，晶胞参数为 A=b=c=127.66，a=b=g=90o，并成功缩减和扩展 (再合并5.8%)。我们现在已经制备了一种新的晶型酵母 Profilin(p65；a=b=58.1，c=151.3E)，并将结构细化到 2.3E分辨率。这种结构被用来设计特定的 将探索肌动蛋白和脯氨酸在体内的作用的突变体 富配体结合。此外，我们最近解决了 人血清白蛋白与L-脯氨酸十聚体结合的结构 提供有关此交互的第一个结构信息，即 负责Profilin的细胞内定位。

Profilin is a ubiquitous 15kD protein required for the normal distribution of filamentous actin structures in vivo. Profilin binds PIP2, suggesting a possible link to signal transduction pathways. Profilins from different organisms have limited sequence homology, but highly homologous 3D structures, indicating an evolutionary pressure to maintain the protein fold. It has been the goal of our laboratory to combine crystallography and in vivo studies to examine the role of profilin in the cell. Two profilin data sets were collected at X9B. Crystals of P2 profilin, (a profilin isoform of Acanthamoeba castellani), which diffracted only to 4.0A using a rotating anode and diffracted to 2.0A at X9B, has a space group of F432 and unit cell parameters of a=b=c=241.28, a=b=g=90o. The atomic structure of P2 profilin will aid in the creation of mutants with which to study the specific lipid binding activity of this protein. Crystals of Saccharomyces cerevisiae profilin which diffracted weakly using a rotating anode, diffracted to 3.0 A at X9B. The S. Cerevisiae crystals are of the P432 space group with unit cell parameters of a=b=c=127.66, a=b=g=90o and were successfully reduced and scaled (Rmerge of 5.8%). We have now prepared a new crystal form of yeast profilin (P65; a=b=58.1, c=151.3E) and have refined the structure to 2.3E resolution. This structure is being used to design specific mutants that will probe the in vivo role of actin and proline rich-ligand binding. Furthermore, we have recently solved the structure of human profilin bound to a decamer of L-proline which gives the first structural information about this interaction that is responsible for the intra-cellular localization of profilin.