STRUCTURE OF THE PORE FORMING FRAGMENT OF COLICIN E1

结肠素E1是一种具有多种功能状态的细菌素，存在于 既是一种可溶性蛋白质，也是一种可以形成电压门控的蛋白质 在膜上形成通道。在体外，这种转化是由pH变化驱动的。 从粘菌素E1的C-末端切割出的18-20kD片段保留 整个分子的所有通道形成特性。这个的水晶 在两个pH处都获得了片段，分别代表了可溶性和 膜形式。晶体的三维晶体结构 将解决高pH(可溶形式)晶体。初步研究 结果表明，这些晶体在2.2A范围内有很强的衍射率，并且具有很强的阻性 在X射线束中衰变。完整的本机数据将收集到2.2 A (目标1，7)。水银衍生品将用单一的 蛋白质的半胱氨酸和具有额外的 半胱氨酸(目标2)。结晶学数据将从这些和 其他衍生品(目标3，4)。阶段将由单项确定 波长分辨-反常相移，多个同构替换， 单一同象取代/密度修饰或分子 更新换代(目标5)，以及建造和改进三维结构 (目标6，7)。完整的结肠素E1分子的结晶和 将改进低pH(通道形式)晶体(AIMS 8，10)。还将确定是否将可溶性物质转化为 膜形式可以在结晶状态下进行(目标9)。这部作品 将不仅导致对这些的结构基础的理解 粘杆菌素E1的转变，但也可能揭示一般机制 电压门控通道、蛋白质转位和毒素作用。

Colicin E1 is a bacteriocin which has multiple functional states, existing both as a soluble protein and as a protein which can form a voltage-gated channel in a membrane. In vitro this conversion is driven by a pH change. An 18-20 kD fragment cleaved from the C-terminal end of colicin E1 retains all the channel forming properties of the whole molecule. crystals of this fragment have been obtained at two pHs which represent the soluble and the membrane form. The three-dimensional crystallographic structure of the high pH (soluble form) crystal will be solved. Preliminary studies indicated that these crystals diffract strongly to 2.2 A and are resistant to decay in the X-ray beam. Full native data will be collected to 2.2 A (Aims 1,7). Mercurial derivatives will be prepared with the single cysteine of the protein and with site-directed mutants having additional cysteines (Aim 2). Crystallographic data will be collected from these and other derivatives (Aims 3,4). Phases will be determined by single wavelength resolved-anomalous phasing, multiple isomorphous replacement, single isomorphous replacement/density modification or molecular replacement (Aim 5), and the three-dimensional structure built and refined (Aims 6,7). Crystallization of the intact colicin E1 molecule and improvements in the low pH (channel form) crystals will be made (Aims 8,10). It will also be determined if the conversion from the soluble to the membrane form can be done in the crystalline state (Aim 9). This work will lead not only to an understanding of the structural basis for these transitions in colicin E1 but may also shed light on the general mechanism of voltage-gated channels, protein translocation, and toxin action.