STRUCTURAL BIOLOGY OF BACTERIOPHAGE P22 DNA PACKAGING/TAIL MACHINE

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 门户蛋白是大的寡聚DNA泵，其将加尾噬菌体的二十面体衣壳连接到病毒尾部。尽管门户蛋白体积大且结构复杂，但它是由一条多肽链自组装形成十二聚体环。我们研究了噬菌体P22的门蛋白，它由12个分子量约为83 KDa的亚基组成。1MDa）。我们已经结晶了全长1 MDa环以及跨越残基1-627和1-602的蛋白质的两个大的C-末端截短片段（分别命名为C（1-627）和C（1-602））。尽管全长P22门蛋白晶体对X射线的衍射能力很差（最多7），但C-末端缺失片段C（1-602）的衍射数据完全达到3.85。我们现在已经获得了硒代甲硫氨酸衍生的C（1-602）P22门蛋白的大晶体（高达0.7 mm），这有望使其能够进行完整的MAD实验。此外，在与舍伍德Casjens教授的合作中，我们表达并纯化了P22尾部辅助因子gp 4、gp 10和gp 26，我们正试图将其与P22门户蛋白复合结晶。该项目的长期目标是确定整个P22 DNA包装/尾机的晶体结构。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Portal proteins are large oligomeric DNA pumps, which connect the icosahedral capsid of tailed bacteriophages to the viral tail. Despite the large size and complex structural organization, portal proteins are formed by a single polipeptide chain that self assembles to yield a dodecameric ring. We have studied the portal protein of bacteriophage P22, which is formed by 12 subunits of ~ 83KDa (overall M.W.~1MDa). We have crystallized the full length 1MDa ring as well as two large C-terminally truncated fragments of the protein spanning residues 1-627 and 1-602 (named C(1-627) and C(1-602), respectively). Whereas full-length P22 portal protein crystals diffract X-rays poorly (7 ¿ at best), complete diffraction data to 3.85 ¿ have been measured for the C-terminal deletion fragment C(1-602). We have now obtained large crystals (up to 0.7 mm) of seleno-methionine derivatized C(1-602) P22 portal protein, which, hopefully, will enable to carry out a complete MAD experiment. In addition, in collaboration with Prof Sherwood Casjens, we have expressed and purified the P22 tail accessory factors gp4, gp10, and gp26, which we are trying to crystallize in complex with the P22 portal protein. The long-term goal of this project is to determine the crystal structure of the entire P22 DNA packaging/tail machine.