CRYSTALS OF 143 KDA BOVINE INTERPHOTORECEPTOR RETINOID BINDING PRO

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。 143 kDa的人/哺乳动物光感受器间类视色素结合蛋白（IRBP）是不寻常的，因为它与其他类视色素结合蛋白没有同源性。 它由四个同源模块组成的单一多肽组成，每个模块约300个氨基酸残基。 大多数对其功能的研究都集中在视周期中视杆细胞和色素上皮细胞之间的11-顺式视黄醇和全反式视黄醇以及脂肪酸的运输。 在脂肪酸配体的存在下，含有全长哺乳动物蛋白质的四个模块首次结晶。 晶体在室温下在内部旋转阳极X射线发生器中显示衍射，但在低温冷却时显示无序。 在用许多冷冻冷却剂进行实验后，PEG 35 K似乎在20%甘油的存在下提供最多的冷冻保护。 我们现在已经从18%的35 K PEG中生长出了晶体。 除了常规的数据收集，我们还建议用国际象棋的Sol Gruner博士开发的高压低温冷却方法进行实验。 格鲁纳博士的团队同意协助我们进行这些实验。

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. The 143 kDa human/mammalian interphotoreceptor retinoid-binding protein (IRBP) is unusual in that it has no homology with other retinoid-binding proteins. It consists of a single polypeptide composed of four homologous modules each ~300 amino acid residues. Most studies into its function have focused on the visual cycle trafficking of 11-cis retinal and all-trans retinol between the rods and pigment epithelium, as well as of fatty acids. The four module containing full-length mammalian protein has been crystallized for the first time, in the presence of a fatty acid ligand. The crystals show diffraction at room temperature at the in-house rotating anode X-ray generator, but disorders on cryo-cooling. After experimenting with a number of cryo-cooling agents, PEG 35K seems to offer most cryo-protection, in the presence of 20% glycerol. We have now grown the crystals from 18% 35K PEG. In addition to routine data collection, we propose also to experiment with the high pressure cryo-cooling method developed by Dr. Sol Gruner at CHESS. Dr. Gruner's group has agreed to assist us in these experiments.