STRUCTURAL STUDY OF MONOMERIC RED FLUORESCENT PROTEINS

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。子项目及 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。 MPlum和mOrange是衍生自DsRed（1）的单体红色荧光蛋白。我们打算收集mPlum、mOrange和选定变体的高分辨率数据。mPlum是发现的红移最大的荧光蛋白，其发射峰和激发峰分别在~ 649 nm和~ 590 nm处。我们先前使用家用X射线源以1.8 <$的分辨率确定了晶体结构，这揭示了掩埋的Glu 16和发色团之间的氢键。我们相信这个氢键是长波长发射的关键。不幸的是，晶体是成熟和未成熟蛋白质的混合物。最近成熟和未成熟的mPlum已分别分离和结晶。在家用X射线源中曝光30分钟后，晶体衍射至1.6 º。我们需要更高分辨率的数据，以解决成熟和未成熟mPlum之间的结构差异，因为它们可能非常小。在化学上，成熟过程涉及蛋白质骨架的氧化，以从正常的反式肽键产生顺式酰胺。原子分辨率数据或更好的是所期望的。 还观察到三种变体E16 Q/H/L蓝移。我们怀疑这些蓝移与是否形成了类似于mPlum的氢键以及氢键的强度有关。因此，我们将在原子分辨率或更高分辨率下研究这些变体。

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. MPlum and mOrange are monomeric red fluorescent proteins derived from DsRed (1). We intend to collect high resolution data for mPlum, mOrange and selected variants. mPlum is the most red-shifted fluorescent protein discovered with emission and excitation peak at ~649nm and ~590nm respectively. We previously determined the crystal structure at 1.8 ¿ resolution using the home x-ray source, which reveals a hydrogen bond between the buried Glu16 and the chromphore. We believe this hydrogen bond is the key to long wavelength emission. Unfortunately, the crystal was a mixture of mature and immature protein. Recently mature and immature mPlum have been separated and crystallized respectively. The crystals diffract to 1.6 ¿ at home x-ray source with 30 minutes of exposure time. We require higher resolution data in order to solve the structural differences between mature and immature mPlum as they may be very small. Chemically, the maturation process involves oxidation of the protein backbone to create a cis-acylamine from a normal trans-peptide bond. Atomic resolution data or better are desired. Also it has been observed that three variants E16Q/H/L are blue-shifted. We suspect that these blue shifts are related to whether a hydrogen bond similar to that of mPlum is made and how strong it is. Therefore, we will study these variants at atomic resolution or higher.