STRUCTURAL STUDY OF MONOMERIC RED FLUORESCENT PROTEINS

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。MPlum和Morange是来源于DsRed(1)的单体红色荧光蛋白。我们打算收集mPlum、Morange和选定变种的高分辨率数据。MPlum是已发现的红移最大的荧光蛋白，其发射峰和激发峰分别位于~649 nm和~590 nm。我们之前使用国产X射线源在1.8？分辨率下测定了晶体结构，它揭示了埋藏的Glu16和发色团之间的氢键。我们认为这种氢键是长波长发射的关键。不幸的是，这种晶体是成熟蛋白质和未成熟蛋白质的混合物。最近，成熟的mPlum和未成熟的mPlum分别被分离和结晶。在曝光时间为30分钟的情况下，晶体在家用X射线源中衍射率为1.6°。我们需要更高分辨率的数据来解决成熟和不成熟mPlum之间的结构差异，因为它们可能非常小。从化学上讲，成熟过程涉及蛋白质骨架的氧化，从正常的反式多肽键生成顺式酰胺。需要原子分辨率数据或更好的数据。此外，还观察到E16Q/H/L三个变异体发生蓝移。我们怀疑这些蓝移与是否形成了类似于mPlum的氢键以及它的强度有关。因此，我们将以原子分辨率或更高的分辨率来研究这些变体。

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.