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.
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