Mizuno H, Sawano A, Eli P, Hama H, Miyawaki A
Laboratory for Cell Function and Dynamics, Advanced Technology Development Center, Brain Science Institute, The Institute of Physical and Chemical Science (RIKEN), 2-1 Hirosawa, Wako-city, Saitama, 351-0198, Japan.
Biochemistry. 2001 Feb 27;40(8):2502-10. doi: 10.1021/bi002263b.
The biochemical and biophysical properties of a red fluorescent protein from a Discosoma species (DsRed) were investigated. The recombinant DsRed expressed in E. coli showed a complex absorption spectrum that peaked at 277, 335, 487, 530, and 558 nm. Excitation at each of the absorption peaks produced a main emission peak at 583 nm, whereas a subsidiary emission peak at 500 nm appeared with excitation only at 277 or 487 nm. Incubation of E. coli or the protein at 37 degrees C facilitated the maturation of DsRed, resulting in the loss of the 500-nm peak and the enhancement of the 583-nm peak. In contrast, the 500-nm peak predominated in a mutant DsRed containing two amino acid substitutions (Y120H/K168R). Light-scattering analysis revealed that DsRed proteins expressed in E. coli and HeLa cells form a stable tetramer complex. DsRed in HeLa cells grown at 37 degrees C emitted predominantly at 583 nm. The red fluorescence was imaged using a two-photon laser (Nd:YLF, 1047 nm) as well as a one-photon laser (He:Ne, 543.5 nm). When fused to calmodulin, the red fluorescence produced an aggregation pattern only in the cytosol, which does not reflect the distribution of calmodulin. Despite the above spectral and structural complexity, fluorescence resonance energy transfer (FRET) between Aequorea green fluorescent protein (GFP) variants and DsRed was achieved. Dynamic changes in cytosolic free Ca2+ concentrations were observed with red cameleons containing yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), or Sapphire as the donor and RFP as the acceptor, using conventional microscopy and one- or two-photon excitation laser scanning microscopy. Particularly, the use of the Sapphire-DsRed pair rendered the red cameleon tolerant of acidosis occurring in hippocampal neurons, because both Sapphire and DsRed are extremely pH-resistant.
对来自一种盘状珊瑚（DsRed）的红色荧光蛋白的生化和生物物理特性进行了研究。在大肠杆菌中表达的重组DsRed呈现出复杂的吸收光谱，其峰值分别位于277、335、487、530和558纳米处。在每个吸收峰处激发都会在583纳米处产生一个主要发射峰，而只有在277或487纳米处激发时才会在500纳米处出现一个次要发射峰。将大肠杆菌或该蛋白在37摄氏度下孵育有助于DsRed的成熟，导致500纳米峰消失，583纳米峰增强。相比之下，在含有两个氨基酸替代（Y120H/K168R）的突变型DsRed中，500纳米峰占主导。光散射分析表明，在大肠杆菌和HeLa细胞中表达的DsRed蛋白形成了稳定的四聚体复合物。在37摄氏度下生长的HeLa细胞中的DsRed主要在583纳米处发射荧光。使用双光子激光（Nd:YLF，1047纳米）以及单光子激光（He:Ne，543.5纳米）对红色荧光进行成像。当与钙调蛋白融合时，红色荧光仅在细胞质中产生聚集模式，这并不能反映钙调蛋白的分布。尽管存在上述光谱和结构复杂性，但仍实现了水母绿色荧光蛋白（GFP）变体与DsRed之间的荧光共振能量转移（FRET）。使用传统显微镜以及单光子或双光子激发激光扫描显微镜，观察到了含有黄色荧光蛋白（YFP）、青色荧光蛋白（CFP）或蓝宝石作为供体且红色荧光蛋白作为受体的红色钙调蛋白传感器中细胞质游离Ca2+浓度的动态变化。特别地，使用蓝宝石 - DsRed对使红色钙调蛋白传感器能够耐受海马神经元中发生的酸中毒，因为蓝宝石和DsRed都具有极强的耐酸性。
