Klink Björn U, Goody Roger S, Scheidig Axel J
Max-Planck-Institut für Molekulare Physiologie, Abteilung Physikalische Biochemie, D-44225 Dortmund, Germany.
Biophys J. 2006 Aug 1;91(3):981-92. doi: 10.1529/biophysj.105.078931. Epub 2006 May 12.
We present a new design for a fluorescence microspectrophotometer for use in kinetic crystallography in combination with x-ray diffraction experiments. The FLUMIX device (Fluorescence spectroscopy to monitor intermediates in x-ray crystallography) is built for 0 degrees fluorescence detection, which has several advantages in comparison to a conventional fluorometer with 90 degrees design. Due to the reduced spatial requirements and the need for only one objective, the system is highly versatile, easy to handle, and can be used for many different applications. In combination with a conventional stereomicroscope, fluorescence measurements or reaction initiation can be performed directly in a hanging drop crystallization setup. The FLUMIX device can be combined with most x-ray sources, normally without the need of a specialized mechanical support. As a biological model system, we have used H-Ras p21 with an artificially introduced photo-labile GTP precursor (caged GTP) and a covalently attached fluorophore (IANBD amide). Using the FLUMIX system, detailed information about the state of photolyzed crystals of the modified H-Ras p21 (p21(mod)) could be obtained. Measurements in combination with a synchrotron beamline showed significant fluorescence changes in p21(mod) crystals even within a few seconds of x-ray exposure at 100 K.
我们展示了一种用于动力学晶体学并与X射线衍射实验相结合的新型荧光显微分光光度计设计。FLUMIX装置（用于监测X射线晶体学中中间体的荧光光谱）专为0度荧光检测而构建，与传统的90度设计荧光计相比具有多个优点。由于空间需求减少且仅需一个物镜，该系统具有高度通用性，易于操作，可用于许多不同的应用。与传统体视显微镜相结合，可直接在悬滴结晶装置中进行荧光测量或反应起始操作。FLUMIX装置可与大多数X射线源组合使用，通常无需专门的机械支撑。作为生物模型系统，我们使用了带有人工引入的光不稳定GTP前体（笼形GTP）和共价连接荧光团（IANBD酰胺）的H-Ras p21。使用FLUMIX系统，可以获得有关修饰后的H-Ras p21（p21（mod））光解晶体状态的详细信息。与同步加速器光束线相结合的测量表明，即使在100 K下X射线照射几秒钟内，p21（mod）晶体中的荧光也会发生显著变化。
