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PDBsum entry 2cl0
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Nucleotide binding protein
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PDB id
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2cl0
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References listed in PDB file
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Key reference
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Title
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A newly designed microspectrofluorometer for kinetic studies on protein crystals in combination with X-Ray diffraction.
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Authors
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B.U.Klink,
R.S.Goody,
A.J.Scheidig.
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Ref.
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Biophys J, 2006,
91,
981-992.
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PubMed id
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Abstract
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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.
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