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PDBsum entry 2owh
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Oxygen storage/transport
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PDB id
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2owh
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References listed in PDB file
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Key reference
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Title
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Time-Resolved crystallographic studies of the heme domain of the oxygen sensor fixl: structural dynamics of ligand rebinding and their relation to signal transduction.
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Authors
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J.Key,
V.Srajer,
R.Pahl,
K.Moffat.
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Ref.
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Biochemistry, 2007,
46,
4706-4715.
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PubMed id
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Abstract
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The FixL protein of Bradyrhizobium japonicum is a dimeric oxygen sensor
responsible for initiating regulation of transcription of genes encoding
proteins involved in nitrogen fixation and oxidative stress. It consists of an
N-terminal heme-bound PAS domain, denoted bjFixLH, and a C-terminal histidine
kinase domain whose enzymatic activity depends on the ligation state of the
heme. To investigate the molecular basis for this dependence and the dynamics
associated with conversion between ligated and unligated states, we have
conducted time-resolved Laue diffraction studies of CO recombination in bjFixLH.
Time-dependent difference Fourier maps from 1 micros to 10 ms after photolysis
of the heme-CO bond show movement of the side chain of Leu236 and the H and I
beta-strands into the ligand binding pocket formerly occupied by CO. Long-range
conformational changes are evident in the protein, driven by relaxation of
steric interactions between the bound ligand and amino acid side chains and/or
changes in heme stereochemistry. These structural changes fully reverse as CO
rebinds to the heme. Spectroscopic measurements of CO recombination kinetics in
bjFixLH crystals relate the behavior of crystalline bjFixLH to solution and
provide a framework for our time-resolved crystallographic experiments. Analysis
of the time-dependent difference Fourier maps by singular value decomposition
reveals that only one significant singular value accounts for the data. Thus
only two structural states are present, the photolyzed and the CO-bound states.
The first left singular vector represents the difference in density between
these two states and shows features common to difference maps calculated from
the static CO and deoxy states. The first right singular vector represents the
time course of this difference density and agrees well with the CO recombination
kinetics measured spectroscopically. We refine the structure of the photolyzed
state present in the early-microsecond time range and find that it does not
differ significantly in conformation from static, deoxy bjFixLH. Thus,
structural relaxation from CO-bound to deoxy bjFixLH is complete in less than 1
micros.
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