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Atomic Scale Structural Studies of Macromolecular Assemblies by Solid-state Nuclear Magnetic Resonance Spectroscopy
Published on: September 17, 2017
Correlation between the OmpG secondary structure and its pH-dependent alterations monitored by FTIR
Filiz Korkmaz-Ozkan1, Stefan Köster, Werner Kühlbrandt
1Institute of Biophysics, Goethe-University, Max-von-Laue-Str. 1, D-60438 Frankfurt am Main, Germany.
Journal of Molecular Biology
|June 22, 2010
Summary
Mutations in the outer-membrane protein G (OmpG) histidine pair His231/His261 lock the channel in an open state, independent of pH. This structural insight reveals key mechanisms of OmpG channel gating.
Area of Science:
- Biochemistry
- Structural Biology
- Microbiology
Background:
- Outer-membrane protein G (OmpG) from Escherichia coli exhibits pH-dependent channel activity.
- The histidine pair His231/His261 is hypothesized to play a critical role in OmpG channel gating.
Purpose of the Study:
- To investigate the function of the His231/His261 histidine pair in OmpG channel opening and closing.
- To elucidate the structural basis of pH-dependent gating in OmpG.
Main Methods:
- Site-directed mutagenesis of His231 and His261 to alanine and cysteine.
- Fourier transform infrared (FTIR) spectroscopy to assess protein conformation.
- Temperature ramp experiments to evaluate protein stability.
- X-ray crystallography to determine the structure of the OmpG mutant.
Main Results:
- OmpG mutants with His231/His261 replaced by alanine or cysteine remained constitutively open, irrespective of pH.
- Mutant OmpG proteins exhibited stability comparable to the open state of wild-type OmpG.
- X-ray structure analysis revealed alterations in the extracellular loop L6 of the alanine mutant, suggesting a role in gating.
Conclusions:
- The His231/His261 histidine pair is essential for the pH-dependent gating of OmpG channels.
- Mutations in this histidine pair lead to a constitutively open channel conformation.
- Conformational changes involving extracellular loop L6 are likely triggered by His231/His261 and regulate OmpG channel activity.
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