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T-wave Ion Mobility-mass Spectrometry: Basic Experimental Procedures for Protein Complex Analysis
Published on: July 31, 2010
Cytoglobin conformations and disulfide bond formation.
Christophe Lechauve1, Cédric Chauvierre, Sylvia Dewilde
1Inserm U779, Universités Paris VI et XI, Le Kremlin-Bicêtre, France.
The FEBS Journal
|June 18, 2010
Summary
Wild-type cytoglobin exhibits a monomeric form despite dimeric hydrodynamic diameter, with flexible N- and C-terminal regions. Ligand binding kinetics are biphasic due to internal histidine competition and potential disulfide bond formation affecting oxygen affinity.
Area of Science:
- Biochemistry
- Structural Biology
- Protein Dynamics
Background:
- Cytoglobin, a member of the globin superfamily, possesses a characteristic globin fold but with unique N- and C-terminal extensions.
- The oligomeric state and ligand-binding kinetics of wild-type cytoglobin are crucial for understanding its function.
- Previous studies have suggested ambiguity regarding cytoglobin's oligomeric status due to its structural features.
Purpose of the Study:
- To elucidate the oligomeric state of wild-type cytoglobin.
- To characterize the kinetics of ligand binding, specifically after carbon monoxide (CO) photolysis.
- To investigate the influence of internal protein ligands and potential structural modifications on cytoglobin's properties.
Main Methods:
- Hydrodynamic diameter measurements to assess oligomeric state.
- Mass spectrometry to determine subunit mass.
- Kinetic assays following CO photolysis to analyze ligand binding.
- Investigation of disulfide bond formation and its impact on protein conformation.
Main Results:
- Cytoglobin presents as a monomer based on mass, despite hydrodynamic diameter suggesting a dimer.
- Ligand binding kinetics are biphasic, indicating competition with an internal E7 distal histidine.
- Formation of an internal disulfide bond was observed, potentially altering distal histidine dissociation rates and leading to conformational changes.
- These conformational changes can modulate oxygen affinity by up to an order of magnitude.
Conclusions:
- Cytoglobin behaves hydrodynamically as a tightly packed globin with flexible termini, rather than a compact globular monomer.
- The internal E7 histidine acts as a competing ligand, influencing CO dissociation kinetics.
- Disulfide bond formation represents a significant post-translational modification that impacts cytoglobin conformation and oxygen-binding properties.
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