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13:26
Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry
Published on: September 13, 2014
Temperature dependence of NO binding modes in human neuroglobin
Florin Trandafir1, Sabine Van Doorslaer, Sylvia Dewilde
1Department of Physics, University of Antwerp, Universiteitsplein 1, Antwerp B-2610, Belgium.
Biochimica Et Biophysica Acta
|October 19, 2004
Summary
Wild-type human neuroglobin (hNgb) primarily binds nitric oxide (NO) through its ferrous form, with the E7-histidine residue influencing NO-heme conformation. Mutations altering this distal histidine facilitate NO binding, revealing its specific role in stabilizing NO-heme isomers.
Area of Science:
- Biochemistry
- Structural Biology
- Biophysics
Background:
- Neuroglobin (Ngb) is a hexacoordinated protein found in vertebrates.
- Wild-type Ngb exhibits ferrous and ferric forms with axial ligation by F8-His and E7-His.
- Nitric oxide (NO) is generated by Escherichia coli under low oxygen conditions.
Purpose of the Study:
- To investigate the interaction of nitric oxide (NO) with wild-type human neuroglobin (hNgb) and its E7-mutants.
- To elucidate the role of the E7-histidine residue in NO binding and heme conformation.
- To characterize the structural basis of NO adduct formation in Ngb.
Main Methods:
- Overexpression of recombinant wild-type and mutant human neuroglobin in Escherichia coli.
- Combined electron paramagnetic resonance (EPR) and optical spectroscopy.
- Temperature-dependent EPR analysis of NO-ligated Ngb proteins and their mutants.
Main Results:
- Wild-type hNgb favors a conformation that limits NO binding.
- Mutating the E7-histidine to Leu or Gln facilitates NO binding, forming the nitrosyl ferrous form.
- EPR studies revealed two distinct NO-heme conformations, with E7-histidine stabilizing one isomer in wild-type hNgb.
- The stabilizing effect is attributed to histidine's specific binding characteristics, not its polarity.
Conclusions:
- The E7-histidine residue plays a crucial role in determining the NO-heme conformation of neuroglobin.
- Specific binding interactions of histidine, rather than its polarity, dictate the stabilization of NO adduct isomers.
- Understanding these interactions provides insight into neuroglobin's function and ligand-binding mechanisms.
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