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Single-Molecule Förster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1
Published on: September 18, 2019
Intramolecular cross-linking in the native JHBP molecule
Dominika Bystranowska1, Zbigniew Szewczuk, Marek Lisowski
1Department of Biochemistry, Wrocław University of Technology, Wrocław, Poland.
Archives of Biochemistry and Biophysics
|November 17, 2011
Summary
Juvenile hormone binding protein (JHBP) exhibits unusual fluorescence due to dityrosine cross-linking, not tryptophan. This cross-linking, detected in native and recombinant JHBP, is likely formed via reactive oxygen species in insect hemolymph.
Area of Science:
- Biochemistry
- Insect Physiology
- Protein Chemistry
Background:
- Juvenile hormone binding protein (JHBP) is essential for insect development, transporting crucial hormones.
- JHBP lacks tryptophan residues but shows unexpected fluorescence at 420nm when excited at 315nm.
Purpose of the Study:
- To investigate the source of unusual optical properties in JHBP.
- To determine the molecular mechanism behind JHBP's fluorescence.
Main Methods:
- Gel filtration chromatography under denaturing conditions
- Electrospray ionization mass spectrometry (ESI-MS)
- UV and Circular Dichroism (CD) spectroscopy
- Immunoblotting
- Peptide mass spectrometry after proteolysis
Main Results:
- ESI-MS and gel filtration ruled out low molecular weight ligands.
- Spectroscopic and immunoblotting studies identified intramolecular dityrosine cross-linking as the cause of fluorescence.
- Dityrosine bridges were found in both native and recombinant JHBP.
- Mass spectrometry pinpointed the dityrosine cross-link between tyrosine residues Y128 and Y130.
- Reactive oxygen species (ROS)-mediated oxidation is proposed as the mechanism for dityrosine formation in Galleria mellonella hemolymph.
Conclusions:
- The unexpected fluorescence of JHBP is attributed to intramolecular dityrosine cross-linking.
- This dityrosine cross-linking occurs between specific tyrosine residues (Y128-Y130).
- ROS-mediated oxidation is the likely pathway for dityrosine bridge formation in insect hemolymph, impacting JHBP structure and function.
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