Intramolecular cross-linking in the native JHBP molecule |
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| Authors: | Bystranowska Dominika Szewczuk Zbigniew Lisowski Marek Sitkiewicz Ewa Dobryszycki Piotr Ożyhar Andrzej Kochman Marian |
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| Institution: | 1. Department of Biochemistry, Faculty of Chemistry, Wroc?aw University of Technology, Wybrze?e Wyspiańskiego 27, 50-370 Wroc?aw, Poland;2. Faculty of Chemistry, University of Wroc?aw, 50-383 Wroc?aw, Poland;3. Institute of Biochemistry and Biophysics, Department of Biophysics, Polish Academy of Sciences, Warsaw, Poland;3. Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Bronx, New York 10461;4. Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461;1. College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, PR China;2. Laboratory of Aquaculture & Artemia Reference Center, Ghent University, Belgium;3. Department of Applied Biological Chemistry, The University of Tokyo, Yayoi, Bunkyo, Tokyo 113-8657, Japan;1. State Key Laboratory of Brain and Cognitive Science, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China;2. University of Chinese Academy of Sciences, Beijing 100049, China;3. Sino-Danish Center for Education and Research, Beijing 100190, China |
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| Abstract: | Juvenile hormone binding protein (JHBP) acts as a shuttle, carrying one of the most crucial hormones for insect development to target tissues. We have found that although the JHBP molecule does not contain tryptophan residues, it exhibits a weak fluorescence maximum near 420nm upon excitation at 315nm. Gel filtration experiments performed in denaturing conditions and ESI-MS analyses excluded the possibility that some low molecular ligand was bound to the protein molecules. Further UV and CD spectroscopy studies, as well as immunoblotting, showed that the unusual JHBP optical properties were due to dityrosine intramolecular cross-linking. These bridges were detected both in native and recombinant protein molecules. We believe that in Galleria mellonella hemolymph the DT generation occurs via ROS-mediated oxidation leading to the formation of cross-linked JHBP monomers. MS analyses of peptides generated after JHBP proteolysis indicated, that the dityrosine bridge occurs between the Y128 and Y130 residues. |
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