Abstract
Brain spectrin enjoys overall structural and sequence similarity with erythroid spectrin, but less is known about its function. We utilized the fluorescence properties of tryptophan residues to monitor their organization and dynamics in brain spectrin. Keeping in mind the functional relevance of hydrophobic binding sites in brain spectrin, we monitored the organization and dynamics of brain spectrin bound to PRODAN. Results from red edge excitation shift (REES) indicate that the organization of tryptophans in brain spectrin is maintained to a considerable extent even after denaturation. These results are supported by acrylamide quenching experiments. To the best of our knowledge, these results constitute the first report of the presence of residual structure in urea-denatured brain spectrin. We further show from REES and time-resolved emission spectra that PRODAN bound to brain spectrin is characterized by motional restriction. These results provide useful information on the differences between erythroid spectrin and brain spectrin.
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Notes
We have used the term maximum of fluorescence emission in a somewhat broader sense here. In every case, we have monitored the wavelength corresponding to maximum fluorescence intensity, as well as the center of mass of the fluorescence emission, in the symmetric part of the spectrum. In most cases, both these methods yielded the same wavelength. In cases where minor discrepancies were found, the center of mass of emission has been reported as the fluorescence maximum.
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Acknowledgments
This work was supported by the Department of Atomic Energy (IBOP project) and the Council of Scientific and Industrial Research, Govt. of India. Ar.C. thanks the Council of Scientific and Industrial Research for the award of a Senior Research Fellowship. M.P. acknowledges the award of a Senior Research Fellowship from the University Grants Commission (India). A.C. gratefully acknowledges support from J.C. Bose Fellowship (Department of Science and Technology, Govt. of India). A.C. is an Adjunct Professor of Jawaharlal Nehru University (New Delhi), Indian Institute of Science Education and Research (Mohali), Indian Institute of Technology (Kanpur) and Honorary Professor of the Jawaharlal Nehru Centre for Advanced Scientific Research (Bangalore). We thank Sourav Haldar for help with the TRES measurements, G. Aditya Kumar for help in making figures, and members of the Chattopadhyay laboratory for their comments and discussions.
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The authors declare that there is no conflict of interest.
Research Involving Human Participants and/or Animals
Sheep brains of freshly sacrificed animals were obtained from a local slaughterhouse for purification of brain spectrin following the guidelines of the Institutional Animal and Bioethics Committee of Saha Institute of Nuclear Physics.
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Madhurima Mitra and Arunima Chaudhuri contributed equally to this work.
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Mitra, M., Chaudhuri, A., Patra, M. et al. Organization and Dynamics of Tryptophan Residues in Brain Spectrin: Novel Insight into Conformational Flexibility. J Fluoresc 25, 707–717 (2015). https://doi.org/10.1007/s10895-015-1556-7
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DOI: https://doi.org/10.1007/s10895-015-1556-7