An Anionic Porphyrin Binds β-Lactoglobulin A at a Superficial Site Rich in Lysine Residues
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Binding of small ligands to globular proteins remains a major research topic in biophysics. We have studied the binding of several photoactive dyes to β-lactoglobulin (BLG), as a model to investigate the photoinduced effects of porphyrins on proteins. A combination of optical spectroscopies (fluorescence, circular dichroism) and molecular docking simulations were used to estimate the pH-dependence of the binding parameters and the docking location for meso-tetrakis(sulfonatophenyl)-porphyrin (TPPS). We have observed that the binding of TPPS is not modulated by the pH-mediated conformational transition of the protein (i.e., Tanford transition). Binding of TPPS appears to occur with some degree of negative cooperativity. Moreover, TPPS remains bound even upon partial denaturation of the protein. These results are consistent with a superficial binding site at a location removed from the aperture of the interior β-barrel. Binding occurs through electrostatic interactions between the negative SO3 − groups of TPPS and positively charged Lys and Arg residues. This is the first study that explores the interaction of an anionic porphyrin with BLGA in a pH range that spans across the Tanford transition. Establishing the location of the binding site will enable us to explain the photoinduced conformational effects mediated by TPPS on BLG.
KeywordsLactoglobulin Porphyrin Fluorescence spectroscopy Binding Photodynamic therapy (PDT)
Fluorescence resonance energy transfer
The research was supported by the 2006 Faculty Research Award of the University of Texas at San Antonio (to L.B.) and by the AFRL/HE grant # FA8650-07-1-6850 (to L.B.). The author would also like to thank Dr. Markandeswar Panda for the use of the CD spectrometer.
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