analyzing thioflavin t binding to amyloid fibrils by an equilibrium microdialysis-based technique分析thioflavin t绑定淀粉样原纤维的平衡microdialysis-based技术.pdfVIP

Analyzing Thioflavin T Binding to Amyloid Fibrils by an Equilibrium Microdialysis-Based Technique 1 1 2,3 1 Irina M. Kuznetsova , Anna I. Sulatskaya , Vladimir N. Uversky *, Konstantin K. Turoverov * 1 Laboratory of Structural Dynamics, Stability and Folding of Proteins, The Institute of Cytology, Russian Academy of Sciences, St. Petersburg, Russia, 2 Department of Molecular Medicine, College of Medicine, University of South Florida, Tampa, Florida, United States of America, 3 Institute for Biological Instrumentation, Russian Academy of Sciences, Pushchino, Moscow Region, Russia Abstract A new approach for the determination of the amyloid fibril – thioflavin T (ThT) binding parameters (the number of binding modes, stoichiometry, and binding constants of each mode) is proposed. This approach is based on the absorption spectroscopy determination of the concentration of free and bound to fibril dye in solutions, which are prepared by equilibrium microdialysis. Furthermore, the proposed approach allowed us, for the first time, to determine the absorption spectrum, molar extinction coefficient, and fluorescence quantum yield of the ThT bound to fibril by each binding modes. This approach is universal and can be used for determining the binding parameters of any dye interaction with a receptor, such as ANS binding to proteins in the molten globule state or to protein amorphous aggregates. Citation: Kuznetsova IM, Sulatskaya AI, Uversky VN, Turoverov KK (2012) Analyzing Thioflavin T Binding to Amyloid Fibrils by an Equilibrium Microdialysis-Based Technique. PLoS ONE 7(2): e30724. doi:10.1371/journal.pone.0030724 Editor: Maria Gasset, Consejo Superior de Investigaciones Cientificas, Spain Rec