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Updated: Jun 3, 2026

Chemical Modification of the Tryptophan Residue in a Recombinant Ca2+-ATPase N-domain for Studying Tryptophan-ANS FRET
Published on: October 9, 2021
Studying the interaction between ANS and the FF domain using fluorescence and amide 15N CEST NMR experiments
Nemika Thapliyal1, S Deepa1, Debajyoti De2
1Tata Institute of Fundamental Research Hyderabad, 36/P, Gopanpally Village, Serilingampally Mandal, Ranga Reddy District, Hyderabad 500046, India.
None:
The four-helix bundle FF domain from human HYPA/FBP11 folds via two intermediates I1 and I2. Here we study the interaction between 8-Anilinonaphthalene-1-sulfonic acid (ANS) and the A17G mutant of the FF domain (A17G FF) at equilibrium under conditions (15 °C) in which the major (visible) folded (F) state is in exchange with three sparsely populated (invisible) states, I1, I2 and the unfolded (U) state. Under these (native) conditions, A17G FF enhances ANS fluorescence showing that ANS interacts with A17G FF. To decipher the details of the interaction between ANS and A17G FF, amide 15N CEST experiments (16.4 T) were carried out using A17G FF samples containing varying (0-0.7 mM) amounts of ANS. A four-state analysis of the amide 15N CEST datasets using information from ANS fluorescence, traditional (amide 1HN-15N HSQC) NMR experiments and prior 15N CEST NMR experiments shows that ANS binds only to folding intermediate I1.
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