New insight on 8-anilino-1-naphthalene sulfonic acid interaction with TgFNR for hydrophobic exposure analysis Kulwant Singh ⁎, Islam Hussain, Vibhor Mishra, Md. Sohail Akhtar ⁎ Molecular and Structural Biology Division, CSIR-Central Drug Research Institute, Sector 10, Jankipuram Extension, Lucknow PIN 226 031, INDIA abstract article info Article history: Received 13 August 2018 Received in revised form 29 October 2018 Accepted 29 October 2018 Available online 31 October 2018 The exposed hydrophobic patches of protein are widely detected through the binding by the fluorescent probes such as 1-anilino-8-naphthalene sulfonate (ANS), Nile Red (NR) and 1-(N-phenylamino) naphthalene, N-(1-Naphthyl) aniline (1NPN). Interestingly, at pH 4, where the Toxoplasma gondii Ferredoxin-NADP(+) reduc- tase (TgFNR) is stable, an exclusive binding and fluorescence emission was observed for ANS. To understand the underlying difference in the binding of ANS, NR and 1NPN; their effect on the protein structure was studied in detail. ANS was found to interact with TgFNR via electrostatic as well as hydrophobic interactions at pH 4. NR and 1NPN did not show any such binding to TgFNR in the similar conditions, however showed strong hydropho- bic interaction in the presence of NaCl or DSS (2, 2-dimethyl-2-silapentane-5-sulfonate). The subsequent struc- tural studies suggest that ANS, NaCl and DSS induced partial unfolding of TgFNR by modulating ionic interactions of the enzyme, leading to the exposure of buried hydrophobic patches amicable for the binding by NR and 1NPN. The induced unfolding of TgFNR by ANS is unique and thus cautions to use the fluorescent dye as simple indicator to probe the exposed hydrophobic patches of the protein or its folding intermediates. © 2018 Elsevier B.V. All rights reserved. Keywords: 1-anilino-8-naphthalene sulfonate (ANS) Nile Red 1NPN FRET Protein stability Folding intermediates 1. Introduction The fluorescent dye, 1-anilino-8-naphthalene sulfonate (ANS) is an amphipathic molecule consisting of a hydrophobic anilinonaphthalene group and a charged sulfonate group [1,2]. ANS has been extensively used as a probe to detect the exposed hydrophobic patches of pro- teins and its folding intermediates, binding pockets of several carrier proteins, misfolded and aggregated conformation of proteins [3–9]. The restricted mobility of ANS upon binding to the non-polar resi- dues results in blue shift of fluorescence emission maxima and the increase of fluorescence intensity [10,11]. ANS is often considered as non-fluorescent probe when bound to the exposed surface of pro- teins because the steady state fluorescence measurement is much less as compared to when it bound to the buried hydrophobic sites [12–14]. Conversely, the role of electrostatic interactions where ion pairs are formed between the sulfonate group of ANS and cationic groups (especially Lys/Arg/His) of proteins and poly amino acids is also reported by measuring the fluorescent lifetimes and even steady-state fluorescence measurements [15–19]. In addition to the identification and characterization of hydrophobic binding sites of proteins, ANS has also been found to induce partial folding to the acid denatured states of Cytochrome C and Pectate lyase C [20,21]. Furthermore, ANS also acts to induce stability and functions as a conformation tightening agent as its binding to the acid expanded and most hydrated conformations of bovine serum albumin and gamma-globulin leads to the extensive molecular compaction [22,23]. The apicoplast and the proteins present therein are specific tar- gets for chemotherapy of apicomplexan parasites. Toxoplasma gondii Ferredoxin-NADP(+) reductase (TgFNR) is a monomeric enzyme localized in the apicoplast and is also an important drug target. The enzyme operates as a general electron switch at the bifurcation step of many different electron transfer pathways [24,25]. The high-resolution structure of TgFNR is yet to be determined; however its structural information has been deduced from the homology modeling with maize root FNR. The overall topology of TgFNR is similar to maize root FNR, having a typical separation between FAD- binding domain and NADP + -binding domain. The primary amino acid sequence of TgFNR contains almost equal number of polar (176) and non-polar (179) amino acids with a theoretical isoelectric point of about 8.5 [26,27]. Under physiological conditions, the recombinant TgFNR is stabilized in an open conformation with optimum activity. The enzyme is also significantly stable over a wide pH range between pH 4-pH 8 [26,27]. International Journal of Biological Macromolecules 122 (2019) 636–643 Abbreviations: ANS, 1-anilino-8-naphthalene sulfonate; NR, Nile Red; CD, Circular Dichroism; DSS, 2,2-dimethyl-2-silapentane-5-sulfonate; 1NPN, 1-(N-phenylamino) naphthalene, N-(1-Naphthyl) aniline; TgFNR, Toxoplasma gondii Ferredoxin-NADP(+) reductase; FRET, Fluorescence resonance energy transfer; CGH buffer, Citrate Glycine Hepes buffer. ⁎ Corresponding authors. E-mail addresses: ksinghcdri@gmail.com (K. Singh), sohail@cdri.res.in (M.S. Akhtar). https://doi.org/10.1016/j.ijbiomac.2018.10.208 0141-8130/© 2018 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac