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The binding of 8-anilino-1-naphthalene sulfonate (ANS) to the nucleotide binding domain (N-domain) of the sarcoplasmic reticulum Ca2 -ATPase (SERCA) was studied. Molecular docking predicted two ANS binding modes (BMI and BMII) in the nucleotide binding site. The molecular interaction was confirmed as the fluorescence intensity of ANS was dramatically increased when in the presence of an engineered recombinant N-domain. Molecular dynamics simulation showed BMI (which occupies the ATP binding site) as the mode that is stable in solution. The above was confirmed by the absence of ANS fluorescence in the presence of a fluorescein isothiocyanate (FITC)-labeled N-domain. Further, the labeling of the N-domain with FITC was hindered by the presence of ANS, i.e., ANS was bound to the ATP binding site. Importantly, ANS displayed a higher affinity than ATP. In addition, ANS binding led to quenching the N-domain intrinsic fluorescence displaying a FRET pattern, which suggested the existence of a Trp-ANS FRET couple. Nonetheless, the chemical modification of the sole Trp residue with N-bromosuccinimide (NBS) discarded the existence of FRET and instead indicated structural rearrangements in the nucleotide binding site during ANS binding. Finally, Ca2 -ATPase kinetics in the presence of ANS showed a partial mixed-type inhibition. The Dixon plot showed the ANS-Ca2 -ATPase complex as catalytically active, hence supporting the existence of a functional dimeric Ca2 -ATPase in sarcoplasmic reticulum vesicles. ANS may be used as a molecular platform for the development of more effective inhibitors of Ca2 -ATPase and appears to be a new fluorescent probe for the nucleotide binding site. [Figure not available: see fulltext.]. © 2020, Springer Science Business Media, LLC, part of Springer Nature.
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The binding of 8-anilino-1-naphthalene sulfonate (ANS) to the nucleotide binding domain (N-domain) of the sarcoplasmic reticulum Ca2%2b-ATPase (SERCA) was studied. Molecular docking predicted two ANS binding modes (BMI and BMII) in the nucleotide binding site. The molecular interaction was confirmed as the fluorescence intensity of ANS was dramatically increased when in the presence of an engineered recombinant N-domain. Molecular dynamics simulation showed BMI (which occupies the ATP binding site) as the mode that is stable in solution. The above was confirmed by the absence of ANS fluorescence in the presence of a fluorescein isothiocyanate (FITC)-labeled N-domain. Further, the labeling of the N-domain with FITC was hindered by the presence of ANS, i.e., ANS was bound to the ATP binding site. Importantly, ANS displayed a higher affinity than ATP. In addition, ANS binding led to quenching the N-domain intrinsic fluorescence displaying a FRET pattern, which suggested the existence of a Trp-ANS FRET couple. Nonetheless, the chemical modification of the sole Trp residue with N-bromosuccinimide (NBS) discarded the existence of FRET and instead indicated structural rearrangements in the nucleotide binding site during ANS binding. Finally, Ca2%2b-ATPase kinetics in the presence of ANS showed a partial mixed-type inhibition. The Dixon plot showed the ANS-Ca2%2b-ATPase complex as catalytically active, hence supporting the existence of a functional dimeric Ca2%2b-ATPase in sarcoplasmic reticulum vesicles. ANS may be used as a molecular platform for the development of more effective inhibitors of Ca2%2b-ATPase and appears to be a new fluorescent probe for the nucleotide binding site. [Figure not available: see fulltext.]. © 2020, Springer Science%2bBusiness Media, LLC, part of Springer Nature.
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