title: Exploring NMR methods as a tool to select suitable fluorescent nucleotide analogues.
abstract: Fluorescent analogues provide important tools for biochemical/biophysical research. However, the analogues contain chemical modifications much larger than those known to affect ligand-binding, such as the inversion of a carbon centre or substitution of an atom. We lack experimental tools and protocols to select the most appropriate fluorescent analogue. Herein, we use several NMR spectroscopy methods, including Saturation Transfer Difference (STD), STD competition and transferred nuclear Overhauser effect spectroscopy (Tr-NOESY), as tools to select appropriate fluorescent probes. Annexin A6 (AnxA6) is a ubiquitous protein that forms in vitro GTP-induced ion channels. We used this protein as a model and screened guanosine triphosphate (GTP) and four fluorescent analogues against AnxA6. STD reported that the GTP moiety of all ligands made similar contacts with the protein, despite additional interactions between the fluorescent tags and AnxA6. Competition STD experiments verified that the analogues and GTP bind to the same site. Tr-NOESY indicated that the bound conformation of the base relative to ribose is altered for some analogues compared to GTP. MANT-GTP or the BODIPY thioester of guanosine 5'-O-(3-thiotriphosphate) are the most suitable fluorescent analogues for AnxA6, according to NMR. These results reveal NMR as a useful technique to select and design proper fluorescent tags for biochemical/biophysical assays.
authors.affiliation: Chemical Biology Division, Instituto de Tecnologia Química e Biológica (ITQB-UNL), Oeiras, Portugal. pgroves@itqb.unl.pt
authors.forename: Patrick
authors.initials: P
authors.lastname: Groves
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Strzelecka-Kiliszek
authors.forename: Anna
authors.initials: A
authors.lastname: Sekrecka-Belniak
authors.forename: Angeles
authors.initials: A
authors.lastname: Canales
authors.forename: Jesús
authors.initials: J
authors.lastname: Jiménez-Barbero
authors.forename: Joanna
authors.initials: J
authors.lastname: Bandorowicz-Pikula
authors.forename: Sławomir
authors.initials: S
authors.lastname: Pikula
authors.forename: F Javier
authors.initials: FJ
authors.lastname: Cañada
mesh_terms: D017317:Annexin A6 / Q000032:analysis / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D009711:Nucleotides / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding
chemical_list: D017317:Annexin A6; D005456:Fluorescent Dyes; D009711:Nucleotides; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate