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ANS binds to the Ca2+-ATPase recombinant N-domain. Fluorescence spectra display a FRET-like pattern upon excitation at a wavelength of 295 nm. NBS-mediated chemical modification of Trp quenches the fluorescence of the N-domain, which leads to the absence of energy transfer (FRET) between the Trp residue and ANS.
The sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) is a P-type ATPase that has been crystallized in various conformations. Detailed functional information may nonetheless be obtained from isolated recombinant domains. The engineered (Trp552Leu and Tyr587Trp) recombinant nucleotide-binding domain (N-domain) displays fluorescence quenching upon ligand binding. An extrinsic fluorophore, namely, 8-anilino-1-naphthalene sulfonate (ANS), binds to the nucleotide-binding site via electrostatic and hydrophobic interactions with Arg, His, Ala, Leu, and Phe residues. ANS binding is evidenced by the increase in fluorescence intensity when excited at a wavelength (λ) of 370 nm. However, when excited at λ of 295 nm, the increase in fluorescence intensity seems to be coupled to the quenching of the N-domain intrinsic fluorescence. Fluorescence spectra display a Föster resonance energy transfer (FRET)-like pattern, thereby suggesting the presence of a Trp-ANS FRET pair, which appears to be supported by the short distance (~20 Å) between Tyr587Trp and ANS. This study describes an analysis of the Trp-ANS FRET pair by Trp chemical modification (and fluorescence quenching) that is mediated by N-bromosuccinimide (NBS). In the chemically modified N-domain, ANS fluorescence increased when excited at a λ of 295 nm, similar to when excited at a λ of 370 nm. Hence, the NBS-mediated chemical modification of the Trp residue can be used to probe the absence of FRET between Trp and ANS. In the absence of Trp fluorescence, one should not observe an increase in ANS fluorescence. The chemical modification of Trp residues in proteins by NBS may be useful for examining FRET between Trp residues that are close to the bound ANS. This assay will likely also be useful when using other fluorophores.
Föster resonance energy transfer (FRET) has become a standard technique for determining the distance between molecular structures after binding or interaction in protein structure and function studies1,2,3,4. In P-type ATPases, FRET has been used to investigate the structure and function of the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA)2,5,6,7,8, e. g., struct....
1. Determination (in silico) of the ANS and SERCA N-domain interaction
Molecular docking shows the binding of ANS to the nucleotide-binding site of the N-domain via electrostatic as well as hydrophobic interactions (Figure 1). Molecular distance (20 Å) between the Trp residue and ANS (bound to the nucleotide-binding site) supports the occurrence of FRET (Figure 1). The designed (engineered) recombinant N-domain was obtained at high purity by affinity chromatography (Figure 2) and was suitable for .......
Fluorescence spectra of the ANS-N-domain complex display a FRET-like pattern when excited at a λ of 295 nm, while the molecular distance (20 Å) between the Trp residue and ANS seems to support the occurrence of FRET (Figure 1). Trp chemical modification by NBS results in a less fluorescent N-domain (Figure 3B, Spectrum f); hence, energy transfer is not possible. The ANS fluorescence spectra are similar to that of the nonmodified N-domain when excited a.......
This work was partially funded by FAI-UASLP grant number C19-FAI-05-89.89 and CONACYT grant number 316463 (Apoyos a la Ciencia de Frontera: Fortalecimiento y Mantenimiento de Infraestructuras de Investigación de Uso Común y Capacitación Técnica 2021). The authors thank the technical assistance of Julian E. Mata-Morales in video edition.
....Name | Company | Catalog Number | Comments |
Acrylamide | Bio-Rad | 1610107 | SDS-PAGE |
Ammonium persulfate | Bio-Rad | 1610700 | SDS-PAGE |
8-Anilino-1-naphthalenesulfonic acid | Sigma-Aldrich | A1028 | Fluorophore |
Bis-acrylamide | Bio-Rad | 1610125 | SDS-PAGE |
N-Bromosuccinimide | Sigma-Aldrich | B81255 | Chemical modification |
N,N-dimethylformamide | J.T. Baker | 9213-12 | Stock solution preparation |
Fluorescein isothiocyanate | Sigma-Aldrich | F7250 | Chemical fluorescence label |
Fluorescence cuvette | Hellma | Z801291 | Fluorescence assay |
Fluorescence Spectrofluorometer | Shimadzu | RF 5301PC | Fluorescence assay |
HisTrap™ FF | GE Healtcare | 11-0004-59 | Protein purification |
IPTG, Dioxane free | American Bionalytical | AB00841-00010 | Protein expression |
Imidazole | Sigma-Aldrich | I5513-25G | Protein purification |
LB media | Fisher Scientific | 10000713 | Cell culture |
Pipetman L P10L | Gilson | FA10002M | Fluorescence assay |
Pipetman L P100L | Gilson | FA10004M | Fluorescence assay |
Pipetman L P200L | Gilson | FA10005M | Fluorescence assay |
Pipetman L P1000L | Gilson | FA10006M | Fluorescence assay |
Pipetman L P5000L | Gilson | FA10007 | Fluorescence assay |
Precision plus std | Bio-Rad | 1610374 | SDS-PAGE |
Sodium dodecyl sulphate | Bio-Rad | 1610302 | SDS-PAGE |
Sodium phosphate dibasic | J.T. Baker | 3828-19 | Buffer preparation |
Sodium phosphate monobasic | J.T. Baker | 3818-01 | Buffer preparation |
Syringe filter 0.2 um | Millipore | GVWP04700 | Solution filtration |
Temed | Bio-Rad | 1610801 | SDS-PAGE |
Tris | Bio-Rad | 1610719 | SDS-PAGE |
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