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., structural fluctuations during the catalytic cycle have been analyzed in the whole protein by FRET7.
FRET donors are diverse, and range from small fluorescent (extrinsic) molecules to fluorescent proteins9,10. Tryptophan (Trp) residues (due to their fluorescence) are useful for identifying structural changes in protein amino acid sequences11,12. The fluorescence intensity of Trp depends substantially on the polarity of its surrounding environment13,14. Ligand binding usually generates structural rearrangements in proteins/enzymes15,16. If Trp is present at or located close to the protein binding site, structural fluctuations frequently affect the degree of Trp exposure to aqueous media13,14; thus, the change in polarity results in quenching of the Trp fluorescence intensity13,14. Hence, the fluorescent property of Trp is useful for performing ligand binding studies for enzymes. Other physical phenomena may also lead to Trp fluorescence quenching17,18,19,20, e. g., FRET and changes in medium polarity. Energy transfer from the excited state of Trp to a fluorophore also has potential applications, e. g., affinity determination of small ligands in proteins21. Indeed, Trp has been primarily used as a fluorescence donor in FRET studies in proteins22,23,24, e. g., in terbium (Tb3+) FRET studies, a Trp residue is used frequently as an antenna for energy transfer to Tb3+ 25,26,27. Trp displays various advantages over other FRET donors due to its inherent constitutive character in the protein structure, which eliminates the need for preparative processes that may affect the function/structure of the studied protein24. Thus, the identification of radiative decays (energy transfer and changes in the medium polarity that are induced by protein structural rearrangements) is important for drawing accurate conclusions regarding ligand binding in protein structural studies13,14,19,28.
In protein structural studies, an extrinsic fluorophore, namely, 8-anilino-1-naphthalene sulfonate (ANS), has been primarily used in experiments related to protein folding/unfolding28,29. ANS binds to proteins/enzymes in the native state, usually in the binding sites of substrates31,32,33; an increase in ANS fluorescence quantum yield (ΦF) (namely, an increase in fluorescence intensity) is induced by exciting the protein at λ=370 nm when suitable interactions of ANS with Arg and His residues in hydrophobic pockets occur34,35,36,37. In various studies, the occurrence of FRET (when exciting at λ within 280-295 nm) between Trp residues (donors) and ANS (acceptor) has been reported, which is based on the following: 1) overlap of the fluorescence emission spectrum of Trp and excitation spectrum of ANS, 2) identification of a suitable distance between one or more Trp residue(s) and ANS for energy transfer, 3) high ANS quantum yield when bound in protein pockets, and 4) characteristic FRET pattern in the fluorescence spectra of the protein in the presence of ANS3,17,27,37,38.
Recently, ligand binding to the nucleotide-binding domain (N-domain) in SERCA and other P-type ATPases have been investigated using engineered recombinant N-domains40,41,42,43,44,45,46. Molecular engineering of the SERCA N-domain has been used to move the sole Trp residue (Trp552Leu) to a more dynamic structure (Tyr587Trp) that is close to the nucleotide-binding site, where fluorescence variations (quenching) may be used to monitor structural changes upon ligand binding34. Experimental results have demonstrated that ANS binds (as ATP) to the nucleotide-binding site in the purified recombinant SERCA N-domain34. Interestingly, the ANS fluorescence increases upon binding to the N-domain upon excitation at a λ of 295 nm, while the intrinsic fluorescence of the N-domain decreases34, thereby producing a FRET pattern that suggests the formation of a Trp-ANS FRET pair.
The use of NBS has been proposed to determine the content of Trp residues in proteins47 by absorbance assay of modified proteins. NBS modifies the highly absorbing indole group of Trp to the less absorbent oxindole47,48. This results in the loss (quenching) of the Trp fluorescent property40. Hence, NBS-mediated chemical modification of Trp residues may be used as an assay to define the role of Trp (as a donor) when FRET is hypothesized.
This protocol describes the chemical modification of the sole Trp residue by NBS in the engineered recombinant N-domain of SERCA as a protein model. Experimental results demonstrate that the ANS fluorescence intensity still increases in the chemically NBS-modified N-domain34, which lacks intrinsic fluorescence. Therefore, the assay is useful for demonstrating the absence of FRET between the Trp residue and ANS when bound to the N-domain34,40,49. Hence, this assay (NBS chemical modification of Trp) is useful in proving the presence of the Trp-ANS FRET pair in proteins.
1. Determination (in silico) of the ANS and SERCA N-domain interaction
2. Expression and purification of the recombinant N-domain
3. Monitor the formation of the ANS-N-domain complex based on ANS and N-domain fluorescence intensity changes.
4. N-domain intrinsic fluorescence titration by Trp chemical modification with NBS.
5. Titrate the NBS-modified N-domain with ANS by recording fluorescence spectra at 25 °C.
6. Evidence of ANS binding to the chemically modified N-domain by excitation at λ=370 nm.
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 experiments. Fluorescence spectra of the ANS-N-domain complex displayed a FRET-like pattern upon excitation at λ=295 nm (Figure 3A). Chemical modification of the Trp residue by NBS led to quenching of the intrinsic fluorescence of the N-domain (Figure 3B). In the chemically NBS-modified N-domain, the experimental results demonstrate that ANS fluorescence increased upon excitation at λ=295 nm (Figure 3C), similar to that observed in the nonmodified N-domain (Figure 3A). Therefore, direct excitation of ANS at λ=295 nm provides the most energy for ANS fluorescence (Figure 3C), as suggested previously28. ANS binding to the chemically modified N-domain is evidenced by an increase in its fluorescence when excited at λ=370 nm (Figure 3D). Therefore, FRET does not occur between the Trp residue and ANS that is bound to the nucleotide-binding site.
Figure 1: Molecular docking of ANS to the nucleotide-binding site of the Ca2+-ATPase N-domain. ANS molecular docking was performed using AutoDock Vina software (http://vina.scripps.edu/) and a generated 3D model of the N-domain40. The engineered N-domain contains mutations Trp552Leu and Tyr587Trp (shown in blue). Amino acid residues that form the nucleotide-binding site are represented as balls and sticks and highlighted in orange. This figure has been modified with permission from Springer Nature: Springer, Journal of Fluorescence. Copyright (2020)34. Please click here to view a larger version of this figure.
Figure 2: SDS−PAGE of the engineered recombinant Ca2+-ATPase N-domain. The N-domain was subjected to affinity purification using a chromatographic column. Fractions that corresponded to absorption (at λ=280 nm) peaks were subjected to SDS−PAGE and visualized by Coomassie blue staining. The ~30 kDa His-tagged N-domain is formed by 27 kDa of N-domain Ca2+-ATPase and 3 kDa of poly-His tag. The Ca2+-ATPase N- domain purity was determined to be ≥95% by densitometry using the ImageJ software (https://imagej.nih.gov/ij/download.html). Please click here to view a larger version of this figure.
Figure 3: NBS-mediated chemical modification of the Trp residue in the N-domain disproves FRET between Trp and ANS that is bound to the nucleotide-binding site. A. FRET pattern of the ANS-N-domain complex upon excitation at λ=295 nm. ANS was added (final concentration in µM: Spectra a, 0; b, 0.2; c, 0.4; d, 0.6; e, 0.8; f, 1.0; g, 1.2; and h, 1.4) to the suspended N-domain (1 µM). B. Fluorescence quenching of the N-domain by NBS (NBS concentration in μM: a, 0; b, 1; c, 2; d, 3; e, 4; and f, 6). NBS mediates chemical modification of the Trp residue. N-domain intrinsic fluorescence was observed upon excitation at λ=295 nm. C. Fluorescence spectra of ANS that is bound to the chemically modified N-domain upon excitation at λ=295 nm. The experimental conditions are as in A. Figures A, B, and C have been modified with permission from Springer Nature: Springer, Journal of Fluorescence. Copyright (2020)34. D. Fluorescence spectra of ANS that is bound to the chemically modified N-domain upon excitation at λ=370 nm. The N-domain was suspended in 1 ml of 50 mM phosphate buffer (pH 8.0) and aliquots of NBS, and ANS was added accordingly, as described in A (ANS) and B (NBS). Please click here to view a larger version of this figure.
Figure 4: ANS fluorescence spectra. ANS (1.4 μM) in 50 mM phosphate buffer with pH 8.0 was excited at λ of 295 and 370 nm; the spectra are presented in black and blue, respectively. Please click here to view a larger version of this figure.
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 at a λ of 295 nm (Figure 3A and C).
Therefore, direct excitation of ANS at a λ of 295 nm is the main source of ANS fluorescence when it is bound to the ATP binding site (Figure 3C), which is in agreement with the mechanism that was proposed by other authors28. Therefore, FRET from the Trp residue to bound ANS does not occur in the N-domain-ANS complex. Nonetheless, NBS-mediated chemical modification of Trp residues in other proteins supports FRET between Trp and ANS, e. g., in the enzymes xylose reductase from Neurospora crassa49, the α-subunit of F1-ATPase from yeast mitochondria58, and thermolysin59.
The assay would perform well in proteins/enzymes with hydrophobic pockets (binding sites) that contain His and Arg residues, as these contribute to the stabilization of the ANS interaction. Additionally, such proteins should ideally contain a sole Trp residue that is located at the protein surface, namely, accessible for rapid reaction with NBS40,41,49.
Alternatively, to analyze the Trp-ANS FRET pair in proteins, chemical modification of His residues by acetylation and succinylation may be used to hamper the ANS interaction in the protein/enzyme binding site60. Deletion of the Trp residue by mutation is another strategy for analyzing FRET. However, this might be time-consuming, and the constructs may exhibit structural differences, thereby affecting ligand binding61. Similarly, mutation of Arg and His residues at the ligand-binding site may generate unforeseen structural changes, thereby rendering the mutated protein unsuitable for experiments62.
With regards to the Trp residue, the performance of the NBS-chemical modification assay would be limited in the following cases: 1) if the Trp residue is buried deeply at the core of a well folded and compact protein; since the NBS moiety would be unable to access the Trp residue due to the absence of large cavities41,48,63, 2) if Trp residues is located in a membrane-embedded structures (transmembrane α-helix), as the aqueous character of NBS will prevent it from entering the hydrophobic medium32,56,64, 3) if the protein structure contains multiple Trp residues; as the variations in accessibility and physicochemical environment may be large, thereby rendering difficult the assignment of a fluorescence signal change to a Trp residue32,41,56, 4) if ANS binding to proteins is due mainly to hydrophobic interaction, as the ANS fluorescence increase is due mainly to electrostatic interactions32,65,66,67, and e) if static quenching of Trp occurs, e. g., in the presence of oxygen68.
NBS mediated chemical modification of Trp residues appears to be a rapid and easy assay for studying FRET between Trp and ANS that is bound to proteins/enzymes. Other Trp-modifying reagents may be used instead of NBS, e. g., hydroxy-5-nitrobenzyl bromide (HNB)69,70. Finally, the assay may be applicable to the detection of proposed FRET pairs of Trp with other flurophores21.
The authors have nothing to disclose.
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.
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 |