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Method Article

Chemical Modification of the Tryptophan Residue in a Recombinant Ca2+-ATPase N-domain for Studying Tryptophan-ANS FRET

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DOI:

10.3791/62770

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October 9th, 2021

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1Instituto de Física, Universidad Autónoma de San Luis Potosí

In This Article

Summary

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.

Abstract

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.

Introduction

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.

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Protocol

1. Determination (in silico) of the ANS and SERCA N-domain interaction

  1. Generate a three-dimensional (3D) structure of the protein (SERCA N-domain) by molecular modeling using the preferred protein modeling software50.
  2. Identify the amino acid residues that form the nucleotide-binding site using the preferred molecular structure software51, and determine the presence of Arg and Lys residues35; these are required for ANS binding and to increase the fluorescence intensity (quantum yield).
  3. Perform molecular docking (using the preferred docking software)52,53,54 to determine the interactions of ATP, fluorescein isothiocyanate (FITC) (which forms a covalent bond with Lys515 labeling the nucleotide-binding site), and ANS with amino acids residues in the nucleotide-binding site (Figure 1).
  4. Calculate the molecular distance (Å) between Trp residue and bound ANS using the measurement tool in the preferred software.
  5. Perform molecular dynamics simulation of ANS-N-domain complex to determine the stability of the interaction52,54. Then, perform the in vitro experiments when the stability of the complex has been confirmed.

2. Expression and purification of the recombinant N-domain

  1. Synthesize the gene coding for N-domain40.
  2. Design and construct the plasmid that contains the synthetic gene that codes for the N-domain40.
  3. Express and purify by affinity chromatography (Ni-NTA), the engineered recombinant N-domain. Perform an SDS-PAGE of the purified protein to determine the purity (Figure 2)40.
  4. Determine the protein concentration by studying the absorbance at λ of 280 nm with the N-domain extinction coefficient (ε= 11,960 M-1·cm-1)40.

3. Monitor the formation of the ANS-N-domain complex based on ANS and N-domain fluorescence intensity changes.

  1. Prepare an ANS stock solution in N,N-dimethylformamide.
    1. Weigh a small amount (1-5 mg) of ANS, and dissolve it in 1 mL of the final volume of N,N-dimethylformamide, e. g., 3.2 mg (10.69 mM final concentration).
    2. Prepare a 100 µM ANS aqueous stock solution using the ANS solution in N,N-dimethylformamide, e. g., add 9.4 µL of the 10.69 mM ANS solution to 990.6 µL of 50 mM phosphate buffer with pH 8.0 to obtain a final volume of 1 mL.
    3. Mix the solutions by vortexing 3 - 5 times for 15 s.
      NOTE: In the following experiment, use only the ANS aqueous stock solution. Freshly prepare the ANS aqueous stock solution before initiating the experiments.
  2. Prepare the NBS stock solution in N,N-dimethylformamide.
    1. Weigh a small amount (1-5 mg) of NBS, and dissolve it in 1 mL of N,N-dimethylformamide, e. g., 5.3 mg in 1 mL (29.78 mM final concentration).
    2. Prepare a 1 mM NBS aqueous stock solution using the NBS solution in N,N-dimethylformamide, e. g., add 3.36 µL of the 29.78 mM NBS solution to 96.64 µL of 50 mM phosphate buffer with pH 8.0 to obtain a final volume 0.1 mL.
    3. Mix the solutions by vortexing 3 - 5 times for 15 s.
      NOTE: Freshly prepare the NBS aqueous stock solution before starting the experiments.
  3. Titrate the N-domain with ANS, and record the fluorescence spectra by excitation at λ=295 nm at 25 °C.
    1. Obtain the fluorescence spectrum baseline.
      1. Place 1 mL of 50 mM phosphate buffer with pH 8.0 in a 1 mL fluorescence quartz cuvette.
      2. Position the cell in the thermo-stated cell chamber (25 °C) of the spectrofluorometer and set the excitation λ to 295 nm.
      3. Record the fluorescence spectrum (305 - 550 nm).
        NOTE: The fluorescence spectrum of the 50 mM phosphate buffer with pH 8.0, which serves as the blank sample, is subtracted from all obtained fluorescence spectra.
    2. Obtain the intrinsic fluorescence spectrum of the N-domain.
      1. Place 900 µL of 50 mM phosphate buffer with pH 8.0 in a fluorescence quartz cuvette.
      2. Add 100 µL of N-domain (10 μM) suspension to obtain a 1 μM N-domain final concentration in a 1 mL final volume.
      3. Gently homogenize using a micropipette 20 times to ensure the homogeneity of the solution.
        NOTE: The protein should be freshly purified to obtain high-quality intrinsic fluorescence spectra, e. g., the purified recombinant N-domain may only be used for a week after purification.
      4. Position the cell in the thermo-stated cell chamber (25 °C) of the spectrofluorometer and set the excitation λ to 295 nm.
      5. Record the N-domain intrinsic fluorescence spectrum (305-550 nm).
    3. Add ANS, and obtain the fluorescence spectrum by excitation at λ=295 nm.
      1. Add a 2 µL aliquot of 100 µM ANS aqueous stock solution to the suspended N-domain (1 μM) to obtain a 0.2 µM ANS final concentration.
      2. Gently homogenize using a micropipette 20 times to ensure the homogeneity of the solution.
      3. Position the cell in the thermo-stable cell chamber (25 °C) of the spectrofluorometer and set the excitation λ to 295 nm.
      4. Record the fluorescence spectrum (305-550 nm).
      5. Repeat the ANS additions and fluorescence spectra recording above 1:1 molar relationship ANS:N-domain.
      6. Subtract the blank spectrum from each spectrum using suitable software.
      7. Plot all the spectra in a single graph.
      8. Determine whether the spectra form a FRET-like pattern. The ANS-N-domain fluorescence spectra form a FRET-like pattern (Figure 3A).

4. N-domain intrinsic fluorescence titration by Trp chemical modification with NBS.

  1. Repeat steps 3.3.1 and 3.3.2.
  2. Add a 1 µL aliquot of 1 mM NBS aqueous stock solution to the suspended N-domain (1 μM) to obtain a final concentration of 1 µM NBS.
  3. Gently homogenize by using a micropipette 20 times to ensure the homogeneity of the solution.
  4. Position the cell in the thermo-stable cell chamber (25 °C) of the spectrofluorometer and set the excitation λ to 295 nm.
  5. Record the fluorescence spectrum (305-550 nm) (Figure 3B).
  6. Repeat the NBS addition and fluorescence spectra recording until minimal N-domain intrinsic fluorescence quenching is observed40. In the N-domain, this usually occurs at a molar ratio of 5-6 NBS/N-domain40.
    NOTE: NBS rapidly quenches (<5 s) the intrinsic fluorescence of the N-domain; a decrease in fluorescence intensity is observed. Proceed immediately to the next step, as NBS may also react with other amino acid residues47.
  7. Subtract the blank spectrum from each spectrum using suitable software.
  8. Plot all spectra in a single graph (Figure 3B).

5. Titrate the NBS-modified N-domain with ANS by recording fluorescence spectra at 25 °C.

  1. Perform Step 3.3.3 using the NBS modified N-domain that was generated in Step 4.
  2. Subtract the blank spectrum from each spectrum using suitable software.
  3. Plot all spectra in a single graph (Figure 3C).
  4. The generated fluorescence spectra (Figure 3C) support or refute the occurrence of FRET, i.e., when FRET occurs, the ANS fluorescence does not increase and vice-versa.

6. Evidence of ANS binding to the chemically modified N-domain by excitation at λ=370 nm.

  1. Perform Step 3.3.3 using the NBS modified N-domain that was generated in Step 4 but changing the excitation λ to 370 nm.
  2. Subtract the blank spectrum from each spectrum using suitable software.
  3. Plot all spectra in a single graph (Figure 3D).
  4. Confirm ANS binding to the N-domain by observing the increase in ANS fluorescence intensity. ANS binding to the N-domain shows a fluorescence increase when excited at λ=370 nm (Figure 3D). As a control, the fluorescence spectrum of ANS (alone) in 50 mM phosphate buffer with pH 8.0 was obtained exciting at λ of 295 and 370 nm (Figure 4, not shown in video).
    NOTE: The stoichiometric relationship of NBS:Trp that is required for chemical modification depends on the degree of burying of the Trp residue(s) in the protein under study46,47,55,56. Therefore, it is recommended to determine the NBS:protein/(Trp) molar ratio, beforehand.

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Results

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 fluor...

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Discussion

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...

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Disclosures

The authors declare that they have no competing financial interests.

Acknowledgements

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.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
AcrylamideBio-Rad1610107SDS-PAGE
Ammonium persulfateBio-Rad1610700SDS-PAGE
8-Anilino-1-naphthalenesulfonic acidSigma-AldrichA1028Fluorophore
Bis-acrylamideBio-Rad1610125SDS-PAGE
N-BromosuccinimideSigma-AldrichB81255Chemical modification
N,N-dimethylformamideJ.T. Baker9213-12Stock solution preparation
Fluorescein isothiocyanateSigma-AldrichF7250Chemical fluorescence label
Fluorescence cuvetteHellmaZ801291Fluorescence assay
Fluorescence SpectrofluorometerShimadzuRF 5301PCFluorescence assay
HisTrap™ FFGE Healtcare11-0004-59Protein purification
IPTG, Dioxane freeAmerican BionalyticalAB00841-00010Protein expression
ImidazoleSigma-AldrichI5513-25GProtein purification
LB mediaFisher Scientific10000713Cell culture
Pipetman L P10LGilsonFA10002MFluorescence assay
Pipetman L P100LGilsonFA10004MFluorescence assay
Pipetman L P200LGilsonFA10005MFluorescence assay
Pipetman L P1000LGilsonFA10006MFluorescence assay
Pipetman L P5000LGilsonFA10007Fluorescence assay
Precision plus stdBio-Rad1610374SDS-PAGE
Sodium dodecyl sulphateBio-Rad1610302SDS-PAGE
Sodium phosphate dibasicJ.T. Baker3828-19Buffer preparation
Sodium phosphate monobasicJ.T. Baker3818-01Buffer preparation
Syringe filter 0.2 umMilliporeGVWP04700Solution filtration
TemedBio-Rad1610801SDS-PAGE
TrisBio-Rad1610719SDS-PAGE

References

  1. Munishkina, L. A., Fink, A. L. Fluorescence as a method to reveal structures and membrane-interactions of amyloidogenic proteins. Biochimica et Biophysica Acta (BBA) - Biomembranes. 1768 (8), 1862-1885 (2007).
  2. Dong, X., Thomas, D. D. Time-resolved FRET reveals the structural mechanism of SERCA-PLB regulation. Biochemical and Biophysical Research Communications. 449 (2), 196-201 (2014).
  3. Szilvay, G. R., Blenner, M. A., Shur, O., Cropek, D. M., Banta, S. A FRET-based method for probing the conformational behavior of an intrinsically disordered repeat domain from Bordetella pertussis adenylate cyclase. Biochemistry. 48 (47), 11273-11282 (2009).
  4. Sun, Y., Wallrabe, H., Booker, C. F., Day, R. N., Periasamy, A. Three-color spectral FRET microscopy localizes three interacting proteins in living cells. Biophysical Journal. 99 (4), 1274-1283 (2010).
  5. Cornea, R. L., et al. High-throughput FRET assay yields allosteric SERCA activators. Journal of Biomolecular Screening. 18 (1), 97-107 (2013).
  6. Gruber, S. J., et al. Discovery of enzyme modulators via high-throughput time-resolved FRET in living cells. Journal of Biomolecular Screening. 19 (2), 215-222 (2014).
  7. Dyla, M., et al. Dynamics of P-type ATPase transport revealed by single-molecule FRET. Nature. 551 (7680), 346-351 (2017).
  8. Corradi, G. R., Adamo, H. P. Intramolecular fluorescence resonance energy transfer between fused autofluorescent proteins reveals rearrangements of the N- and C-terminal segments of the plasma membrane Ca2+ pump involved in the activation. The Journal of Biological Chemistry. 282 (49), 35440-35448 (2007).
  9. Piston, D. W., Kremers, G. -J. Fluorescent protein FRET: The good, the bad and the ugly. Trends in Biochemical Sciences. 32 (9), 407-414 (2007).
  10. Ma, L., Yang, F., Zheng, J. Application of fluorescence resonance energy transfer in protein studies. Journal of Molecular Structure. 1077, 87-100 (2014).
  11. Chen, Y., Barkley, M. D. Toward understanding tryptophan fluorescence in proteins. Biochemistry. 37 (28), 9976-9982 (1998).
  12. Zelent, B., et al. Tryptophan fluorescence yields and lifetimes as a probe of conformational changes in human glucokinase. Journal of Fluorescence. 27 (5), 1621-1631 (2017).
  13. Callis, P. R. Binding phenomena and fluorescence quenching. I: Descriptive quantum principles of fluorescence quenching using a supermolecule approach. Journal of Molecular Structure. 1077, 14-21 (2014).
  14. Callis, P. R. Binding phenomena and fluorescence quenching. II: Photophysics of aromatic residues and dependence of fluorescence spectra on protein conformation. Journal of Molecular Structure. 1077, 22-29 (2014).
  15. Agarwal, P. K., Geist, A., Gorin, A. Protein dynamics and enzymatic catalysis: Investigating the peptidyl-prolyl cis-trans isomerization activity of cyclophilin A. Biochemistry. 43 (33), 10605-10618 (2004).
  16. Deng, H., Zhadin, N., Callender, R. Dynamics of protein ligand binding on multiple time scales: NADH binding to lactate dehydrogenase. Biochemistry. 40 (13), 3767-3773 (2001).
  17. van de Weert, M. Fluorescence quenching to study protein-ligand binding: common errors. Journal of fluorescence. 20 (2), 625-629 (2010).
  18. van de Weert, M., Stella, L. Fluorescence quenching and ligand binding: A critical discussion of a popular methodology. Journal of Molecular Structure. 998 (1-3), 144-150 (2011).
  19. Stella, L., van de Weert, M., Burrows, H. D., Fausto, R. Fluorescence spectroscopy and binding: Getting it right. Journal of Molecular Structure. 1077, 1-3 (2014).
  20. Credi, A., Prodi, L. Inner filter effects and other traps in quantitative spectrofluorimetric measurements: Origins and methods of correction. Journal of Molecular Structure. 1077, 30-39 (2014).
  21. Lee, M. M., Peterson, B. R. Quantification of small molecule-protein interactions using FRET between tryptophan and the pacific blue fluorophore. ACS Omega. 1 (6), 1266-1276 (2016).
  22. Zhang, Y., et al. Comparison of FÖrster-resonance-energy-transfer acceptors for tryptophan and tyrosine residues in native proteins as donors. Journal of Fluorescence. 23 (1), 147-157 (2013).
  23. Xie, Y., Maxson, T., Tor, Y. Fluorescent ribonucleoside as a FRET acceptor for tryptophan in native proteins. Journal of the American Chemical Society. 132 (34), 11896-11897 (2010).
  24. Ghisaidoobe, A. B. T. T., Chung, S. J. Intrinsic tryptophan fluorescence in the detection and analysis of proteins: A focus on Förster resonance energy transfer techniques. International Journal of Molecular Sciences. 15 (12), 22518-22538 (2014).
  25. Goryashchenko, A. S., et al. Genetically encoded FRET-sensor based on terbium chelate and red fluorescent protein for detection of caspase-3 activity. International Journal of Molecular Sciences. 16 (7), 16642-16654 (2015).
  26. Arslanbaeva, L. R., et al. Induction-resonance energy transfer between the terbium-binding peptide and the red fluorescent proteins DsRed2 and TagRFP. Biophysics. 56 (3), 381-386 (2011).
  27. Di Gennaro, A. K., Gurevich, L., Skovsen, E., Overgaard, M. T., Fojan, P. Study of the tryptophan-terbium FRET pair coupled to silver nanoprisms for biosensing applications. Physical Chemistry Chemical Physics. 15 (22), 8838-8844 (2013).
  28. Hawe, A., Poole, R., Jiskoot, W. Misconceptions over Förster resonance energy transfer between proteins and ANS/bis-ANS: Direct excitation dominates dye fluorescence. Analytical Biochemistry. 401 (1), 99-106 (2010).
  29. Ghosh, U., Das, M., Dasgupta, D. Association of fluorescent probes 1-anilinonaphthalene-8-sulfonate and 4,4´-dianilino-1,1´-binaphthyl-5,5´-disulfonic acid with T7 RNA polymerase. Biopolymers. 72 (4), 249-255 (2003).
  30. Vreuls, C., et al. Guanidinium chloride denaturation of the dimeric Bacillus licheniformis BlaI repressor highlights an independent domain unfolding pathway. The Biochemical Journal. 384, 179-190 (2004).
  31. Möller, M., Denicola, A. Study of protein-ligand binding by fluorescence. Biochemistry and Molecular Biology Education. 30 (5), 309-312 (2002).
  32. Chang, L., Wen, E., Hung, J., Chang, C. Energy transfer from tryptophan residues of proteins to 8-anilinonaphthalene-1-sulfonate. Journal of Protein Chemistry. 13 (7), 635-640 (1994).
  33. Togashi, D. M., Ryder, A. G. A fluorescence analysis of ANS bound to bovine serum albumin: Binding properties revisited by using energy transfer. Journal of Fluorescence. 18 (2), 519-526 (2008).
  34. Dela Cruz-Torres, V., Cataño, Y., Olivo-Rodríguez, M., Sampedro, J. G. ANS interacts with the Ca2+-ATPase nucleotide binding site. Journal of Fluorescence. 30 (3), 483-496 (2020).
  35. Gasymov, O. K., Glasgow, B. J. ANS fluorescence: Potential to augment the identification of the external binding sites of proteins. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics. 1774 (3), 403-411 (2007).
  36. Matulis, D., Lovrien, R. 1-anilino-8-naphthalene sulfonate anion-protein binding depends primarily on ion pair formation. Biophysical Journal. 74 (1), 422-429 (1998).
  37. Samukange, V., Yasukawa, K., Inouye, K. Interaction of 8-anilinonaphthalene 1-sulphonate (ANS) and human matrix metalloproteinase 7 (MMP-7) as examined by MMP-7 activity and ANS fluorescence. Journal of Biochemistry. 151 (5), 533-540 (2012).
  38. Qin, J., et al. Selective and sensitive homogenous assay of serum albumin with 1-anilinonaphthalene-8-sulphonate as a biosensor. Analytica Chimica Acta. 829, 60-67 (2014).
  39. Malik, A., Kundu, J., Karmakar, S., Lai, S., Chowdhury, P. K. Interaction of ANS with human serum albumin under confinement: Important insights and relevance. Journal of Luminescence. 167, 316-326 (2015).
  40. Páez-Pérez, E. D., De La Cruz-Torres, V., Sampedro, J. G. Nucleotide binding in an engineered recombinant Ca2+-ATPase N-domain. Biochemistry. 55 (49), 6751-6765 (2016).
  41. Sampedro, J. G., Nájera, H., Uribe-Carvajal, S., Ruiz-Granados, Y. G. Mapping the ATP binding site in the plasma membrane H+-ATPase from Kluyveromyces lactis. Journal of fluorescence. 24 (6), 1849-1859 (2014).
  42. Abu-Abed, M., Millet, O., MacLennan, D. H., Ikura, M. Probing nucleotide-binding effects on backbone dynamics and folding of the nucleotide-binding domain of the sarcoplasmic/endoplasmic-reticulum Ca2+-ATPase. The Biochemical Journal. 379, Pt 2 235-242 (2004).
  43. Abu-Abed, M., Mal, T. K., Kainosho, M., MacLennan, D. H., Ikura, M. Characterization of the ATP-binding domain of the sarco(endo)plasmic reticulum Ca2+-ATPase: probing nucleotide binding by multidimensional NMR. Biochemistry. 41 (4), 1156-1164 (2002).
  44. Sazinsky, M. H., Mandal, A. K., Argüello, J. M., Rosenzweig, A. C. Structure of the ATP binding domain from the Archaeoglobus fulgidus Cu+-ATPase. Journal of Biological Chemistry. 281 (16), 11161-11166 (2006).
  45. Liu, L., et al. Crystallization and preliminary X-ray studies of the N-domain of the Wilson disease associated protein. Acta Crystallographica Section F: Structural Biology and Crystallization Communications. 65 (6), 621-624 (2009).
  46. Banci, L., et al. The binding mode of ATP revealed by the solution structure of the N-domain of human ATP7A. Journal of Biological Chemistry. 285 (4), 2537-2544 (2010).
  47. Spande, T. F., Witkop, B. Determination of the tryptophan content of proteins with N-bromosuccinimide. Methods in Enzymology. 11, 498-506 (1967).
  48. Spande, T. F., Green, N. M., Witkop, B. The Reactivity toward N-bromosuccinimide of tryptophan in enzymes, zymogens, and inhibited enzymes. Biochemistry. 5 (6), 1926-1933 (1966).
  49. Rawat, U. B., Rao, M. B. Purification, kinetic characterization and involvement of tryptophan residue at the NADPH binding site of xylose reductase from Neurospora crassa. Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology. 1293 (2), 222-230 (1996).
  50. Zaki, M. J., Bystroff, C. Protein Structure Prediction. , Humana Press. Totowa, NJ. (2008).
  51. Wang, Z., et al. Comprehensive evaluation of ten docking programs on a diverse set of protein-ligand complexes: The prediction accuracy of sampling power and scoring power. Physical Chemistry Chemical Physics. 18 (18), 12964-12975 (2016).
  52. Pagadala, N. S., Syed, K., Tuszynski, J. Software for molecular docking: A review. Biophysical Reviews. , 91-102 (2017).
  53. Dolatkhah, Z., Javanshir, S., Sadr, A. S., Hosseini, J., Sardari, S. Synthesis, Molecular Docking, Molecular Dynamics Studies, and Biological Evaluation of 4 H -Chromone-1,2,3,4-tetrahydropyrimidine-5-carboxylate Derivatives as Potential Antileukemic Agents. Journal of Chemical Information and Modeling. 57 (6), 1246-1257 (2017).
  54. Forli, S., et al. Computational protein-ligand docking and virtual drug screening with the AutoDock suite. Nature Protocols. 11 (5), 905-919 (2016).
  55. Lindahl, E. R. Molecular dynamics simulations. Molecular Modeling of Proteins. Methods in Molecular Biology. 443, 3-23 (2008).
  56. Turk, T., Maček, P., Gubenšek, F. The role of tryptophan in structural and functional properties of equinatoxin II. Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular. 1119 (1), 1-4 (1992).
  57. Peterman, B. F., Laidler, K. J. Study of reactivity of tryptophan residues in serum albumins and lysozyme by N-bromosuccinamide fluorescence quenching. Archives of Biochemistry and Biophysics. 199 (1), 158-164 (1980).
  58. Divita, G., Goody, R. S., Gautheron, D. C., Di Pietro, A. Structural mapping of catalytic site with respect to α-subunit and noncatalytic site in yeast mitochondrial F1-ATPase using fluorescence resonance energy transfer. Journal of Biological Chemistry. 268 (18), 13178-13186 (1993).
  59. Horrocks, W. D., Holmquist, B., Vallee, B. L. Energy transfer between terbium (III) and cobalt (II) in thermolysin: a new class of metal-metal distance probes. Proceedings of the National Academy of Sciences of the United States of America. 72 (12), 4764-4768 (1975).
  60. Chakraborty, J., Das, N., Halder, U. C. Unfolding diminishes fluorescence resonance energy transfer (FRET) of lysine modified β-lactoglobulin: Relevance towards anti-HIV binding. Journal of Photochemistry and Photobiology B: Biology. 102 (1), 1-10 (2011).
  61. Sirangelo, I., Malmo, C., Casillo, M., Irace, G. Resolution of Tryptophan-ANS Fluorescence Energy Transfer in Apomyoglobin by Site-directed Mutagenesis. Photochemistry and Photobiology. 76 (4), 381-384 (2007).
  62. Ribeiro, A. J. M., Tyzack, J. D., Borkakoti, N., Holliday, G. L., Thornton, J. M. A global analysis of function and conservation of catalytic residues in enzymes. Journal of Biological Chemistry. 295 (2), 314-324 (2020).
  63. Eftink, M. R., Ghiron, C. A. Exposure of tryptophanyl residues in proteins. Quantitative determination by fluorescence quenching studies. Biochemistry. 15 (3), 672-680 (1976).
  64. Eftink, M. R., Ghiron, C. A. Fluorescence quenching of indole and model micelle systems. The Journal of Physical Chemistry. 80 (5), 486-493 (1976).
  65. Kinsley, N., Sayed, Y., Mosebi, S., Armstrong, R. N., Dirr, H. W. Characterization of the binding of 8-anilinonaphthalene sulfonate to rat class Mu GST M1-1. Biophysical Chemistry. 137 (2-3), 100-104 (2008).
  66. Mohsenifar, A., et al. A study of the oxidation-induced conformational and functional changes in neuroserpin. Iranian Biomedical Journal. 11 (1), 41-46 (2007).
  67. Gonzalez, W. G., Miksovska, J. Application of ANS fluorescent probes to identify hydrophobic sites on the surface of DREAM. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics. 1844 (9), 1472-1480 (2014).
  68. Eftink, M. R., Ghiron, C. A. Fluorescence quenching studies with proteins. Analytical Biochemistry. 114 (2), 199-227 (1981).
  69. Poulos, T. L., Price, P. A. The identification of a tryptophan residue essential to the catalytic activity of bovine pancreatic deoxyribonuclease. The Journal of biological chemistry. 246 (12), 4041-4045 (1971).
  70. Hu, J. -J., He, P. -Y., Li, Y. -M. Chemical modifications of tryptophan residues in peptides and proteins. Journal of Peptide Science An Official Publication of the European Peptide Society. 27 (1), 3286(2021).

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Tryptophan ModificationANS BindingFRET AssayN Bromosuccinimide QuenchingSERCA N DomainFluorescence SpectroscopyProtein Fluorescence QuenchingMolecular DockingATP Binding SiteRecombinant Protein Purification
Chemical Modification of the Tryptophan Residue in a Recombinant Ca2+-ATPase N-domain for Studying Tryptophan-ANS FRET
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