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JoVE Journal Biochemistry

Biochemistry

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

Published: October 9, 2021 doi: 10.3791/62770
Automatic Translation
1, 1
1Instituto de Física, Universidad Autónoma de San Luis Potosí

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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Representative 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 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
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
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
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
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.

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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 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.

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Disclosures

The authors declare that they have no competing financial interests.

Acknowledgments

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.

Materials

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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Chemical Modification Tryptophan Residue Recombinant Ca2+-ATPase N-domain ANS Binding Studies FRET Fluorescence NBS Nucleotide Binding Domain SERCA ANS Fluorescence Intensity Rapid Assay In Silico Determination Molecular Modeling Protein Structure Amino Acid Residues Arginine Lysine Residues
Chemical Modification of the Tryptophan Residue in a Recombinant Ca<sup>2+</sup>-ATPase N-domain for Studying Tryptophan-ANS FRET
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Sampedro, J. G., Cataño, Y.… More

Sampedro, J. G., Cataño, Y. Chemical Modification of the Tryptophan Residue in a Recombinant Ca2+-ATPase N-domain for Studying Tryptophan-ANS FRET. J. Vis. Exp. (176), e62770, doi:10.3791/62770 (2021).

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