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

These assays may help to clarify the role of tryptophan residues in proteins when performing ANS binding studies. The ANS fluorescence increases upon binding to specific structural sites in proteins. Sometimes ANS binding site locates close to a tryptophan residue with a distance that suggests the existence of FRET between the tryptophan and ANS.

NBS mediates chemical modification of tryptophan residues in proteins, quenching its fluorescence. Therefore, these assays allow determining whether tryptophan residues contribute to the increase of ANS fluorescence intensity by FRET. In this work, we use an engineered recombinant nucleotide binding domain from SERCA containing mutations, aiming to locate a tryptophan close to the nucleotide binding site where ANS also binds.

This assay is rapid and easy to perform, hence its main advantage. Determination in silico of the ANS and SERCA N-Domain interaction. Generate a three-dimensional structure of the protein by molecular modeling using the preferred protein-modeling software.

Identify the amino acid residues that form the nucleotide binding site using the preferred molecular structural software and determine the presence of arginine and lysine residues. These are required for ANS binding and increase the fluorescence intensity. Because of the presence of arginine and lysine residues in the nucleotide binding site of SERCA, we hypothesize the binding of ANS to the N-Domain.

In the 3D model, the amino acid residues forming the ATP binding site are identified and highlighted in orange. Similarly, the tyrosine-to-tryptophan mutation is located and highlighted in blue. Perform molecular docking with ATP, FITC, and ANS.

Docking results shows that ANS fits well in the nucleotide binding site, similarly to ATP and FITC. FITC labels the nucleotide binding site. Calculate the molecular distance between the tryptophan residue and ANS.

20 angstroms seems to be a proper distance for FRET. Perform molecular dynamic simulation of the ANS N-Domain complex to determine the stability of the interaction. A 10 nanosecond simulation time shows the complex's stability.

Proceed to perform the in vitro experiments. Expression and purification of the recombinant N-Domain. Express and purify, by affinity chromatography, the engineered recombinant N-Domain.

Perform an SDS-PAGE of the purified protein to determine the purity. Determine the protein concentration by absorbance at a wavelength of 280 nanometers with the N-Domain extinction coefficient. Monitor the formation of the ANS N-Domain complex based on ANS and N-Domain fluorescence intensity changes.

Prepare an ANS stock solution in N, N-Dimethylformamide. Weigh a small amount of ANS, between one to five milligrams. Dissolve ANS in one mL of final volume of N, N-Dimethylformamide.

Prepare a 100 micromolar ANS stock solution using the ANS solution in N, N-Dimethylformamide. Mix the solution by vortexing three to five times. Prepare the NBS stock solution in N, N-Dimethylformamide.

Weigh a small amount of NBS, one to five milligrams. Dissolve the NBS in one mL of N, N-Dimethylformamide. Prepare a one millimolar NBS stock solution using the NBS solution in N, N-Dimethylformamide.

Mix the solution by vortexing three to five times. Titrate the N-Domain with ANS and record the fluorescence spectra for excitation at a wavelength of 295 nanometers at 25 degrees. Obtain the fluorescence spectrum baseline.

Place one mL of 50 millimolar phosphate buffer with pH eight in a one mL fluorescence quartz cuvette. Position the cell in the thermostated cell chamber of the spectrophotometer and set the excitation wavelength to 295 nanometers. Record the fluorescence spectrum.

The fluorescence spectrum of the blank shows the peak of water, which has to be subtracted later. Obtain the intrinsic fluorescence spectrum of the N-Domain. Place 900 microliters of 50 millimolar phosphate buffer with pH 8 in a fluorescence quartz cuvette.

Add 100 microliters of N-Domain suspension to yield a one micromolar N-Domain final concentration. Gently homogenize using a micropipette to ensure the homogeneity of the solution. Position the cell in the thermostated chamber of the Spectrophotometer.

Set the excitation wavelength to 295 nanometers. Record the N-Domain intrinsic fluorescence spectrum. Add ANS and obtain the fluorescence spectrum by excitation at a wavelength of 295 nanometers.

Add a two microliter aliquot of 100 micromolar ANS stock solution to the suspended N-Domain to obtain a 0.2 micromolar ANS final concentration. Gently homogenize, using a micropipette to ensure the homogeneity of this solution. Record the fluorescence spectrum.

Repeat the ANS additions and fluorescence spectral recording as described above for a 1:1 ANS N-Domain molar relationship. Subtract the blank spectrum from each spectrum using software. Plot all the spectra in a single graph Determine whether a FRET-like pattern is formed by the spectrum.

The ANS N-Domain fluorescence spectra form a FRET-like pattern. N-Domain intrinsic florescence titration by tryptophan chemical modification with NBS. Obtain again the intrinsic fluorescence spectrum of the N-Domain.

Add a one microliter aliquot of one millimolar NBS stock solution to the suspended N-Domain to obtain a final concentration of one micromolar NBS. Gently homogenize using a micropipette to ensure the homogeneity of the solution. Record the fluorescence spectrum.

Repeat the NBS addition and fluorescence spectral recording until minimal N-Domain intrinsic fluorescence quenching is observed. Gently homogenize using a micropipette to ensure the homogeneity of the solution. Subtract the blank spectrum from each spectrum using software.

Plot all the spectra in a single graph. Titrate the NBS modified N-Domain with ANS by recording fluorescence spectra at 25 degrees. Add ANS and obtain the fluorescence spectrum by excitation at a wavelength of 295 nanometers.

Gently homogenize using a micropipette to ensure the homogeneity of the solution. Record the fluorescence spectrum. Subtract the blank spectrum from each spectrum using software.

Plot all the spectra in a single graph. The generated fluorescence spectra support or refute the occurrence of FRET. Namely when FRET occurs, the ANS fluorescence doesn't increase and vice-versa.

Evidence of ANS binding to the chemically modified N-Domain by excitation at a wavelength of 370 nanometers. Add ANS to the NBS modified N-Domain and record the N-Domain intrinsic fluorescence spectrum by excitation at a wavelength of 370 nanometers. Subtract the blank spectrum from each spectrum using software.

Plot all the spectra in a single graph. Confirm ANS binding to the N-Domain by the increasing ANS fluorescence intensity. ANS binding to the N-Domain shows a fluorescence increase when excited at a wavelength of 370 nanometers.

Results overview. Panel A.ANS N-Domain complex display a FRET-like pattern when exciting at a wavelength of 295 nanometers. Panel B.NBS quench the N-Domain intrinsic fluorescence by chemically modifying the sole tryptophan residue in the chemically modified N-Domain.

Panel C.The ANS fluorescence increases when exciting at a wavelength of 295 nanometers. Panel D.The ANS fluorescence also increases when exciting at a wavelength of 370 nanometers. Therefore, direct excitation of ANS at a wavelength of 295 nanometers is the main source of ANS fluorescence when it is bound to the ATP binding site.Conclusions.

These assays may be used to determine the existence of FRET between tryptophan residues and ANS in proteins. The clarification of FRET, confirmation or not, between tryptophan and ANS will allow to draw better conclusions in protein structural studies. The assay may also be useful when using other fluorophores.