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

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 studies^1,2,3,4. 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....

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)

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 .......

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.......

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