Fluorescent Probes and Quenchers in Studies of Protein Folding and Protein-Lipid Interactions

Abstract

Fluorescence spectroscopy provides numerous methodological tools for structural and functional studies of biological macromolecules and their complexes. All fluorescence-based approaches require either existence of an intrinsic probe or an introduction of an extrinsic one. Moreover, studies of complex systems often require an additional introduction of a specific quencher molecule acting in combination with a fluorophore to provide structural or thermodynamic information. Here, we review the fundamentals and summarize the latest progress in applications of different classes of fluorescent probes and their specific quenchers, aimed at studies of protein folding and protein-membrane interactions. Specifically, we discuss various environment-sensitive dyes, FRET probes, probes for short-distance measurements, and several probe-quencher pairs for studies of membrane penetration of proteins and peptides. The goals of this review are: (a) to familiarize the readership with the general concept that complex biological systems often require both a probe and a quencher to decipher mechanistic details of functioning and (b) to provide example of the immediate applications of the described methods.

Keywords: FRET; environment-sensitive; intrinsic and extrinsic fluorescent probe; protein-membrane interactions; short-distance fluorescence quenching.

Figure 1.

(a) Amino acid sequence of the membrane-proximal external region (MPER) of membrane-interacting C-terminal domain of the HIV-1 gp41 fusion protein. (b-c) Tryptophan fluorescence of the MPER peptide in various membranous environment: the MPER peptide in solution (black), added to preformed POPC vesicles (MPER + LUV, blue) and coextruded with POPC vesicles (MPER/LUV, red). (d-g) Fluorescence titration of the unlabeled MPER peptide (d-e) and the NBD-labeled W666C mutant of the MPER peptide (f-g) with POPC LUV in 50 mM phosphate buffer solution at pH 8.^[46]

Figure 2.

(a) Schematic representation of the Shaker K1 channel monomer illustrating the positions of the LBT (between transmembrane, TM, segments S3 and S4) and of the four 6-His tags tested. Displayed is the voltage sensing domain, comprised of TM segments S1-S4, and the pore region, segments S5-pore loop-S6, of the adjacent subunit. Inset shows the LBT site with a bound Tb³⁺ ion. (b-c) Tb³⁺ luminescence decay of Shaker channel constructs with the LBT in the S3-S4 loop with and without a 6-His tag close to S1 and S3 for Ni²⁺ and Cu²⁺, respectively. Adapted from^[103]. Copyright © 2007. The Biophysical Society. Published by Elsevier Inc. All rights reserved.

Figure 3.

Application of lipid-attached probes as FRET acceptors to the protein donor fluorescence to study membrane binding. (a) Scheme of FRET-binding assay, in which peptide binding to a bilayer is monitored by FRET occurring between a donor-dye-labeled pHLIP and Dansyl-PE acceptor-doped bilayer. (b) FRET measurements between Trp (donor) present in pHLIP–P20G and POPC-LUV containing 2% Dansyl-PE (acceptor) by gradual increasing LUV concentration.^[38]

Figure 4.

(a) Schematic presentation of the excited state intramolecular proton transfer (ESIPT) reaction in 3-hydroxyflavones (3HF). (b) Dual-band fluorescence spectra of 3-hydroxyflavone dye. (c) Dual-fluorescence fluorescent conjugates, nonnatural amino acids and alkyl-functionalized membrane probes based on 3HF-dyes: 6-acetamido-4′-(dimethylamino)-3-hydroxyflavone (MFL), 3-[2-(4-methoxyphenyl)-3-hydroxy-4-oxo-4H-chromen-6-yl]-$L$-alanine (M3HFaa), 3-[2-(2-furyl)-3-hydroxy-4-oxo-4H-chromen-6-yl]-L-alanine (3HCaa).

Figure 5.

Application of FRET measurements to study conformational changes in a protein selectively labeled with an Alexa probe and a conjugated Fluorescent Protein. (a) Bcl-xL construct labeled by AlexaFluor-488-mCherry FRET dyes. (b) Experimental FRET set-up is based on the presence of FRET when the mCherry acceptor is close to the AlexaFluor-488 donor and the lack of FRET in the refolded/inserted form of Bcl-xL due to the increase in distance between the donor-acceptor pair. (c) Steady state measurements show a progressive increase in donor A488 intensity at 518 nm accompanied by a decrease in the acceptor mCherry intensity at 605 nm as a function of pH. (d) The smFRET efficiency is characterized by a progressive shift of the distributions to lower FRET efficiencies as a function of pH.^[132,189]

Figure 6.

Example of the distance measurements during conformational switching in a protein by using a combination of long-range and short-range fluorescence. (a) pH-triggered histidine protonation of the T-domain result in structural changes in proximity of residues Q369 (C-terminal helix TH9) and W206 (N-terminal helix TH1) in membrane-incompetent W-state (cyan) and membrane-competent W⁺ -state (yellow), as estimated by MD simulations. (b) Trp-to-bimane proximity changes (black and red arrows in panel a) are monitored by fluorescence decay, which is strongly quenched in W-state (pH 8, black), but not in W⁺ -state (pH 5.5, red). (c) Long-range FRET measurements between two energy donors, W206 and W281, and an Q369C-AEDANS energy acceptor, demonstrating the retention of the overall compact conformation of the T-domain.

Figure 7.

Fluorescence quenching of bimane-labeled peptide by transition metal ions (Cu²⁺ and Ni²⁺). (a) Molecular models of a Bi–C2H6H10 peptide composed of a bimane dye attached to an ideal a-helical by the cysteine residue is shown in blue, whereas the two histidines comprising a metal binding site and Cu²⁺ ion are shown in red and green, respectively. The three panels correspond to the three representative dihedral angles of the bimane linker, estimated by molecular modelling. The arrows show the center-to-center distances between the centers of a bimane dye and a metal ion. Steady-state (b) and time-resolved (c) fluorescence quenching of bimane-labeled peptide C2H6H10 using transition by metal ions.^[209]

Figure 8.

(a) Structure of soluble diphtheria toxin T-domain with highlighted NBD-labeled residues L350C-NBD and P378C-NBD. FRET quenching of NBD by LysoUB within quenching radius R, as illustrated by a shaded circle. (b) Application of NBD/LysoUB FRET quenching method for distinguishing topologies of the interfacial (IF) intermediate and final transmembrane (TM) conformations of helixes TH8-TH9 of the membrane-inserted T-domain.

Figure 9.

Depth-dependent fluorescence quenching of NBD–PE in a lipid bilayer. (a–b) The steady-state and lifetime fluorescence quenching of NBD–PE by Tempo–PC in POPC-LUVs, as shown for the samples containing different quencher content. (c-d) Distribution analysis (DA) of depth-dependent fluorescence quenching profiles of NBD–PE obtained with a series of the six spin-labeled lipids. (c) Quenching profiles of steady-state fluorescence quenching of NBD–PE in LUVs containing the different quencher concentrations. (d) Application of DA methodology to lifetime quenching (squares) and “differential” quenching (circles). See the text and refs^{[223,234-235]} for more detail.

Figure 10.

Illustration of the difference in membrane penetration of helices of the central hairpin of Bcl-xL (left panels, helix α6^[132]) and the translocation domain of diphtheria toxin (right panels, helix α9^[235]). Differential depth-dependent quenching profiles of NBD probe selectively attached to a single cysteine residue at position 161 (a) or 169 of Bcl-xL (b), and at position 378 (c) or 364 (d) of the diphtheria toxin T-domain (see the text for more detail), (e) Schematic representation of the membrane topology of a-helix 6 of Bcl-xL and a-helix 9 of the T-domain (corresponding $h_m$ parameters are given in parenthesis). Reproduced with permission from^[240]. © 2023 The Authors. Published by Elsevier B.V.

Scheme 1.

Fluorescent amino acids. Native amino acids: phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp). Unnatural fluorescent amino acids; 5-fluorotryptophan (5F-Trp), 7-azatryptophan (7ATrp), 2-amino-3-(5-(dimethyl-amino)-naphthalene-1-sulfonamide)propanoic acid (dansylalanine), (7-hydroxy-coumarin-4-yl)ethyl-L-glycine (7-HCaa), Anap (3-(6-acetylnaphthalen-2-yl-amino)-2-aminopropanoic acid),^[28] N-[4-(N,N-dimethylamino)-phthalimidyl]-L-alanine (4-DMAP).^[29]

Scheme 2.

Thiol-reactive probes for site-selective labelling: monobromobiname, BODIPY FL N-(2-aminoethyl) maleimide, IANBD ester (N-((2-(iodoacetoxy) ethyl)-N-methyl)-amino-7-nitrobenz-2-oxa-1,3-diazole), 7-diethylamino-3-[N-(2-maleimido-ethyl)-carbamoyl] coumarin (MDCC) and 5-(N-iodoacetyl-aminoethyl)-naphthylamine-1-sulfonic acid (IAEDANS).

Scheme 3.

Structure of some thiol-reactive AlexaFluor dyes.

Scheme 4.

Structure of some popular FPs: Enhanced cyan fluorescent protein (ECFP, PDB 1CV7), wild-type green fluorescent protein (GFP, PDB 4KW4), enhanced yellow fluorescent protein (EYFP, PDB 1YFP), mCherry red fluorescent protein (mCherry, PDB 6YLM). The secondary structure of a FP carrier is shown as β-barrel and a chromophore is shown in vdW representation. For clarity, the structure of the chromophore alone is given by a licorice model. Excitation (Ex), emission (Em) maxima and fluorescent quantum yield (Qy) are given for comparison.

Scheme 5.

Fluorescent probes, fatty acids and amines: dibutylamino stilbazolium butylsulfonate (di-4-ASPBS), 1-[6-(dimethylamino)naphthalen-2-yl]propan-1-one (Prodan), 6-lauroyl-2-(dimethylamino)-naphtalene (Laurdan), tryptophan octyl ester (TOE), 4-(hexadecylamino)-7-nitrobenz-2-oxa-1,3-diazole (NBD-C₁₆),1,1′-dioctadecyl-3,3,3,3′-tetramethyl-indocarbocyanine (DiI-C₁₈).

Scheme 6.

Fluorescent lipids:1-palmitoyl-2-(2-pyrenedecanoyl)-sn-glycero-3-phosphocholine (Pyr₁₀-PC), 1-palmitoyl-2-(12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]-dodecanoyl)-sn-glycero-3-phosphocholine (C₁₂-NBD-PC), 1-palmitoyl-2-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl)-sn-glycero-3-phosphocholine (C₆-NBD-PC), 2-(4,4-difluoro-5-butyl-4-bora-3a,4a-diaza-s-indacene-3-octanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (BODIPY-PC), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-ben-zoxa-diazol-4-yl) (NBD-PE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanol-amine-N-(lissamine rhodamine B sulfonyl) (ammonium salt) (rhodamine-PE), Texas Red 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (Texas Red PE).

Scheme 7.

Dye-labeled Lyso-phospholipids, designed for measurements of membrane topology of proteins and peptides. LysoMC (N-(7-hydroxyl-4-methylcoumarin-3-acetyl)-1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanol-amine) and LysoUB (UniBlue-A-1-palmitoyl-2-hydroxy-sn-glycero-3-phos-phoethanolamine) are FRET quenchers (acceptors) for Trp and NBD, respectively.

Scheme 8.

Structures and transverse depths of lipid-attached fluorescence quenchers. (Top) Dibrominated phospholipids: 1-palmitoyl-2-(n,n-dibromo)-stearoyl-sn-glycero-3-phosphocholine (n,n-Br-PC). (Bottom) Paramagnetic spin-labeled phospholipids: Tempo-PC (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(TEMPO)-choline), n-Doxyl-PC (1-palmitoyl-2-stearoyl-(n-Doxyl)-sn-glycero-3-phospho-choline) spin-labeled lipids. The numbers below the structures correspond to the average immersion depth (e. g., distance from the bilayer center) for each of the quenchers.