Fostering protein–calixarene interactions: from molecular recognition to sensing

Abstract

Two isomeric bis-calixarene-carbazole conjugates (CCC-1 and CCC-2) endowed with carboxylic acid functions at their lower rims have been found to display a high sensing ability (K_SV up to 6 × 10⁷ M⁻¹) and selectivity toward cytochrome c, a multi-functional protein, in an aqueous-based medium. After targeting basic amino acid residues on the protein surface residing near the prosthetic heme group through electrostatic and hydrophobic interactions, a rapid photoinduced electron transfer ensues between the integrated transduction element (aryleneethynylene chromophore) of CCCs and the iron-oxidized heme of cytochrome c, enabling direct detection of the protein at nanomolar levels. Our results show that CCCs are capable of efficiently discriminating heme proteins (cytochrome c vs. myoglobin) and non-heme proteins (lysozyme) in an aqueous medium. Studies performed in two solvent systems (organic and aqueous) strongly suggest that in an organic medium a Förster-type resonance energy transfer is responsible for the observed reduction in CCCs emission upon contact with heme proteins while in an aqueous medium a specific photoinduced electron transfer mechanism prevails.

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Introduction

As one of the most studied supramolecular hosts,¹ calixarenes have attracted in recent years an increased considerable interest in biochemistry, biology and medicine, as artificial protein binders able to interfere with cellular processes and inhibit the cellular signal transduction pathways involved in disease development.²

Through in-depth fundamental studies it has been demonstrated that calixarenes bearing anionic derivatives, typically sulfonates, phosphates and carboxylates, at their lower and/or upper rims are most apt to develop strong interactions with complimentary charged species such as amino acids, peptides and proteins.³ It has also been shown that the recognition events occurring between the basic amino acids (either alone or integrated in peptide chains) and several calixarene hosts are mainly driven by electrostatic interactions modulated by hydrophobic interactions, being their extension (binding affinity) governed by multivalent (cooperative) interactions.^2a,c,4 In particular, calixarenes containing carboxylic acid functions at their lower rims have been studied for their complexing abilities toward single amino acids,⁵ cationic⁶ and neutral proteins.⁷ The last reports besides disclosing suitable extraction methods of cytochrome c,^6a hemoglobin^7a and myoglobin^7b into an organic phase, have also found that the calixarene–protein complexes are able to perform biocatalysis in organic media, exhibiting pseudo peroxidase activity.^6a,7

Direct detection of proteins by conjugated polymers in aqueous media, in particular polyelectrolytes, has been an intensive field of research.⁸ However, several studies have pointed out severe limitations on the use of polyelectrolytes as biosensors due to nonspecific interactions with proteins and other biomolecules.⁹ Actually, since most of the developed sensing schemes rely on fluorescence turn-off mechanisms (photoinduced electron transfer and/or resonance energy transfer), any other molecular event not related with the specific protein targeting by the host (e.g. aggregation and/or conformational changes of the polymer chains induced by any species present in the media) will tend to reduce the overall emission of the polyelectrolyte, resulting in poor substrate selectivity.

Recognition and sensing of proteins has also been achieved by tetraphenylporphyrin-,^10a tetrabiphenylporphyrin-^10b and anthracene-based^10c receptors possessing multi-carboxylic/carboxylate residues attached to central fluorogenic cores with high sensitivity. The high binding affinities attained with some of these receptors toward cytochrome c in aqueous buffered solutions (pH = 7.4) were attributed to the number of carboxylate residues in the periphery of the receptor, as well as their inner distances, and to hydrophobic interactions developed between the receptor cores and hydrophobic sites of the proteins.

Expedite fluorescence methods for the direct sensing of proteins by suitable calixarene compounds are, on the other hand, practically inexistent. Coming as an exception, calix[4]arenes endowed with carboxylphenyl sub-units at their upper rim turned to be excellent fluorescence-based sensors for cytochrome c (from bovine heart) in organic medium.¹¹ The other works that we are aware had involved the use of resorcinarene scaffolds: a cyclophane-based resorcinarene trimer (with 21 carboxylate residues) possessing an appended dansyl group as the fluorescence signaling element^12a and a rotaxane-type resorcinarene tetramer (with 28 carboxylate residues) having fluorescein or rhodamine derivatives (axle components) as transduction reporters.^12b Both showed remarkable sensing selectivity for histone in aqueous media.

We have recently communicate on the synthesis and structural characterization of new homoditopic bis-calix[4]arene-carbazole conjugates (CCC-1 and CCC-2, Fig. 1), both armed with hydrophilic carboxylic acid functions at their lower rims but differing on the type of substitution at the carbazole rings.¹³ It was demonstrated that these fluorophores are able to function as highly sensitive sensors toward electron-deficient aromatic compounds (picric acid and 2,4,6-trinitrotoluene) used in explosives' compositions in solution as well as serve as structural motifs in the construction of supramolecular polymers in organic solution phase.¹³ These new synthetic receptors were also designed having in mind their potential as biosensors.¹³ Given the spatial distribution and multitude of the potential binding sites provided by the calixarene hosts bearing carboxylic/carboxylate functions and the integrated fluorescence transduction element incorporating the bis-carbazole-derived aryleneethynylene central unit with good electron donor ability,¹⁴ we thought that strong interactions might be developed with H-bond receptors and cationic guests residing in target proteins (particularly those positively charged amino acids distributed near the heme edge region), which synergistically would foster the overall sensing capabilities toward specific heme-containing proteins, allowing for an efficient differentiation between heme and non-heme proteins.

Fig. 1 Molecular structures of bis-calix[4]arene-carbazole conjugates (CCCs); CCC-1, CCC-2 and CCC-3 optimized conformers with the calixarene moieties disposed in a syn arrangement around the central aryleneethynylene rings. Hydrogens omitted for clarity.

Within this framework, the present contribution details our studies toward the selective recognition of heme proteins by CCCs. Cytochrome c (h-cyt c) and myoglobin (myo), both from horse heart, were chosen as models for iron-oxidized (Fe(III)) porphyrin proteins, while lysozyme (lys) from chicken egg white was selected as a model for cationic proteins lacking the prosthetic center. For comparison, a bis-calixarene analogue devoid of carboxylic functionalities (CCC-3, Fig. 1)¹⁴ was tested in order to ascertain the specific role of each contributing structural element of the calixarene host to the overall sensory capabilities of proteins.

A particular issue to be addressed in this work relates to the influence of media (aqueous vs. organic) in the sensing ability of CCCs toward heme proteins. As will be demonstrate, the mechanism by which the fluorescence of CCCs is quenched in organic medium is diverse from that occurring in aqueous medium owing to the disruption of particular ligands around the protein redox center in the former medium which negatively affects the electron transfer reactivity of the heme and concomitantly the sensing response.

Experimental section

Materials

CCC-1,¹³ CCC-2 ¹³ and CCC-3 ¹⁴ were synthesized according to our reported procedures and fully characterized by FT-IR, ¹H/¹³C NMR, and microanalysis. Cytochrome c from equine heart (≥95% (SDS-PAGE), Aldrich C2506), myoglobin from equine heart (≥90% (SDS-PAGE), Aldrich M1882), lysozyme from chicken egg white (≥90%, Aldrich L6876) were purchased from Sigma-Aldrich Corp., EUA, and used as received. Chloroform (CHCl₃) (99.99%) was acquired from Fisher Chemicals, UK, N,N-dimethylformamide (DMF) (≥99.9%) from Carlo Erba, France and 9,10-diphenylanthracene (9,10-DPA) (scintillation grade) from Nuclear Enterprises, Ltd., EUA, and used as such. Aqueous buffered solutions (50 mM phosphate buffer (pH = 7.2), 20 mM HEPES buffer (pH = 8.2) and 20 mM glycine buffer (pH = 9.8)) were prepared by standard methods, using (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES, >99%) purchased from Fisher BioReagents, China, glycine (>99.7%) from Riedel-deHäen, Germany, and sodium dihydrogen phosphate monohydrate (>98%) and disodium hydrogen phosphate heptahydrate (>98%) from Panreac, Spain. Ultrapure water (Milli-Q, Millipore) was used throughout the experiments.

Instruments and methods

UV-vis spectra were recorded on a Nicolet Evolution 300 spectrophotometer (Thermo Fisher Scientific Inc.) or on a Jasco J-815 CD spectrometer (Jasco Analytical Instruments) using 1 cm quartz cells. Steady-state fluorescence spectra were recorded on a Perkin-Elmer LS45 fluorimeter (Perkin-Elmer Inc.) using a 1 cm quartz cuvette in right angle geometry at 25 °C in air-equilibrated conditions. Electronic circular dichroism (CD) and UV-vis spectra were recorded simultaneously on a Jasco J-815 CD spectrometer using 1 cm quartz cells at 20 °C, using the Jasco Peltier type accessory CDF-426S/426L as temperature controller.

Fluorescence quantum yields (QYs) were measured using 9,10-DPA as reference standard (Φ_F = 0.72 in ethanol)¹⁵ at 25 °C. To prevent inner filter effects during QY measurements the optical density (OD) of the samples and reference were kept below 0.05 at the excitation wavelength (360 nm); solutions of CCCs were prepared in DMF while that of 9,10-DPA in ethanol. Fluorescence spectra were recorded with the same operating settings. QYs were determined using the equation Φ_F = Φ_Fr × F/F_r × OD_r/OD × n²/n_r²,¹⁶ where Φ is the quantum yield, F is the integral of fluorescence emission intensity, OD denote the optical density at the specified wavelength, with the subscript r referring to the reference; n and n_r are the refractive indices of DMF and EtOH, respectively.

Concentrated stock solutions of CCCs (1.0–1.3 × 10⁻⁴ M) were prepared in DMF or in 50 mM phosphate buffer solutions (pH = 7.2) containing specific amounts of DMF (buffer solution : DMF ratios ranging from 0 : 10 to 9 : 1; see text for particular uses). Stock protein solutions were prepared in ultrapure water, in 20 mM HEPES buffer (pH = 8.2) or in 20 mM glycine buffer (pH = 9.8) at c = 2.0 × 10⁻³ M, and in 50 mM phosphate buffer solution (pH = 7.2) at c = 1.0 × 10⁻⁶ M. Their specific use (UV-Vis, fluorescence, CD or titration measurements) is specified along the text.

For titration experiments, sample solutions were prepared in 5 mL volumetric flasks with the CCC : protein molar ratio adjusted for each set of experiments, which were varied according to the magnitude of the association constant of the complex formed. In organic medium, the host concentration was set to 1.0 × 10⁻⁷ M while in phosphate buffer : DMF (9 : 1) to 1.0 × 10⁻⁶ M, in order to compensate the lower fluorescence quantum yield of CCCs in this solution and thus increase the signal-to-noise ratio during the experiments.

The sample solutions were kept at 25 ± 1 °C for 30 min prior to spectrofluorometric determination. Fluorescence intensities corresponding to the wavelength near the emission maximum were measured by exciting at 360 nm, except otherwise stated. In all the experiments it is assumed that the quantum yield is proportional to the intensity peak fluorescence, since the spectral shape does not vary appreciably during the titration.

To derive the association constant (K_a) from spectrofluorometric titration experiments using a nonlinear curve-fitting approach, assuming a 1 : 1 stoichiometry in the host : guest complexation, the following equation was used:¹⁷

ΔF = 1/2{Δε_F([H]₀ + [G]₀ + 1/K_a) ± [Δε_F²([H]₀ + [G]₀ + 1/K_a)² − 4Δε_F²[H]₀[G]₀]^1/2} (1)

where ΔF and Δε_F are the changes in fluorescence intensity and molar fluorescence intensity of the host upon complexation, and [H]₀ and [G]₀ denote the initial concentrations of the host (CCCs) and the guest (proteins), respectively. Regression analysis was performed by the SOLVER function in Microsoft Excel.¹⁸

Computational studies

Conformational searches on CCCs were carried out with Monte Carlo method using a molecular mechanics model (MMFF94 force field). After full-geometry re-optimizations (PM3 semi-empirical model) of the lowest-energy conformers, single-point energy calculations were then done with a hybrid density functional model (B3LYP) using a 6-31G* basis set. Docking was performed by manual assembling an XRD structure of h-cyt c with the syn conformer of CCC-1 at the surface of the protein near the heme edge, monitoring the specific interactions (H-bonding) developed between Lys8 and Lys86 and the acid residues of CCC-1. All calculations were performed in Spartan'14.¹⁹

Results and discussion

Physical and optical properties of CCCs

Given their amphiphilic nature, CCC-1 and CCC-2 are freely soluble in polar aprotic organic solvents (e.g. DMF) as well as in less polar and apolar solvents such as CH₂Cl₂, CHCl₃ and toluene. On the contrary, they are only sparingly soluble in alcohols (MeOH, EtOH) and are nearly insoluble in water and buffered solutions. For this reason, DMF was chosen as the primary solvent in the first set of binding assays, since it was found that in the concentration range used in the experiments the guest proteins are also completely soluble providing they are previously dissolved in water/buffered solutions and then diluted to the desired concentration with DMF.

The absorption and fluorescence spectra of CCC-1 and CCC-2 in two distinct solvent systems are shown in Fig. 2. In CHCl₃, the absorption profiles of both hosts present absorption maxima between 354–359 nm with several perceptible shoulders. In CCC-1, the shoulder appearing at longer wavelength (ca. 394 nm) is considerably more pronounced than in CCC-2 likely reflecting a higher π-conjugation length of the main chromophore (aryleneethynylene segment). A larger molar extinction coefficient is also exhibited by CCC-1 (ε_max = 6.84 × 10⁴ M⁻¹ cm⁻¹ at λ = 354 nm) in comparison to CCC-2 (ε_max = 5.64 × 10⁴ M⁻¹ cm⁻¹ at λ = 359 nm), due to the higher oscillator strength achieved by CCC-1 during the π–π* transition.

Fig. 2 Absorption and emission spectra of CCC-1 (a) and CCC-2 (b) in CHCl₃ (dashed line) and DMF (solid line); λ_exc = 360 nm.

When dissolved in DMF, a significant change in the absorption profile of both compounds occurs. Indeed, the shoulder previously appearing at around 390 nm became the most prominent band in their spectra, while the maxima of the higher energy bands are kept around their former values. Furthermore, the molar absorptivities of CCC-1 and CCC-2 also experience a sharp rising of their values (ε_max(CCC-1) = 9.05 × 10⁴ M⁻¹ cm⁻¹ at λ = 394 nm and ε_max(CCC-2) = 6.69 × 10⁴ M⁻¹ cm⁻¹ at λ = 391 nm). The huge increase in intensity of the low energy band may be traced to the rise of the chromophore population attaining a more coplanarized structure, owing to the conformational reorganization induced by the more polar solvent. The observed solvatochromism can also be appreciated in the fluorescence spectra although to a lesser degree. Indeed, ongoing from CHCl₃ to DMF, the spectrum of CCC-1 kept two well discernible emission bands at 404 nm and 424 nm while for CCC-2 the lower wavelength band became an almost imperceptible shoulder at around 408 nm; for both compounds a considerable broadening occurs at the low energy edge of the spectra.

CCC-1 and CCC-2 are blue emitters with high fluorescence quantum yields (Φ_F = 0.78 and 0.68, respectively) in DMF solution under air-equilibrated conditions.

Recognition and sensing evaluation in organic medium

Our primary goal was to determine the recognition abilities and sensory power (sensitivity and selectivity) of calix[4]arene-carbazole conjugates (CCC-1 and CCC-2) toward cytochrome c, a model multi-functional protein. Cytochromes c (cyts c) are small (<12.4 kDa) single-domain globular proteins playing significant and crucial roles as electron carriers in mitochondrial respiratory system of living organisms as well as in apoptosis after being released from the inter-membrane of mitochondria into the cytosol.²⁰ Mitochondrial cyts c from different species typically comprise 104 ± 10 amino acid residues arranged in different sequences. Nineteen lysine and two arginine residues lend the protein surface positively charged. The tertiary structure of h-cyt c,²¹ highlighting several lysine and arginine residues near the heme edge, is depicted in Fig. 3a. These residues can become engaged in strong electrostatic interactions with complimentary charged receptors, namely with the carboxylic/carboxylate multiple functionalities of CCCs. Fig. 3b illustrates a putative complex formed between a low-energy conformer CCC-1 ²² and the h-cyt c crystal structure²¹ after manual docking, where the carboxylic acid residues in both calixarene sub-units directly interact with the Lys8 and Lys86 basic residues of the protein. In a configuration like this, one can foresee that additional hydrophobic interactions could be established between the CCCs' central core and the solvent exposed region of the prosthetic heme group which besides enhancing the overall binding simultaneously provides a suitable pathway for electron transfer to occur between the partners. Indeed, this binding region (which also includes Lys13, Lys72 and Lys87 residues) has been identified as operating in complex formation between cytochrome c peroxidase and cytochrome c, revealing possible electron transfer pathways for this redox pair.²³

Fig. 3 (a) Crystal structure of h-cyt c²¹ (α-helical and random coil regions coloured by residue), showing positively charged lysine (purple) and arginine (cyan) residues near the top of the protein pocket, and the location of heme (shown in CPK format) binding residues Cys14, Cys17 (light blue), His18 (red) and Met80 (light green); (b) A manually docked complex of CCC-1 ²² and h-cyt c²¹ showing a possible mode of host–guest interaction (CCC-1 conformer displayed with the calixarene moieties oriented in a syn fashion around the central chromophore unit, pointing the carboxylic acid functionalities downward).

The interaction of CCCs with target proteins was followed by spectrofluorometric titration. Titration of CCC-1 (1.0 × 10⁻⁷ M) with h-cyt c using previously prepared solutions with increasing host : guest molar ratios (up to 66 molar equiv. of protein, in turn prepared from a stock solution in water, pH_app ∼ 7) in DMF bring about an efficient quenching of CCC-1 emission (Fig. 4). At this apparent pH, h-cyt c is positively charged (isoelectric point, pI = 9.6).²⁴

Fig. 4 Fluorescence spectra of CCC-1 (1.0 × 10⁻⁷ M) in the presence of increasing amounts of h-cyt c at 25 °C in DMF (pH_app ∼ 7). Inset (a): change in fluorescence intensity ratio at 402 nm as a function of h-cyt c concentration (λ_exc = 360 nm); inset (b): Job plot after monitoring the CCC-1 emission at 425 nm as a function of the mole fraction of h-cyt c, keeping the total concentration of the two species constant at 6.0 × 10⁻⁷ M.

The continuous variation method (Job analysis) was used to determine the main stoichiometry attained in the CCC-1:h-cyt c complex.²⁵ As shown in Fig. 4 (inset b), the plot confirms the formation of n : n complex, from which a 1 : 1 stoichiometry was derived by applying a reported methodology.²⁶

A nonlinear curve-fitting of the fluorescence data to a 1 : 1 binding model (Fig. 5) gave an association constant (K_a) of 4.6 × 10⁵ M⁻¹ for the CCC-1:h-cyt c complex (cf. Experimental section for details). The good curve-fitting plot obtained for this system further points to a 1 : 1 stoichiometry in the host : guest complexation.

Fig. 5 Curve-fitting of fluorescence titration data of CCC-1 with h-cyt c at 25 °C in DMF (pH_app ∼ 7). The experimental data was fitted to eqn (1) (see Experimental section) for estimation of K_a.

Using the same methodology to derive the association constant, a lower K_a (3.1 × 10⁵ M⁻¹) was found for the interaction of the isomeric CCC-2 with h-cyt c.

The observed quenching can be quantified by the Stern–Volmer (S–V) formalism. When moderate to strong interactions occur between a fluorophore and a quencher in their ground-state, the resulting complex is non-emissive since upon irradiation it immediately decays to the ground-state by non-radiative processes. In this situation, only the fraction of the total fluorophore population that remains uncomplexed will emit. Under this static quenching mechanism, the fluorescence decrease is expressed by eqn (2) which is derived from the association constant for the formation of the complex, assuming a 1 : 1 stoichiometry:²⁷

F₀/F = 1 + K_SV[Q], (2)

where F₀ and F are the fluorescence intensities corresponding to the fluorophore concentrations in the absence and presence of the quencher, [Q] is the quencher concentration and K_SV is the static Stern–Volmer constant, which for pure static mechanisms should coincide with the association constant.

From the Stern–Volmer plot of CCC-1-h-cyt c pair depicted in Fig. 4 (inset a), a K_SV of 4.3 × 10⁵ M⁻¹ (R² = 0.9962) was extracted, considering only the low regime quencher concentrations (up to 4.0 µM) that provide a F₀/F linear dependence. Indeed, an upward curvature is immediately apparent in the plot at higher quencher concentrations which can be associated to a sphere-of-action quenching mechanism and/or from a collisional quenching contribution, in either case providing positive deviations to the S–V plot. A similar S–V plot was obtained for CCC-2 (Fig. S1†), which incidentally give a K_SV = 4.0 × 10⁵ M⁻¹ (R² = 0.9983; [h-cyt c] up to 2.8 µM).

These values show that the K_SV are in acceptable agreement with the association constants previously calculated by the nonlinear curve-fitting analysis, particularly for the CCC-1:h-cyt c pair. In either approach, CCC-1 emission is quenched more efficiently than that of its CCC-2 isomer by h-cyt c.

This last result was at first somewhat surprising since at this stage we expected that the main operating fluorescence quenching mechanism would be originated by a photoinduced electron transfer (PET) from the excited single occupied molecular orbital (¹SOMO*) of the receptors to the h-cyt c lowest unoccupied molecular orbital (LUMO). Solely based in this parameter, and given that the LUMO energy of CCC-2 (−1.65 eV)²⁸ is higher than that of CCC-1 (−1.99 eV),²⁸ one should have expected a more exergonic ET to the protein (E_LUMO = −4.76 eV in its native state)²⁹ from the former, which in turn will presumably lead to a higher K_SV for CCC-2:h-cyt c complex. Clearly other factors, such as a larger steric hindrance on complex formation in the case of CCC-2, might have contributed to the observed values, given that this kind of quenching requires an orbital contact between the host and the guest. Besides, a Förster-type resonance energy transfer (RET) mechanism may also be considered in order to explain the observed reduction in the emission for these systems under the described conditions (using DMF as primary solvent). To further shed some light on this point and establish the possible occurrence of RET mechanism we studied the efficiency of energy transfer between CCC-1/CCC-2 and h-cyt c in DMF. According to Förster resonance energy transfer theory,³⁰ the efficiency of the energy transfer (E; eqn (3)) between a donor (D) and an acceptor (A) molecule depends on their distance (r) and the critical energy transfer distance or Förster distance (R₀):

E = R₀⁶/(R₀⁶ + r⁶) (3)

At this distance, half of D molecules decay by RET and the other half by radiative and non-radiative decays, thus the efficiency (E) is 50%. Typical Förster distances fall in the range of 20 to 60 Å.³⁰ The spectral properties of D and A molecules define the magnitude of R₀, which is given by the relation:

R₀⁶ = 8.79 × 10⁻⁵(κ²n⁻⁴Φ_DJ(λ)) (in Å⁶) (4)

where κ² is the spatial orientation factor expressing the orientation of the interacting transition dipoles of the donor and acceptor, n is the refractive index of the medium, Φ_D is the fluorescence quantum yield of D (in the absence of A) and J(λ) is the overlap integral of the fluorescence emission spectrum of D and the absorption spectrum of A (expressed in M⁻¹ cm⁻¹ nm⁴). The overlap integral is given by the following expression:

J(λ) = ∫F_D(λ)ε_A(λ)λ⁴dλ (5)

where F_D(λ) is the corrected fluorescence of D in the wavelength range λ to (λ + Δλ) with the total intensity (area under the curve) normalized to unity and ε_A(λ) is the extinction coefficient of A at λ.

Values of the overlap integrals for the D–A pairs corresponding to the absorption and fluorescence spectra shown in Fig. 6, computed according to eqn (5),³¹ fluorophores' quantum yields in DMF and calculated Förster distances are gathered in Table 1.

Fig. 6 Fluorescence emission of CCC-1 and CCC-2 (λ_exc = 360 nm) and absorption spectra of h-cyt c in DMF.

Table 1 Critical energy transfer distances (R₀) and D–A distances (r) for RET from CCC-1 and CCC-2 to h-cyt c and metmyo in DMF

Donor	Acceptor	ΦD^a	J (M^−1 cm^−1 nm^4)	R0^b (Å)	r^c (Å)	E
a The fluorescence quantum yields of CCC-1 and CCC-2 were determined in DMF using 9,10-diphenylanthracene as reference (ΦF = 0.72, ethanol)^15 in air-equilibrated conditions (λexc = 360 nm).b R0 was calculated according to eqn (4) using n = 1.4305 (DMF) and κ^2 = 2/3 (considering a dynamic random averaging of the D–A pair).c r was calculated from eqn (3) using energy transfer efficiencies (E) for 4.0 µM protein solutions.
CCC-1	h-cyt c	0.78	7.466 × 10^14	44.9	40.8	0.64
CCC-2	h-cyt c	0.68	7.185 × 10^14	43.5	39.2	0.65
CCC-1	Metmyo	0.78	1.240 × 10^15	48.8	49.5	0.48
CCC-2	Metmyo	0.68	1.229 × 10^15	47.6	51.9	0.37

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The larger Förster distance attained for CCC-1 in comparison to CCC-2 is mainly due to its larger quantum yield and also a better overlap integral. This may explain why the CCC-1 fluorescence is reduced more efficiently in comparison to its isomer since the energy transfer could occur at longer D–A distances.

Knowing the Förster distance, the average distance (r) at which the RET between the D–A pair occurs can be calculated using eqn (3); for that one needs to first determine the transfer efficiency which can be accomplished by taking the emission intensities of the donor in the absence (F₀) and presence (F) of acceptor (eqn (6)):

E = 1 − F₀/F (6)

The above expression strictly applies to D–A pairs separated by fixed distances.³⁰ In situations where the donor and acceptor are not part of the same molecule, and are randomly distributed in solution, the calculated efficiency for the non-radiative excitation energy transfer process is dependent on the acceptor concentration. Thus, for the CCC-protein pairs listed in Table 1, the r distances were calculated using proteins at 4.0 µM.

The foregoing data strongly suggests the likelihood occurrence of a successful RET process in DMF between CCC-1/CCC-2 and h-cyt c.

The behavior of myoglobin, another heme-containing protein, was next considered. Myoglobin (16.95 kDa)³² is also a globular protein containing a single peptide chain of 153 amino acid residues arranged in eight α-helical regions and a Fe-protoporphyrin IX heme group tethered in the hydrophobic pocket by histidine (His93). A second distal histidine residue (His64 not bound to the heme group) controls the oxygen access to the iron center. Its mainly primary physiological role is to maintain an intracellular oxygen storage and supply in skeletal muscle tissues and heart cells of vertebrates.

The experiments were performed with myoglobin in its oxidized form (metmyoglobin). The presence of characteristic bands of metmyoglobin (metmyo)³³ at 409, 504 and 635 nm was verified by UV-Vis analysis (Fig. S2†).

Metmyo tertiary structure³⁴ is shown in Fig. 7 featuring the positively charged lysine residues and two histidines around the oxidized heme center.

Fig. 7 Structure of metmyo,³⁴ presenting positively charged lysine (purple) and histidine (red) residues near the heme.

Although globally neutral at pH ∼ 7 (pI = 6.8–7.4),²⁴ the probable interaction with CCCs via charged lysine residues near the heme center resulted in fluorescence quenching upon excitation at 360 nm. The binding constants for complex formation with both hosts were derived as before by nonlinear curve-fitting of the emission data assuming a 1 : 1 stoichiometry for the complex. Representative results for CCC-1 are illustrated in Fig. 8 (see Fig. S3† for CCC-2:metmyo pair).

Fig. 8 Fluorescence spectra of CCC-1 (1.0 × 10⁻⁷ M in DMF) upon addition of metmyo at 25 °C (pH_app ∼ 7). Inset: binding isotherm monitored at 404 nm (λ_exc = 360 nm).

The association constants obtained for the two receptors (K_a(CCC-1) = 1.9 × 10⁵ M and K_a(CCC-2) = 1.5 × 10⁵ M) unveil a lower binding affinity (less than half) toward metmyo as compared to h-cyt c, while keeping a binding ascendant of CCC-1 over CCC-2. These results seem to indicate that even in organic medium a significant dependence of the binding affinities on the partners' electrostatic interactions exists, reflected in this case by the higher basic nature of h-cyt c.

The calculated Förster and D–A pair distances for the systems CCCs-metmyo are summarized in Table 1. It can be seen that the Förster distances are larger for these systems as compared to those with h-cyt c, certainly due to the greater overlap integrals for CCCs-metmyo pairs, meaning that the RET would be allowed at even longer donor–acceptor distances. However, since the electrostatic interactions between the partners should be significantly lower for metmyo, D and A interact at longer distances yielding energy transfer efficiencies notoriously reduced in comparison to h-cyt c. In addition, if one conceives that besides RET a complementary PET mechanism is also operating in these systems, although with a reduced expression in organic medium (see below), the higher electron affinity of h-cyt c compared to metmyo³⁵ could also help to explain the larger quenching efficiency of the former.

To confirm the effect of the overall charge of the protein on the binding affinity, stock solutions of h-cyt c were prepared in 20 mM HEPES buffer (pH = 8.2) and 20 mM glycine buffer (pH = 9.8) and then titrated in DMF solution with CCC-1. At an apparent pH of 8.2, a substantial reduction on K_a ensues (K_a = 2.45 × 10⁵ M, pH = 8.2), decreasing slightly as the pH is further raised (K_a = 2.13 × 10⁵ M, pH = 9.8).

To probe the relevance of the structural properties of CCC-1 in the binding process to the protein, a bis-calixarene analogue lacking the carboxylic functionalities (CCC-3; Fig. 1) was next evaluated following similar experimental protocols and data treatment. On h-cyt c titration in DMF (pH_app ∼ 7), an association constant of 1.46 × 10⁵ M could be retrieved from fluorescence data curve-fitting (not shown). This result, while clearly attributing a specific role to the acidic substituents in the calixarene lower rims of CCC-1 and CCC-2 toward the h-cyt c surface recognition leading to an enhanced binding constant which amounts for more than threefold when CCCs with the same type of linkage in the carbazole rings are compared (CCC-1 and CCC-3), it also reveals that in the absence of such functionalities a noteworthy binding affinity subsists between CCC-3 and h-cyt c.

To assess the receptors' selectivity toward heme proteins, the chicken egg white lysozyme (lys),³⁶ composed by 129 amino acid residues (14.33 KDa), and showing glycoside hydrolase activity in response to bacterium, was selected as a highly basic protein model (pI = 10.5–11).³⁷ At the neutral working pH in DMF, the protein should be in its highly charged form prompting for strong electrostatic interactions with the receptors. We anticipate however that given the absence of any prosthetic group in the protein capable of interacting with CCCs either via PET or RET mechanisms, no measurable transduction signal from the putative interaction should result. We test this hypothesis by titrating a solution of CCC-1/CCC-2 in DMF with lysozyme (pH_app ∼ 7.3; lys previously dissolved in water). No evidence of fluorescence quenching promoted by the protein was observed for any of the calixarene receptors in the same concentration range used for the other proteins; actually, ongoing up to 40 molar equiv. of protein (4 µM), the emission intensity of CCCs only varies around ±4% of its initial value.

Sensory abilities in aqueous buffered solutions containing DMF

The foregoing results demonstrated the potential of CCC-1/CCC-2 receptors as highly selective sensors for heme proteins in organic media, where the observed fluorescence quenching effects were chiefly assigned to RET processes. As stated earlier, a photoinduced electron transfer from the excited state of CCCs to the heme centres was expected to be the dominant quenching mechanism given the thermodynamically allowed ET. So why is this not happening? In order to clarify this issue, we conducted a series of experiments to evaluate to which point the medium used in the preceding assays could modulate the overall binding and sensing efficiency of heme proteins by CCCs.

The rationale behind this comes from the knowledge that the electron transfer properties of cyt c are affected by the protein conformation around the heme group. The heme binding residues Cys14, Cys17 (both covalently bound to the heme c prosthetic group through thioether bridges), His18 and Met80, are largely preserved in most cyts c.²⁰ In native form, His18 and Met80 are the axial ligands of Fe(III) heme in h-cyt c. Met80 is thought to be responsible for the high redox potential of cyt c owing to the enhanced stabilization of the ferrous over the ferric state provided by the good electron–acceptor character of the sulfur atom in the axial ligation Met80-Fe(III).³⁸ Replacement of the native axial methionine ligand by exogenous donor ligands can lead to the disruption of the heme Fe(III)-Met80 coordination bond leading to significant changes in the protein conformation around the heme pocket and substantial decreases in the protein reduction potential,³⁹ deeply affecting its ability as an electron acceptor. This might have had significant implications when we were dealing with the sensing of heme proteins in non-aqueous media.

The progressive disruption of such bond can be monitored by electronic circular dichroism (CD) spectra in the Soret band region (λ_{max abs} = 409 nm).⁴⁰ Fig. 9 presents the CD spectra of h-cyt c in several solvent mixtures.

Fig. 9 CD spectra of h-cyt c (c = 1.0 × 10⁻⁵ M) at 20 °C in (a) water, (b) 50 mM phosphate buffer : DMF (9 : 1), (c) 50 mM phosphate buffer : DMF (8 : 2), and (d) DMF solutions. Inset: CD and UV-Vis spectra of equimolar solutions (c = 1.0 × 10⁻⁵ M) of CCC-1 and h-cyt c in 50 mM phosphate buffer : DMF (9 : 1). Solutions in DMF were prepared from a stock solution of protein in water (c = 2.0 × 10⁻³ M) followed by dilution with DMF to the desired concentration.

As easily perceived, the CD spectra of h-cyt c in water (pH ∼ 7) and aqueous buffered : DMF (9 : 1) solution (pH ∼ 7.2) mixture are similar, showing a negative couplet centered at around 408 nm. On rising the DMF content, the negative bisignate Cotton effect (peaking at 420 nm) progressively decreases in intensity (8 : 2 mixture) and completely vanishes in DMF solution, ending up with a positive monosignate Cotton effect at 409 nm. In this context it is worth mentioning that the CD spectrum of equimolar solutions (1.0 × 10⁻⁵ M) of CCC-1 and h-cyt c in 50 mM phosphate buffer : DMF (9 : 1) shows a profile very similar to that of the native state of h-cyt c, showing that the conformation of the protein near the heme group is not altered by the presence of the receptor (cf. inset Fig. 9).

The preceding data unequivocally demonstrate that when DMF is used as primary solvent, a profound change around the heme cleft occurs. The Fe(III)-Met80 coordination bond is broken and the stabilization effect on the putative ferrous state is lost.

The change in h-cyt c conformation in this region can also be corroborated by taking into consideration the relative emission of tryptophan residue (Trp59). In h-cyt c native form (solutions in water, phosphate buffer or phosphate buffer : DMF (9 : 1)), the fluorescence of Trp59 (λ_em near 330 nm) is highly reduced due to strong resonance energy transfer to the heme (Fig. S4†).⁴¹ When the Met80-heme ligation is disrupted in DMF solution, the Trp59-heme interaction also falls apart and its emission is red shifted to around 345 nm with a concomitant large increase in fluorescence intensity (Fig. S4†).

These two facts taken in conjunction definitively indicate that the pristine structure of the protein is severely changed in DMF. Hence, water-based solvent systems, that are simultaneously compatible with CCCs solubility and preserve the native conformation and redox properties of the protein, seem to be essential for binding assays in order to fully exploit the h-cyt c potential for ET mediated events.

After finding that CCC-1 and CCC-2 receptors were soluble in all the aqueous mixtures containing DMF mentioned above, their UV-Vis and fluorescence spectra were measured. Representative spectra from CCC-1 are shown in Fig. 10.

Fig. 10 Absorption and emission spectra of CCC-1 (c = 1.0 × 10⁻⁵ M) in various solvent mixtures: (a) DMF, (b) 50 mM phosphate buffer : DMF (8 : 2) and (c) 50 mM phosphate buffer : DMF (9 : 1); λ_exc = 360 nm.

By analysis of UV-Vis spectra it results clear that an aggregation of CCC-1 molecules takes place as the water content raises in the mixture, as may be witnessed by the gradual broadening of the chromophore band in the long wavelength region. Additionally, a reduction in the overall absorption intensity is observed, especially in the low energy band region. In aqueous buffered solutions, the emission spectrum of CCC-1 (1.0 µM) is dominated by an emission at 454–456 nm with shoulders at around 403 and 424 nm (Fig. 10). This situation does not parallel that of CCC-2 which has an unstructured emission with a major band peaking at 437 nm. The observed effects are likely related to the stronger relaxation of the excitation energy of CCC-1 (which possesses a larger dipole moment) promoted by the more polar solvent system.⁴² This is accompanied by a strong reduction of the quantum yield of CCC-1. We estimate from fluorescence data that in phosphate buffer : DMF (9 : 1) solution the QY of CCC-1 is around 0.16 (ca. 20% of its original value in DMF; both solutions [CCC-1] = 1.0 × 10⁻⁵ M).

The following set of spectrofluorometric titrations where then conducted in phosphate buffer : DMF (9 : 1) at 25 °C (pH = 7.2), using a concentration of the host [CCC-1] = 1.0 µM. Fig. 11 depicts the results obtained.

Fig. 11 Fluorescence spectra of CCC-1 (1.0 × 10⁻⁶ M) in the presence of increasing amounts of h-cyt c in phosphate buffer : DMF (9 : 1) solution at 25 °C (pH = 7.2). Inset: Stern–Volmer plot after monitoring the change in fluorescence intensity at 454 nm (λ_exc = 360 nm).

In the low concentration regime (up to 32 nM of h-cyt c), the Stern–Volmer plot (Fig. 11, inset) yields a straight line from which an S–V constant (K_SV) of 6.0 × 10⁷ M⁻¹ (R² = 0.986) could be retrieved. For the titration of CCC-2 receptor with h-cyt c in the same concentration range, only a slightly lower K_SV (4.7 × 10⁷ M⁻¹; R² = 0.989) was obtained (Fig. S5†). Moreover, no photobleaching/photodegradation of CCCs was detected in this medium under the experimental conditions.

These are among the highest constants reported for the fluorescence quenching of artificial receptors/sensors with h-cyt c.⁴³ For example, a former disclosed calix[4]arene receptor containing four peptide loops in its upper rim, with a total of eight free carboxylic acid groups in the periphery, strongly bounds to cyt c with a K_a of 3.05 × 10⁶ M⁻¹, as determined by kinetic measurements of ascorbate reduction of Fe(III)-cyt c at pH = 7 in aqueous buffer.⁴⁴ It is worth mentioning that the binding of cyt c by this receptor is strong enough to compete with its natural partners cyt c peroxidase and Apaf-1;⁴⁵ our current receptors bound to h-cyt c over one order of magnitude higher in aqueous buffered solutions at pH = 7.2.

Recall that the association constants (expressed as K_SV) previously determined in DMF solution were in the range 4.0–4.3 × 10⁵ M⁻¹ for both hosts. We believe that the astonishing increase in K_SV in aqueous medium is a result of a change in the quenching mechanism. Indeed, keeping the intrinsic electron acceptor properties of h-cyt c almost intact in the solvent mixture (phosphate buffer : DMF (9 : 1)), a very efficient ET process could now ensue owing to the excellent electron donating capabilities of excited CCCs.

To support this view, a titration experiment of CCC-1 with metmyoglobin was carried out under the same conditions, keeping the protein concentration at nanomolar levels. No quenching activity was observed whatsoever (Fig. S6a†). This outcome is consistent with the low electron transfer capabilities of metmyo.⁴⁶ Moreover, since the overall quenching efficiency is dependent on the host–guest complex formation, which in turn is driven by electrostatic interactions, the fact that at the experimental pH of 7.2 metmyo presents a net charge near zero further justifies the absence of fluorescence quenching of CCC-1 by metmyo. To exclude the possibility that the observed quenching was due to further aggregation of CCC-1/CCC-2 upon addition of h-cyt c with concomitant self-quenching, a control experiment with the highly basic protein lysozyme was performed. As expected, no signs of fluorescence quenching of CCC-1 were observed under otherwise identical conditions (Fig. S6b†).

These findings reveal the noteworthy sensing ability and selectivity of our calixarene-based sensors CCC-1 and CCC-2 toward cyt c. This is particularly relevant as several reports have shown that nonspecific interactions and signal responses dominate several protein sensory schemes.^9,47

Conclusions

The bis-calix[4]arene-carbazole conjugates CCC-1 and CCC-2 are exceedingly able to target basic residues on the surface of cytochrome c by means of electrostatic and hydrophobic interactions near the heme cleft providing a simple pathway for electron transfer between the complexed partners and thus a useful signaling response for protein sensing in aqueous-based medium (pH = 7.2). These synthetic receptors display a high binding affinity toward h-cyt c and a remarkable selectivity for this protein in aqueous medium, as was demonstrate by spectrofluorometric experiments involving other heme (myoglobin) and non-heme (lysozyme) proteins.

Through a series of experiments conducted in organic and aqueous media, it became evident that the mechanisms by which the fluorescence of the receptors was reduced upon contact with heme proteins are of different origin and strongly dependent on the media which in turn modulate the electron accepting properties of the guest proteins, as was evidenced by circular dichroism and fluorescence analysis of diagnostic signals coming from specific amino acid residues around the heme environment of h-cyt c. Thus, while in the former medium a Förster-type resonance energy transfer seems to dominate the events with both proteins, with binding constants for h-cyt c and metmyo in the same order of magnitude (K_a in the range of 1.5–4.6 × 10⁵ M for the two receptors), the observed quenching in aqueous solvent systems result from a photoinduced electron transfer which is only highly effective (K_SV of 6.0 × 10⁷ M and 4.7 × 10⁷ M for CCC-1 and CCC-2, respectively) and specific for h-cyt c owing to its larger reductive capabilities.

It is expected that the high sensitivity and selectivity attained by our bis-calixarene receptors (CCC-1 and CCC-2) toward cyt c could positively contribute for the future design and development of calixarene-based biosensors for in vitro and in vivo applications.

Acknowledgements

We thank Fundação para a Ciência e a Tecnologia/MEC (Portugal) for financial support (PEst-OE/EQB/UI0702/2011-2014). Dedicated to Professor Ana M. Lobo on the occasion of her 70th birthday.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Fluorescence spectra, binding isotherms and Stern–Volmer plots of spectrofluorometric titrations, and absorption spectrum of metmyoglobin. See DOI: 10.1039/c5ra19887a

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