Trichromophoric pentapeptide: impact of β-sheet conformation on dual path to excimer emission and sensing of BSA

Abstract

We are reporting two mechanisms for excimer emission in a designed trichromophoric pentapeptide wherein the triazolo aromatic amino acid scaffold (^ArTAA) nucleates β-sheet conformation. The designed unnatural fluorescent pentapeptide shows an excimer emission either via FRET from the scaffold (^ArTAA) acting as a donor or via direct excitation of an acceptor chromophore, ^TPyAla_Do. Moreover, it serves as an effective fluorescence light-up probe for studying protein–peptide interactions.

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Fluorescent/fluorescently labelled peptides/proteins serve as sensitive probes for visualizing intracellular events and understanding molecular interactions inside a cell.¹ Many of such aspects have been studied either by exploiting intrinsic fluorescence of a protein or with the help of extrinsic fluorescent labels. However, these labels suffer from several shortcomings and thus are unable to fulfill all the research needs.² Therefore, there is a strong demand for generating a small protein tag³ or site-specific incorporation of unnatural fluorescent amino acids⁴ into a protein.^3,4 Such fluorescent proteins or small peptides are currently attracting much research interest to gain a deeper insight into the nature, regulation and functions inside living cells.⁵ Over the various available strategies, fluorescence spectroscopic technique is a powerful tool for studying such biological events because of its high sensitivity, excellent temporal resolution and good reproducibility.^2a,3a Furthermore, among the various fluorescence photophysical phenomena, Förster resonance energy transfer (FRET)⁶ and excimer⁷ emission find widespread applications in elucidating such events of proteins/peptides.^6,7

Moreover, nucleating β-sheet conformation is of great importance because it not only plays a scaffolding role, but also serves as a key recognition motif in many important biological processes.⁸ Efforts have been undertaken to study the protein–protein or other interbiomolecular interactions utilizing a fluorescent protein/fluorescently labeled peptide. However, no or little attention has been paid in the design of a fluorescent peptide which in a particular conformation, such as β-sheet, can impact on the fundamental aspects of photophysics. Investigating such impact might find widespread applications in studying peptide–protein or protein–protein or peptide–DNA interactions which ultimately would help developing peptide therapeutics.

As a part of our continuous research efforts in designing fluorescent biomolecular building blocks and probes of fundamental importance, we report herein the design of a conceptual trichromophoric β-sheet pentapeptide which in that particular conformation showed an excimer emission via two mechanism.^4a,b We envisaged that the mechanism of excimer/exciplex formation via FRET would become possible by judicious designing and proper positioning a donor/acceptor pair with respect to a third chromophore in a probe wherein a pair of chromophores involve in π–π stacking interaction. Thus, we designed an aromatic triazolo amino acid scaffolded pentapeptide 2 wherein the scaffold (1, ^ArTAA, Fig. 1) itself is a chromophore. The fluorescent triazolylpyrene (TPy) unnatural amino acids (^TPyAla_Do) were chosen as other two chromophores attached to the N-and C-terminus, respectively, of the scaffold via an intervening natural amino acid, leucine. We thought that excitation at the scaffold would led to an energy transfer to a second chromophore, ^TPyAla_Do, which would then form π-stacked complex (excimer) with the proximally positioned third chromophore, ^TPyAla_Do. To the best of our knowledge, we are the first to introduce a new generation peptide-probe as a dual path system to excimer emission (Fig. 1). As an application we exploited the probe in switch-on fluorescence sensing of BSA protein.

Fig. 1 Chemical structures of the scaffold 1, the trichromophoric fluorescent pentapeptide 2 showing the photophysical aspects and a leucine–enkephalin analogue peptide 3.

To establish the fundamental concept of entry to excimer emission either via FRET and/or via direct excitation of a FRET acceptor, triazolylpyrene (TPy) of ^TPyAla_Do, we first synthesised the triazolo aromatic amino acid (1, ^ArTAA) scaffolded fluorescent pentapeptide 2 via a solution phase peptide coupling protocol following our earlier reports (ESI, Schemes 1–3†).^4a,b The peptide 3, a Leu-enkephalin analogue, was also synthesised in a similar way to study the conformation induced by the scaffold 1 (ESI, Scheme 4†).⁹

The secondary structure elucidation of peptide 3 via CD spectroscopy revealed the presence of a negative peak at 225 nm and a positive peak at 195 and 236 nm (characteristic of tyrosine) in ethanol indicating a predominant β-sheet like structure (ESI, Section 4.1†).¹⁰ The fluorescent pentapeptide 2 was also found to adopt predominant β-sheet conformation (Fig. 2a). While the tripeptide 18 (ESI, Scheme 2†) containing a single TPy showed a positive induced CD signal (ESI, Section 4.1†), the pentapeptide 2 showed a negative Cotton effect in the absorption range of TPy indicating π–π-stacking interaction between two terminal TPy units.^10c,d These observations indicated that the aromatic triazolo amino acid scaffold (^ArTAA) in the turn conformation acts as a turn mimetic β-sheet nucleator (Fig. 2a).^10a,b

Fig. 2 (a) CD spectra of pentapeptide 2 and UV traces of pentapeptide 2, scaffold 1 and dipeptide 16 containing only one TPy [10 μM, ethanol]. (b) MD simulated cluster of conformers within 21 kJ mol⁻¹ from the global minima of pentapeptide 2.

The presence of intramolecular H-bonding through amide NHs was evident from both IR and variable temperature NMR (VT NMR) spectroscopy (ESI, Section 4.2–4.3†).^4a,b,11 Fourier self-deconvolution (FSD) trace of IR spectra of peptide 2–3 showed amide I band absorptions at 1689–1685 cm⁻¹ and 1644–1635 cm⁻¹, respectively, supported the predominant β-sheet conformation along with a contribution from turn reflecting in the FSD trace at 1665–1670 cm⁻¹.^11d While all the amide NHs of pentapeptide 2 were strongly H-bonded, the peptide 3 showed H-bonding involving the amide NH of C-terminal-Leu only (ESI, Section 4.3†).^4a,b,11c The study of solution conformation using NOESY and ROESY spectra of both the peptides 2–3 revealed that the scaffold itself adopted hairpin shape with overall turn like conformation. The peptide 3 showed no interaction between Phe and Tyr (ESI, Section 4.4†). The spatial proximity between two terminal TPy units in peptide 2 is evident from both NOESY and ROESY spectra. Moreover, peptide 2 showed interactions among the aromatic hydrogens of two terminal triazolylpyrenes (TPy) suggesting their close proximity as well as rigidity in a single conformation and hence a possibility of photophysical interaction among the scaffold, ^ArTAA, and the C-terminal triazolylpyrene (TPy) in pentapeptide 2 (ESI, Section 4.4.1†) which was also supported by a molecular dynamics (MD) simulation (Fig. 2b).^4a,b,12

To test our hypothesis, we, next, examined the possible photophysical interaction behaviour among the terminal triazolyl unnatural amino acids and the scaffold in fluorescent pentapeptide 2 in ethanol. Based on our designing concept we found that the fluorescence spectrum of the aromatic triazolo amino acid scaffold (1, ^ArTAA) overlapped significantly with the absorption spectrum of ^TPyAla_Do containing dipeptide, 16 (ESI, Scheme 2†) indicating a possibility of FRET process to occur (ESI, Section 6.4†). Moreover, the peptide 2 could selectively be excited at 290 nm (λ^max_abs of ^ArTAA) where there is very low absorbance of ^TPyAla_Do. With this observation we turned our attention to study the FRET process in detail.⁶ When excited at absorption maximum of the donor, ^ArTAA (λ_ex = 290 nm), the pentapeptide 2 showed three emission bands at 330, 405 and 486 nm corresponding to emission from scaffold, ^ArTAA, monomer emission from ^TPyAla_Do and excimer emission from the π-stacked excited state complex between two terminal ^TPyAla_Do, respectively. Moreover, we observed that the fluorescence intensity of the acceptor, ^TPyAla_Do, increased from that of the free acceptor emission by almost two times in presence of donor (Fig. 3a). On the other hand, the fluorescence intensity of the donor, ^ArTAA, in pentapeptide 2 decreased almost two times of that of the free donor fluorescence in presence of an acceptor, ^TPyAla_Do (Fig. 3a). These observations revealed the visual evidence of FRET process from ^ArTAA to ^TPyAla_Do in peptide 2 which was also supported from a FRET process in tripeptide 18 (ESI, Scheme 2†) wherein a single TPy was attached with the scaffold via an intervening Leu (Fig. 3a).^6d The phenomena were also reflected in the change and differences in intensities of fluorescence images under UV-irradiation of various peptides at λ_ex = 290 and 350 nm, respectively (Fig. 3b).

Fig. 3 (a) Steady state fluorescence spectra of donor chromophore (scaffold ^ArTAA_Do) in absence or in presence of acceptor (^TPyAla_Do in peptide 16) showing a FRET and FRET mediated excimer emission in pentapeptide 2. (b) Photographs of various peptides under UV irradiation (λ_ex = 290 and 350 nm).

Further evidence of FRET process came from a time resolved fluorescence study wherein we observed a decrease in life time of both the components of donor (^ArTAA; λ_ex = 293 nm, λ_em = 330 nm) from 3.7 to 2.0 ns and 8.9 to 6.4 ns. While monitoring the decay at excimer emission (λ_em = 485 nm), the life time of TPy excimer was found to increase from 22.4 ns (λ_ex = 336 nm) to 25.9 ns (λ_ex = 293 nm) (ESI, Section 6.5†).^6e,10c,d All these observations evident our hypothesis of FRET mediated excimer emission. Based on 2D NMR observation we can propose that the FRET occurred between the scaffold (^ArTAA) and the C-terminal TPy of ^TPyAla^Do. The FRET mediated excimer emission is a new concept and might find wide applications in chemical biology.

To establish the second path of excimer emission, i.e. via direct excitation of a FRET acceptor, ^TPyAla^Do, we excited the pentapeptide 2 at the absorption maxima of ^TPyAla^Do (λ_max = 350 nm) and in reality we observed both the emission-the monomer emission at 405 nm as well as the excimer emission at 486 nm which was also supported from a time resolve fluorescence experiment (ESI, Section 6.5†). Therefore, we established our concept of dual mechanism of excimer emission-either via FRET or via direct excitation of the FRET acceptor. To the best of our knowledge this fundamental phenomenon is new and will attract scientists for designing probes for application in chemical biology.

Next, we explored the novel dual door entry system to excimer emissive pentapeptide 2 as a possible probe for sensing BSA protein. We envisaged that the trichromophoric fluorescent pentapeptide would sense the interaction with BSA via the generation of an enhanced fluorescence signal-either via FRET mediated monomer emission from triazolylpyrene or excimer emission. Analysis of UV-visible absorption of probe peptide 2 in phosphate buffer showed a structureless absorption at around 360 nm corresponding to triazolyl pyrene (TPy) absorption. Addition of an increasing concentration of BSA to the probe solution resulted in a hyperchromicity in absorbance along with a blue shift of wavelength (13 nm) indicating a strong binding interactions between BSA and probe 2 in the hydrophobic region (ESI, Section 7.2†).

The probe exhibited a broad excimer emission at around 470 nm along with an overlapped monomer emission at 410 nm in phosphate buffer. Upon gradual addition of an increasing amount of BSA, the monomer emission intensities of the probe 2 increased when excited at the absorption maximum of TPy (350 nm, ESI, Section 7.2†) indicating a strong interaction among the probe and hydrophobic pocket of BSA. On the contrary, upon excitation at 280 nm (absorption of the scaffold, ^ArTAA and BSA), a gradual increase in TPy monomer emission intensity at 385 nm was observed (ESI, Section 7.2, Fig. S34†). However, a minimal change in excimer/monomer intensity indicated a hindrance in the formation of π–π-stacked excited state complex between two TPy as the concentration of BSA increased. Moreover, TPy moieties got accommodated in the hydrophobic pocket of BSA leaving aside peptide chain on the surface which ultimately resulted in an increased monomer emission. This was supported from a solvent induced emission study of the probe peptide 2 wherein an enhancement of fluorescence intensity was observed while going from polar to hydrophobic solvent (ESI, Fig. S24†). Furthermore, a large enhancement of fluorescence anisotropy from 0.02 to 0.15 (monitored at 385) indicated that the TPy moiety of probe 2 bound strongly inside the hydrophobic pocket of BSA and experienced a highly restricted rotational motion (ESI, Section 7.6†).^13a,14 However, a small change in anisotropy corresponding to the excimer (from 0.01 to 0.04; monitored at 472) indicated a less perturbation of the excimer emission intensity of the probe 2 upon gradual addition of BSA. The slight change in the % α-helicity of BSA observed in the CD spectra in presence of the probe-peptide 2 indicated a possible conformational adjustment of BSA upon association in the hydrophobic region (ESI, Section 7.7†).^9,12

The spectroscopic study indicated that the probe might involve in energy transfer (FRET) with Trp unit of hydrophobic sub-domain that was again suggested from a overlapped emission spectrum of BSA and absorption spectrum of the probe (ESI, Section 7.4, Fig. S37†).¹³ Interestingly, excitation at the BSA absorption (280 nm) we observed a FRET from Trp to TPy with a concomitant decrease in intensity of donor Trp emission at 345 nm and two to three times enhancement of emission of TPy monomer at 385 nm acting as an acceptor (Fig. 4a). The excimer intensity at 470 nm was also increased slightly. Both the FRET and excimer emissions were also reflected in the differences in intensities of fluorescence images of pentapeptide 2 in presence of BSA under UV-irradiation at λ_ex = 280 and 365 nm, respectively (Fig. 4b). The FRET event in pentapeptide 2 upon binding with BSA was also evident from a time resolve fluorescence study. Thus, we observed a decrease in both the components of donor life time (Trp; λ_ex = 293 nm, λ_em = 350 nm) from 4.0 to 2.8 ns and 7.0 to 6.4 ns. While monitoring the decay at excimer emission (λ_em = 470 nm), the life time of TPy excimer remained almost unchanged (ESI, Section 7.4†).

Fig. 4 (a) Steady state emission of BSA in absence or in presence of pentapeptide 2 showing a FRET process and FRET mediated excimer emission. Dotted lines represent the resolved spectra. (b) Emission photographs of pentapeptide 2 in presence or absence of BSA under UV-irradiation at λ_ex = 280 and 365 nm.

The association constant of probe with BSA determined by Benesi–Hildebrand plot (ESI, Section 7.3†) was found to be 1.8 × 10⁵ M⁻¹ with an experimental free energy of binding, ΔG = −7.16 kcal mol⁻¹. The binding thermodynamics was also supported by an isothermal calorimetry (ITC)¹⁵ measurement (Fig. 5a and ESI, Section 7.3†). The close proximity of TPy of probe 2 and Trp of BSA and hence the possibility of occurrence of FRET process was also supported by a molecular docking calculation with Autodoc programme¹⁶ which clearly showed that C-terminal TPy moiety of the probe-peptide 2 was located in the vicinity of tryptophan (Trp-134) and remained surrounded by other hydrophobic amino acids of the hydrophobic pocket of subdomain IB of site I of BSA (Fig. 5b). The free energy of binding (ΔG) was found to be in (−7.10 kcal mol⁻¹) excellent agreement with our experimental value.

Fig. 5 (a) Integrated heat profile of the ITC with non-linear-squares fit to a two-site binding model. (b) Docking pose of pentapeptide 2 in presence of BSA.

Next, the detection limit of BSA detection was calculated as three times the standard deviation of the background noise.¹⁷ The linear fit line of plot (I_min − I)/(I_min − I_max) vs. log[BSA] was extended till it crossed the ordinate axis. A good linear relationship (R² = 0.977) in the range of BSA concentrations from 0.06 μg ml⁻¹ to 1.86 μg ml⁻¹ indicated that our probe peptide 2 could be utilized for the detection of the BSA in the submicrogram concentration range. The crossing point is the detection limit which came in the region of 0.054 μg ml⁻¹ with S/N ratio of 8.7 (ESI, Fig. S45†).

All these observations clearly proved the occurrence of excimer emission in the triazolo aromatic amino acid scaffolded trichromophoric β-sheet fluorescent pentapeptide 2 both via FRET as well as through excitation of a FRET acceptor. Furthermore, the probe was found to be efficient in sensing BSA protein via the generation of an enhanced TPy fluorescence via FRET and FRET-mediated TPy–TPy excimer emission. The present study of BSA–peptide interaction exploiting our conceptually new dual door entry to excimer emissive probe would expectedly shed light for the development of probe of higher generation for the detection and studying of peptide–protein interactions which ultimately would provide a deeper insight into the nature, regulation, and functions inside living cells.

Finally, we tested our probe peptide 2 for sensing another protein, α-amylase.¹⁸ Thus, from a fluorescence titration experiment it was observed that the probe could sense α-amylase with very low fluorescence enhancement at 385 nm (TPy emission) as compared to the detection of BSA when the probe peptide 2 was excited at α-amylase/scaffold (λ_ex = 280 nm). The phenomenon of FRET emission and the FRET mediated excimer emission is conserved similar to the case of BSA detection indicating the reserved conformation of the probe peptide 2 (Fig. 6a and ESI Section 8†). However, when excited at TPy absorption of 350 nm, the increase in α-amylase concentration did not change the fluorescence intensity of the probe which is contrary to the result observed in case of BSA. Furthermore, in contrary to the BSA, gradual addition of α-amylase did not shift the absorption spectra to short wavelength region indicating a very weak association between the probe and α-amylase (ESI Section 8†). All the spectroscopic studies evident that the interaction between the probe and α-amylase is very weak compared to BSA. This fact might be attributed to the difference in hydrophobicity between BSA and α-amylase.¹⁸ BSA is known to be more hydrophobic than α-amylase. The α-amylase lacks of suitable hydrophobic clefts in order to bind hydrophobic molecule/moiety and hence α-amylase rightly showed very lower binding affinity for the probe peptide 2 compared to BSA.¹⁸ Our experiment showed that the probe could sense BSA with much more enhancement in fluorescence intensity compared to α-amylase. It was observed that the probe peptide 2 could sense BSA with 685% enhancement in fluorescence intensity (at 385 nm) compared to 390% enhancement in case of α-amylase (Fig. 6b). Therefore, the large fluorescence enhancement is quite good for practical applications for sensing of BSA and discrimination from α-amylase.

Fig. 6 (a) Fluorescence emission titration spectra (λ_ex = 280 nm) of probe peptide 2 in absence and presence of increasing concentration of α-amylase. (b) Comparison of fluorescence (λ_em = 385 nm) intensity changes [(I − I₀)/I₀ × 100%] of probe peptide 2 upon addition of BSA and α-amylase, respectively. I₀ is fluorescent emission intensity of free probe and I is the maximized fluorescence intensity after adding BSA/α-amylase.

In summary, the newly designed trichromophoric β-sheet pentapeptide represents an important and fundamental discovery of a dual door entry system for excimer emission. The newly designed and novel axially chiral aromatic triazolo amino acid scaffold (^ArTAA) in the turn conformation acts as a turn mimetic β-sheet nucleator which would be exploited further in the peptide based drug design. Both the processes of excitation of TPy of ^TPyAla^Do-either energy transfer from excited scaffold amino acid, ^ArTAA (FRET) to TPy or direct excitation of TPy-led to the excimer emission in pentapeptide 2. Moreover, the novel probe of dual door entry to excimer emission system was found to be an effective fluorescence light-up probe for detecting and studying protein–peptide interactions in solution. This study would provide fundamental guidelines to design such conceptual fluorescent probe of dual door entry system to excimer emission which would find wide applications in the field of chemical biology.

Acknowledgements

This work is financially supported by Department of Biotechnology [DBT: BT/PR5169/BRB/10/1065/2012], Govt. of India. SJ and MK are thankful to CSIR and UGC, respectively, New Delhi, for their fellowships.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Synthesis, characterisation data, spectroscopic data, macromodel study and ¹H and ¹³C NMR spectra. See DOI: 10.1039/c6ra14084j

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