Li-Yang
Lin
,
Po-Chiao
Huang
,
Deng-Jie
Yang
,
Jhen-Yan
Gao
and
Jin-Long
Hong
*
Department of Materials and Optoelectronic Science, National Sun Yat-Sen University, Kaohsiung 80424, Taiwan. E-mail: jlhong@mail.nsysu.edu.tw; Tel: +886-7-5252000, ext 4065
First published on 12th October 2015
In this study of the amphiphilic polypeptide TP-PPLG-g-MEO2 containing a hydrophobic tetraphenylthiophene (TP) terminal, with aggregation-induced emission (AIE) properties, and hydrophilic ether side groups, it was found that secondary structures (α-helix, β-sheet and random coil) of the peptide chains affected the AIE-related emission of the TP terminal. The amphiphilic TP-PPLG-g-MEO2, prepared from a ring-opening polymerization and the following click reaction, contained a secondary structure mostly composed of α-helical chains. The main α-helical chain nevertheless can be converted into a β-sheet structure by adding NaCl into the aqueous solution of TP-PPLG-g-MEO2 and by doing so, the solution emission can be enhanced. In addition, the coil chain generated in the alkaline solution was found to emit efficiently with the highest emission intensity among all three conformations. The relationship between the AIE property and the secondary structure of the peptide chains can therefore be evaluated. All three secondary structures were also used as luminescent sensors to test their sensitivity for bovine serum albumin (BSA).
Synthetic polypeptides have been under considerable investigation regarding their potential applications in various scientific fields and their close relationship to proteins.22–32 The secondary structures (α-helix, β-sheet and random coil) of peptide chains are cornerstones for the construction of the well-defined tertiary structure of proteins, therefore, a study on the synthetic polypeptide and its chain conformations in organic solvents illustrated one of the major efforts previously dedicated to understanding the complicated protein–protein interactions. In this respect, several traditional luminogens (such as carbazolyl,33 dansyl34 and pyrene35) had been chemically incorporated with synthetic polypeptides in order to monitor the chain conformation of polypeptides in solution. For example, no excimer emission was detected for the pyrene-labeled polypeptide35 since pyrene groups were well separated from each other by the polypeptide chains.
AIEgens were previously incorporated with synthetic polypetides36,37 with the purpose of detecting conformational changes of the peptide chain by AIE-related emission. An AIEgen of tetraphenylthiophene (TP) served as the terminal and central units of poly(benzyl-L-glutamate)s (PBLGs) in the synthetic polypeptides of TP1PBLG and TP2PBLG,36 respectively. Because the intermolecular aggregation of TP centers in TP2PBLG is sterically blocked by the large α-helical chains of TP2PBLG, a solution of TP2PBLG is lower in emission than a solution of TP1PBLG, whose TP terminal can be readily approached by other TP terminals to result in aggregates with a high emission intensity. Adding trifluoroacetic acid (TFA) to a solution of TP2PBLG converted the rigid α-helical chain into a flexible random coil; after that, the emission intensity of TP2PBLG was greatly increased due to the easy aggregation of the TP centers in the flexible coil chains. In contrast to the hydrophobic TP1PBLG and TP2PBLG, a water-soluble, TP-terminated polypeptide containing ionic sulfonate pendent groups37 was synthesized later and characterized, exhibiting a pH-induced helix-to-coil transition in an alkaline aqueous solution. Again, the emission of this ionic polypeptide in an alkaline solution was enhanced to a great extent due to the easy aggregation of the TP terminals in the coil chains.
The above examples36,37 suggested crucial roles for the random coil and α-helix structures in enhancing and weakening the AIE-related emission, respectively, of synthetic polypeptides. Despite early efforts, a correlation between the β-sheet structure and AIE activity is still in demand due to the lack of a reliable way to generate a peptide chain composed mostly of a β-sheet structure. This question can nevertheless be solved soon since we found that the fraction of β-sheet conformations can be greatly enhanced by adding NaCl salt into an aqueous solution of the amphiphilic polypeptide TP-PPLG-g-MEO2 (Scheme 1). With a hydrophobic TP terminal and hydrophilic methyl bis(ethylene oxide) (MEO2) etheric side groups, the target polypeptide TP-PPLG-g-MEO2 is amphiphilic and can be prepared by a two-step reaction procedure including a ring-opening polymerization (ROP) of the PLG-NCA monomer initiated by the amino-functionalized AIEgen TP-NH2 and the following click reaction for the introduction of the MEO2 side groups. The amphiphilic TP-PPLG-g-MEO2, composed mostly of α-helical chains, can be dispersed in salt and in alkaline aqueous solutions to generate the respective β-sheet and random-coil chains for correlating their AIE-related emission behavior with the secondary structures of the peptide chains. This correlation is meaningful considering that the aggregation tendency and corresponding AIE-related emission of the TP terminal in TP-PPLG-g-MEO2 are essentially controlled by the conformation of the peptide chain connecting to the TP terminal; that is, aggregation of TP terminals is affected by neighboring peptide chains of different chain conformations and so is the aggregation-related AIE emissive behavior. Besides theoretical correlation between the conformational change and AIE activity, all three secondary structures generated in this study were further employed as luminescent sensors for the natural protein bovine serum albumin (BSA). Corresponding luminescence responses can be used to evaluate how well the individual secondary structure complexed to BSA. The complexation tendency is strongly related to the aggregation level of TP terminals in the large peptide matrix of BSA.
Scheme 1 Syntheses of MEO2-N3, TP-NH2, and the PLG-NCA monomer for the ring-opening polymerization by TP-NH2 and the following click reaction to obtain TP-PPLG-g-MEO2. |
Sample | Molecular weight | Secondary structure | |||||
---|---|---|---|---|---|---|---|
M na | PDIa | M nb | DPb | A helixc | A sheet | A rodc | |
a Obtained from GPC. b Determined from 1H NMR (calculated from the peak ratio between resonances Hb and Hg in TP-PPLG and between Hb′ and Hg′ in TP-PPLG-g-MEO2). c Determined from deconvoluting the infrared absorption peaks in Fig. 4. | |||||||
TP-PPLG | 53000 | 1.15 | 17838 | 104.5 | 72.7 | 15.2 | 12.1 |
TP-PPLG-g-MEO2 | 156000 | 1.22 | 32999 | 105.2 | 89.4 | 4.9 | 5.7 |
Primarily, FTIR analysis was applied to confirm the success of the click reaction. Fig. 1 characterizes the absorption changes of the azido and acetylene functions in MEO2-N3 and TP-PPLG, respectively. The signals at 2130 cm−1, representative of the alkyne group of TP-PPLG, and 2105 cm−1, representative of the azido group of MEO2-N3, are absent in the spectrum of TP-PPLG-g-MEO2. A successful click reaction, which is efficient in converting azido and acetylene groups into triazole rings, should take place preferentially.
We further compared 1H NMR spectra of TP-PPLG and TP-PPLG-g-MEO2 in Fig. 2, to demonstrate the success of the click reaction. In the spectrum of TP-PPLG (Fig. 2a), the alkyne protons Ha resonated at 2.47 ppm as a singlet, overlapping with resonances of alkyl protons Hc and Hd, and solvent peaks in the range of 2.17 to 2.73 ppm. The click reaction transformed the alkyne group into triazine and therefore, the corresponding protons Hb′ of TP-PPLG-g-MEO2 resonated at 5.17 ppm (Fig. 2b). The resonance of CH2 protons Hb, neighboring the alkyne group of TP-PPLG, originally were located at 4.70 ppm but after the click reaction, the CH2 protons Hb′ now neighbored the triazole ring of TP-PPLG-g-MEO2 and therefore resonated at a lower field of 5.17 ppm. A successful click reaction was therefore verified from the 1H NMR spectra.
By calculating the intensity ratio of resonances Hb to Hg, the average molecular weight (Mn, 17840 g mol−1, Table 1) of TP-PPLG was conveniently evaluated (Table 1) but for TP-PPLG-g-MEO2, the aromatic multiplets of Hg′ cannot be detected in the spectrum (Fig. S4b†) conducted in pure CD2Cl2. Aromatic multiplets can only be resolved in the spectrum (Fig. 2b) conducted in d6-DMSO containing small amounts of DCl. In this case, acidic DCl acted to rupture intramolecular hydrogen bonds (H bonds) existing in the rigid α-helical chains, converting rigid helical chains into flexible random coils. The coil-linked TP terminals of TP-PPLG-g-MEO2 chains were no longer hampered in rotation, as the original helical chains were before adding DCl, therefore rendering detectable NMR signals due to the enhanced molecular motion in acidic solution. Therefore, the molecular weight of TP-PPLG-g-MEO2 can be calculated from the intensity ratio of Hb′ to Hg′ and the result indicated a Mn of 33000 g mol−1. The calculated degrees of polymerization (DPs) of TP-PPLG and TP-PPLG-g-MEO2 are in close proximity to 105; therefore, the conversion of the click reaction must be quantitative in order to result in similar values for the DPs.
The Mn values measured from the GPC analysis are also included in Table 1, which are much higher than the values obtained from 1H NMR. The high value from the GPC analysis is due to the early elution of the rigid polymer chains, which is especially true for the large α-helical chains. With a large diameter, α-helical chains spend little if any time in the pores of the gels packed in GPC columns; therefore, they elute quickly to result in Mn values that are much higher than the polymer standard (polystyrene) of the same Mn value. The early elution also resulted in Mn values much higher than the more realistic Mn values determined from 1H NMR.
Fig. 3 Curve-fitting of the FTIR spectra of (a) TP-PPLG and (b) TP-PPLG-g-MEO2 in the spectral range of 1500 to 1800 cm−1. |
Fig. 4 Solution emission spectra of (a) TP-PPLG-g-MEO2 in dichloromethane of different concentrations and in (b) solution mixtures of dimethyl foramide (DMF)/diethyl ether (λex = 340 nm). |
The aggregation of TP terminals in solvent/nonsolvent mixtures was also used in this study to identify the AIE effect. In this respect, diethyl ether (Et2O) was employed as a nonsolvent to induce solution aggregation of TP terminals in a good solvent of dimethylforamide (DMF). Upon irradiation at 340 nm, a dilute solution (3 × 10−6 M) of TP-PPLG-g-MEO2 in DMF already emitted with discernible intensity (Fig. 4b) and adding Et2O (while keeping the concentration of TP-PPLG-g-MEO2 at 3 × 10−6 M) resulted in progressive intensity gains in both the monomer and aggregate emissions. Upon adding Et2O, TP terminals tend to be more associated together and therefore are more hampered in rotational motion, thereby promoting the AIE-related emission.
Scheme 2 Conformational changes of TP-PPLG-g-MEO2 from an α-helix to a β-sheet in an aqueous salt solution and from an α-helix to a random coil in an alkaline aqueous solution, respectively. |
Most of the TP-PPLG-g-MEO2 chains in the aqueous solution are in the α-helical conformation and because of that they can be conveniently converted into β-sheets and random coil chains in NaCl salt and alkaline solutions, respectively. The induced helix-to-sheet and helix-to-coil transitions and the corresponding emission variations will be identified and discussed separately below.
Fig. 5 shows the emission enhancement caused by adding NaCl to an aqueous solution of TP-PPLG-MEO2. With increasing salt content in the solution, both the monomer and aggregate emissions gradually gained in intensity. The intensity ratio of aggregate and monomer emissions (Ia/Im) was then calculated and is summarized in Table 2, which indicates that Ia/Im increased from 0.677 to 0.825 as the salt content was increased from 0 to 0.154 M. A higher value of Ia/Im that correlates with a greater aggregation level of the TP terminals was therefore involved in the newly-developed secondary structure induced by NaCl. The enhanced aggregation is supposed to be responsible for the observed enhanced emission of the aqueous salt solution.
Fig. 5 Emission spectra of an aqueous solution of TP-PPLG-g-MEO2 (1.5 × 10−5 M) containing different amounts of NaCl (λex = 340 nm). |
We then recorded circular dichroism (CD) spectra (Fig. 6) to characterize the corresponding conformations responsible for the enhanced emissions of the salt solutions. The α-helical structure was characterized by a triply inflected spectrum,39 corresponding to two negative bands at 208 and 222 nm and a strong positive band at 192 nm. The β-sheet structure was characterized by a negative minimum band near 218 nm and a positive maximum near 198 nm. The random coil structure was characterized by a small positive band at 218 nm and a negative band near 200 nm. Fig. 6 shows the gradual transformation of the α-helical conformation, in correlation to the slight change of the ellipticity ratio ([θ]222/[θ]208), upon increasing NaCl content from 0 to 0.154 M. Disregarding the slight change of the ellipticity ratio, a significant spectral variation occurred in the short-wavelength region in which the main band shifted from 192 nm, characteristic of α-helical chains, to 198 nm, typical of β-sheet chains. The spectra were then fitted, using the Spectra Manager program, to resolve the percentages of each secondary structure and the results are illustrated in the inset of Fig. 6, which indicate that the content of α-helical chains decreased from 72.9% to 23.3%, corresponding to an increase in β-sheet chains from 3% to 54.1% (also, refer to the calculated results in Table S1†). Therefore, the high fraction of 3D α-helical chains were transformed into 2D β-sheet chains by NaCl salts added in the solution. The intimately-packed β-sheet chains (Scheme 2) are therefore responsible for the better emission of the salt solution but what is the driving force causing this interesting helix-to-sheet transition? The 1H NMR analysis of the salt solutions may give us a clue to evaluate the potential cause leading to the helix-to-sheet transition.
It was envisaged that NaCl additives complexed to both amide groups of the main chain and heteroatoms (nitrogen and oxygen) of the side chain, pulling both chain segments close enough to form intimately-packed β-sheet chains. Such a structural transformation was indirectly demonstrated by the 1H NMR band shape analysis of solutions of TP-PPLG-MEO2 in D2O containing different amounts of NaCl (Fig. 7). The band shape analysis was previously performed in a study of rotation-induced conformational changes.51 The main principle behind this analysis is that fast conformational exchanges caused by fast molecular rotations would result in sharp resonance peaks, whereas slower exchanges due to restricted molecular rotations broaden resonance peaks. Moreover, in many polymer systems,52,53 the rotational restriction imposed by polymer chains is so effective that the corresponding resonance peaks become weaker or disappear. In the present study, the concentration of TP-PPLG-g-MEO2 was kept at a constant value of 1.5 × 10−5 M but the resonance peaks due to protons in the main (Hb) and side (Hc–k) groups still broadened and weakened with increasing NaCl from 0 to 0.154 M. Presumably, sodium cations complexed to amide CO groups of the main chains and heteroatoms (nitrogen and oxygen) of the MEO2 side chains and hampered the rotations of the main and side chain groups to result in the broadening and weakening of the resonance peaks, due to the clumsy response of the corresponding chemical bonds towards the stimuli of the external magnetic field.
Fig. 7 1H NMR spectra of TP-PPLG-g-MEO2 (= 1.5 × 10−5 M) in D2O containing different amounts of NaCl. |
In the salt solution, intramolecular H bonds, originally prevalent in α-helical chains, were ruptured and replaced by intermolecular ionic bonds, between sodium cations and amide main chain groups, present in the β-sheet structure. Complexation of sodium cations and MEO2 side groups also functioned to hold the neighbouring MEO2 side groups at closer distances, which is beneficial for the intimately-packed β-sheet structure. When linked by the intimately-packed β-sheet chains, TP terminals associate together more easily, resulting in an emission with a higher efficiency than the helix-linked TP terminals, whose mutual approaches were seriously blocked by large helical rods.
Fig. 8 Emission spectra of aqueous TP-PPLG-g-MEO2 (= 10−5 M) solutions at different pH values (λex = 340 nm). |
The conformational transformation was also traced by CD spectra (Fig. 9). By increasing the pH from 2 to 11, there are only slight variations in the ellipicity ratio of [θ]222/[θ]208. A major conformational transformation occurred only at pH > 12 according to the spectral change where triply inflected peaks reduced to a small doubly inflected dichroic spectrum. At this stage, the majority of α-helical chains should have converted into random coils by NaOH.
Fig. 9 Circular dichroism spectrum of aqueous TP-PPLG-g-MEO2 (= 10−5 M) solutions at different pH values. |
Conformational transformation of peptide chains affected the extent of aggregation and the AIE-related emission of peptide-linked TP terminals. As illustrated in Scheme 2, with mobile segmental motion, the flexible peptide chains easily moved their TP terminals to a closer range to become a highly-aggregated TP phase, which emitted with a strong aggregate emission at an intensity much higher than TP units linked by the less-mobile α-helical or β-sheet structure.
Experimentally, α-helix, random coil and β-sheet chains prepared from the respective neutral, alkaline and salt solutions were used for complexing with different amounts of BSA. The results suggested that solution emissions of random coil (Fig. 10a) and β-sheet chains (Fig. 10b) are both decreased by the inclusion of BSA, which is distinctly different from the intensity gain observed in the emission spectra (Fig. 11) from the α-helical chain. Considering that the AIE-related emission is closely related to the aggregation level of the luminogenic TP, the emission reductions observed in alkaline and salt solutions should be correlated with the dissociation of aggregated TP terminals. Particularly, regarding the large reduction of the aggregate emission (Fig. 10a) in the alkaline solution, dissociation of TP aggregates in random coil chains must be significant in order to correlate with the large spectral change. Comparatively, that the emission loss (Fig. 10b) is minor for the salt solution indicated that there is only a slight dissociation of TP terminals during the complexation of β-sheet chains with BSA. The emission gain (Fig. 11) observed for α-helical chains is therefore correlated with the enhanced rotational restriction of TP terminals.
Fig. 11 Emission spectral variation of aqueous TP-PPLG-g-MEO2 (10−5 M) solutions in response to different amounts of BSA from 0 to 4 × 10−5 M (λex = 340 nm). |
The large emission reduction observed in alkaline solutions suggested a preferable complexation of random coil chains in BSA; most likely, the flexible coil chains readily diffused into the interior domain of the large BSA chains, to result in an exclusive reaction responsible for the large reduction of the aggregate emission shown in Fig. 10a. With low structural stability, flexible coil chains were subjected to a significant conformational change when complexed with BSA. It was then envisaged that stable chains with structural integrity are less prone to undergo conformational change during complexation with BSA. When complexed to BSA, the α-helical chains, with structural integrity maintained by intramolecular H bonds, and the β-sheet conformation, stabilized by intermolecular H bonds, are therefore suggested to undergo minor structural changes and less emission variations compared to the unstable random coil. When in the BSA domain, the stable α-helical rod remained in the rigid conformation and rotational restriction of the TP terminals was further reinforced by the viscous matrix of BSA, thereby resulting in the enhanced emission observed in Fig. 11. According to the small emission variation shown in Fig. 10b, the less stable β-sheet chain may undergo slight conformational change when complexed with BSA. Some intermolecular H bonds in the β-sheet chains may be ruptured, resulting in the dissociation of TP aggregates and the slight emission reduction.
In an aqueous solution, the majority of α-helical chains of TP-PPLG-g-MEO2 can be converted into β-sheet and random coil structures by NaCl salt and NaOH base, respectively. With an improved aggregation tendency, TP terminals linked by 2D β-sheet chains emitted with a higher emission intensity than TP terminals linked by large 3D helical rods. TP terminals linked by flexible random coils are the most mobile ones, rendering highly-aggregated TP terminals with an emission intensity higher than TP terminals linked by less-mobile helix and β-sheet chains. The role of the secondary structure of the peptide chain in determining the aggregation tendency and AIE-related emission behavior was therefore evaluated.
The complexation of BSA resulted in an emission enhancement of helix-linked TP terminals due to the reinforced rotational restriction imposed by BSA. In contrast, a large emission reduction was observed in TP terminals linked by random coil chains and most likely, a significant dissociation of the aggregated TP terminals occurred during the complexation of the coil chains with BSA. Comparatively, a structural change in β-sheet chains is small due to the slight reduction in the emission intensity upon complexing with BSA.
Footnote |
† Electronic supplementary information (ESI) available: Table S1 and Fig. S1–S9. See DOI: 10.1039/c5py01485a |
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