DOI: 10.1002/chem.201501243
Communication
& Self-Assembly
The Two-Step Assemblies of Basic-Amino-Acid-Rich Peptide with a Highly Charged Polyoxometalate Teng Zhang, Hong-Wei Li, Yuqing Wu,* Yizhan Wang, and Lixin Wu*[a] Abstract: Two-step assembly of a peptide from HPV16 L1 with a highly charged europium-substituted polyoxometalate (POM) cluster, accompanying a great luminescence enhancement of the inorganic polyanions, is reported. The mechanism is discussed in detail by analyzing the thermodynamic parameters from isothermal titration calorimetry (ITC), time-resolved fluorescent and NMR spectra. By comparing the actions of the peptide analogues, a binding process and model are proposed accordingly. The driving forces in each binding step are clarified, and the initial POM aggregation, basic-sequence and hydrophobic C termini of peptide are revealed to contribute essentially to the two-step assembly. The present study demonstrates both a meaningful preparation for bioinorganic materials and a strategy using POMs to modulate the assembly of peptides and even proteins, which could be extended to other proteins and/or viruses by using peptides and POMs with similar properties.
Polyoxometalates (POMs), as a typical class of metal–oxygen anionic clusters,[1] have been found to possess activity in many biological systems such as antibacterial, antivirus, anticancer, and so forth.[2] Recently, POMs have been recognized to possess the capacity to induce cell apoptosis[3] and inhibit the aggregation of amyloid peptides.[4] Thus, clarification for the interaction of POMs with biomolecules is critical for understanding their biofunctions. The known investigations have demonstrated that the binding is mainly dependent on favorable electrostatic attraction and cluster size effects. However, there is little information for specific interactions in virus-related protein systems because in many cases general proteins, for example, human serum albumin (HSA) and bovine serum albumin (BSA), have been employed. In addition, few studies have dealt with the selective recognition of highly charged POMs to define binding site or peptidic segments which can affect the assembled structures relate to the function of proteins.[5] Therefore, elucidating the association mechanisms of POMs with representative peptides would be beneficial for the deter[a] T. Zhang, Dr. H.-W. Li, Prof. Dr. Y. Wu, Dr. Y. Wang, Prof. Dr. L. Wu State Key Laboratory of Supramolecular Structure and Materials Jilin University, No. 2699, Qianjin Street, Changchun, 130012 (China) E-mail: [email protected] [email protected] Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201501243. Chem. Eur. J. 2015, 21, 9028 – 9033
mination of binding sites to proteins and for controlling their assemblies. As a family of non-enveloped viruses human papillomavirus (HPVs) can easily infect epithelial cells in the skin and mucosa. Both epidemiologic and biochemical studies indicate that infection with certain high-risk types is directly associated with anogenital malignancies.[6] The interactions of several arginineand lysine-rich peptides of HPV-16 and HPV-18 capsid proteins, called nuclear localization signals (NLS), with the negatively charged cell surface receptor heparan/heparin have been reported to be involved in the infection of virus particles in host cells.[7] To analyze the binding-generated change of protein structure and to find a possible POM inhibitor, we herein investigated the highly charged POM-binding NLS peptides toward revealing the selective recognition to HPVs capsid protein for possible antiviral mechanism clarification in future work. Different from simple POMs with fewer charges used in previous studies,[8] a europium decatungstate (K13[Eu(SiW10MoO39)2]·28 H2O, EuSiWMo) that has a sandwich structure is used, accompanied by its analogue, Na9[EuW10O36]·32 H2O (EuW10) for comparison. Typical NLS peptides from high-risk PV types, HPV-16 and -18 capsid proteins (Table 1) were chosen. The binding site and interaction model between EuSiWMo and NLS peptides were used to evaluate the biological activity of the inorganic cluster. Interestingly, the synergistic assembly of the two components that induce a great enhancement of luminescence of the POM and a twostep assembly of the peptide is observed, being different from published results. The present study also opens an alternative method for the use of new fluorescent bioinorganic materials with increased biocompatibility. EuSiWMo (30 mm) in buffer solution shows luminescent peaks that are assigned to the transitions of 5D0 !7F1 (591 nm) and 5D0 !7F2 (614 nm) of Eu3 + .[9] Upon successive addition of HPV16Ctb, the luminescent intensity undergoes a dramatic enhancement (ca. 57-time; Figure 1 a). For a dilute POM solution (10 mm), a much higher enhancement (68-times) is achieved (Figure S1 in the Supporting Information). In contrast, similar but much lower expansion (8-times) was found for the binding of this POM with basic amino acids (AAs) electrostatically.[10] Therefore, enhancement can be well attributed to the stronger electrostatic combination, which expels water molecules from the secondary coordination sphere of Eu3 + , and leads to an increase in luminescence. A similar binding can be expected for the peptide and the enhanced luminescence can be explained as the synergistic performance of AAs in the peptides.
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Communication tion geometry around Eu3 + for both peptides, but different Peptide Amino acid (AA) sequence Description binding kinetics after this ratio. Apparently, the two peptides HPV16Ctb SSTSTTAKRKKRKL 492–505 of HPV16 L1 follow different routes when HPV16Ctb-F SSTSTTAKRKKRKL-F F added at C terminus of HPV16Ctb HPV16Ctb-21 SSTSTTG-SSTSTTAKRKKRKL SSTSTTG added at N terminus of HPV16Ctb the concentration is increased HPV16Ctb-Scr SKSRTKSRTKTAKL sequence scrambled HPV16Ctb above than the turning point. HPV18Ctb SSKPAKRVRVRARK 555–568 of HPV18 L1 The time-resolved fluorescence of EuSiWMo was performed before and after binding with HPV16Ctb to explain the intensity ratio changes of luminescence. The fitting to the lifetime (Figure S2 in the Supporting Information) shows a bi-exponential decay. The short phase can be attributed to the POM bearing coordinated water molecules,[11] while the longer phase should come from the peptide-induced assembly and change of chemical environments. A previous study indicates that an Eu3 + -containing POM, EuW10, shows a similar transition from a mono-exponential decay to a bi-exponential process after binding with histone H1 or BSA.[12, 13] As illustrated in Figure 2 and Table S1 in the Supporting Information, Figure 1. The luminescent spectra of EuSiWMo (30 mm) in MES/NaOH buffer solution (10 mm, pH 6.0) upon graduupon increasing the ratio of al addition of: a) HPV16Ctb, and c) HPV18Ctb, as representative of three repeated experiments. b) The averaged intensity changes at 591 nm of HPV16Ctb; inset: photographs of EuSiWMo (left) and HPV16Ctb/EuSiWMo (3:1; peptide, there is an obvious deright) mixture in quartz cuvettes. d) The averaged intensity ratios (I591/I614) of EuSiWMo versus the concentration of crease of the shorter-lifetime peptide. component (t1) and a concomitant increase of the longer-lifeThe fluorescence titration for POM displays a concentrationtime phase (t2), suggesting the tight influence of peptide for dependence of peptide while the saturation appears at 86 mm the chemical environment of POM cluster. Upon further addi(Figure 1 b). The luminescence can be visualized under UV light tion of HPV16Ctb, the longer luminescent decay time increases irradiation (right cell in inset), being much stronger than that dramatically. The ratio of longer lifetime segment reaches its of pure EuSiWMo (left in inset). A basic-AAs-rich peptide from maximum at component ratio 0.3 of the peptide to EuSiWMo another high-risk HPV, 18L1 (HPV18Ctb, Table 1) also shows en(Figure 2 b), indicating the contribution of the state with this hancement (Figure 1 c), indicating a possible general ability for lifetime to the luminescence. The lifetime changes are perfectly basic-AA-rich peptides to promote POM luminescence. In comconsistent with that occurring in the I591/I614 plot (Figure 1 d). Both should be attributed to the reduced coupling of water to parison, under identical condition, isolated peptide solutions POM when the amount of HPV16Ctb increases. do not show any apparent luminescence (data not shown). To clarify the binding process of peptides to POMs, isotherThe microenvironment of Eu3 + during binding HPV16Ctb mal titration calorimetry (ITC) measurement was conducted. and HPV18Ctb was assayed by means of fluorescent intensity Monitoring the time-dependent luminescence intensity of Euratio at I591/I614 (Figure 1 d). Dramatic changes are clearly seen SiWMo after the addition of HPV16Ctb indicates that a quick upon addition of very little peptide (peptides/EuSiWMo < 0.3). binding maximum can be reached within 3 min (Figure S3 in For the POM alone, the ratio I591/I614 is calculated to be 0.831, the Supporting Information), confirming the usefulness of ITC. whereas for the mixed systems the values increase to 1.397 Figure 3 a shows a calorimetric heat-flow trace and the correand 1.102 at a ratio of 0.3 for HPV16Ctb and HPV18Ctb to sponding titration curve obtained by adding HPV16Ctb into POM, respectively. Over this concentration range, the ratio rea solution of EuSiWMo. Differing from previously reported mains constant for HPV18Ctb, but for HPV16Ctb it first declines combinations of HPV16Ctb with heparin[7b] and that of and then reaches a stable state after molar ratios above 2.4. The turn at 0.3 implies the similar transformation of coordinaHPV18Ctb with EuSiWMo (Figure 3 b), a definite two-step bindTable 1. The sequences and origins of HPV L1 peptides used in this study.
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Figure 2. a) The molar ratio-dependent longer lifetimes (t2) of EuSiWMo, which reached maximum at a value of 1:3 for HPV16Ctb/EuSiWMo and then decreased. b) Plot of longer-lived component ratio of t2 versus the molar ratio of HPV16Ctb/EuSiWMo.
ing model can be well fitted to the titration plot of the integrated heat for HPV16Ctb and the POMs. The detailed thermodynamic parameters calculated from the Gibbs free energy change (DG) is summarized in Table S2 in the Supporting Information. Considering the contribution of enthalpy and entropy to the processes, in step I, the former mainly drives the strong binding of HPV16Ctb and EuSiWMo, while the latter is apparently favorable for step II, as well as the assembly of HPV18Ctb and POMs.[7b, 14] To track the binding kinetics, calorimetric titrations for HPV16Ctb and HPV18Ctb were performed at 5, 15 and 25 8C (Figure S4 in the Supporting Information). The slopes of the DH values versus temperature indicate the change of constant pressure heat capacity (DCp) for the interaction of peptide with POMs. It is known that the DCp is correlated with the binding-induced burial of surface area. A negative DCp value refers to a reduction of number for nonpolar surface-exposed residues, while a positive DCp suggests a reduction in the number of polar surface-exposed residues.[15] Here, the negative DCp emerging in step I of HPV16Ctb can be described in terms of the surface polarity increase due to its electrostatic and hydrophobic interactions with POMs. In contrast, the positive DCp values in step II of HPV16Ctb and the binding of HPV18Ctb can be ascribed to the surface polarity decrease involved in electrostatic and hydrogen-bonding interactions. The binding constant for step II of HPV16Ctb, calculated from the data in Table S2 in the Supporting Information, is at Chem. Eur. J. 2015, 21, 9028 – 9033
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Figure 3. Binding isotherms for the interaction of EuSiWMo with: a) HPV16Ctb, and b) HPV18Ctb. The calorimetric traces (top panels) are recorded by titrating EuSiWMo (20 mm) in a cell with HPV16Ctb or HPV18Ctb (450 mm, in syringe) at 25 8C. The bottom panels plot the integrated heat (squares) and the fitted lines (solid) of the bindings.
a magnitude of approximately 108 m¢1, being much (at least two orders) higher than many of the reported values.[11a, 16] Meanwhile, the binding constant for step I of this peptide is in the order of about 109 m¢1, being even tenfold higher than step II and that of HPV18Ctb. A reverse titration of EuSiWMo to the HPV16Ctb solution was conducted and a similar inflexion to that of step II was found (Figure S5 in the Supporting Information). At component ratio of 0.30 for EuSiWMo to HPV16Ctb, a binding state bearing an almost identical binding constant is found at the reciprocal ratio of HPV16Ctb to EuSiWMo (2.75) in the normal titration. The consistent transition in the two titration processes reveals that step II is in its thermodynamic equilibrium state. 1 H NMR spectra were used to determine the binding sites (Figure 4) and the chemical shifts of amide NH protons of
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Communication HPV16Ctb in the absence and presence of POM clusters were assigned based on the published results.[17] According to the reported explanations, the sensitivity of six cationic residues in the peptide to the addition of POMs should be the source of the electrostatic interactions.[18] The weakening-to-vanishing of peaks is attributed to NH (8.20–8.42 ppm) and the broadening of peaks deriving from guanidinium of arginine (7.15– 7.23 ppm) point to the binding sites in the peptide. Notably, a large change (0.031 ppm) of chemical shift at 8.05 ppm from leucine (Leu14), the hydrophobic residue at the C terminus, is observed. This result implies the hydrophobic interaction occurring between Leu14 with POMs upon the interaction of peptide, as supported by the negative DCp for step I of HPV16Ctb (Figure S4 in the Supporting Information). Beside electrostatic interactions, the hydrogen bonding inferred from ITC results can be supported by the chemical-shift change of amide protons of Ser4 at 8.39 ppm, which should come from the interaction between polar NH group and oxygen atoms of POM clusters. In addition, there was no obvious change for Thr6 at 8.12 ppm, Ala7 at 8.24 ppm, Thr3 and -5 at 8.30– 8.32 ppm,[17] illustrating either nonbinding between these sites and POMs or that the binding is too weak to be detected by NMR spectroscopy under the present conditions.
each assembly, indicating a hybrid composition. The morphology of pure HPV16Ctb solution shows that the peptide self-assembles into spheres with an average size of approximately 300 nm (Figure S8 in the Supporting Information). Hence, a complicated process may occur when mixing the two components together. The z-potentials present more detailed binding information for the synergistic self-assembly when using HPV16Ctb titrating EuSiWMo solution (Figure S9 in the Supporting Information). As expected, the interaction of the positively charged peptides with negatively charged EuSiWMo causes obvious changes of surface charge. The z-potential values of EuSiWMo increase gradually with the addition of peptide from negative, as that of POM cluster, to neutral and then to the positive threshold, similar to the peptide. These results further elucidate the electronic neutralization of the polyanionic cluster and the domination of the peptide on the surface of the final assemblies.
Figure 5. High resolution TEM images of EuSiWMo (30 mm) in the: a) absence, and presence of different molar ratios of HPV16Ctb to EuSiWMo at: b) 0.25:1 (insets: scaled-up views); c) 1:1; d) 1:1 scaled-up views.
Figure 4. 1H NMR spectra of 1.2 mm HPV16Ctb at molar ratios of HPV16Ctb/ EuSiWMo: a) 1:0, b) 1:0.01, c) 1: 0.02, d) 1: 0.05, and e) 1: 0.1 in solution (90 %H2O/10 %D2O). The spectral intensity at 7.10–7.25 ppm is multiplied by 2.
The TEM results demonstrate pure EuSiWMo (30 mm) existing in a small aggregate (~ 4 nm) comprised of 3–5 clusters (Figure 5 a). After the addition of HPV16Ctb with a molar ratio to EuSiWMo lower than 0.25:1, strip-like aggregates are found (Figure 5 b) in bearing with the high resolution lattice structure of POMs. When their ratio is increased to 1:1, spherical assemblies are observed (Figure 5 c and d). Further increasing the ratio to 3:1 (Figure S6 in the Supporting Information), causes the assemblies to associate while maintaining the spherical morphology. Energy dispersive X-ray (EDX) spectroscopy attached to TEM and SEM (Figure S7 in the Supporting Information) confirmed the presence of europium, silicon, tungsten, molybdenum, oxygen, nitrogen and carbon elements within Chem. Eur. J. 2015, 21, 9028 – 9033
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Based on the above results, a binding model and assembly procedure during the titration are proposed in Scheme 1. At the initial stage, the EuSiWMo in small aggregates associate with a small amount of HPV16Ctb through electrostatic and hydrophobic interaction, which breaks the peptide assembly; this leads to the formation of a small constitution unit for further self-assembly. The increase of peptide ratio results in the formation of strip-like co-assembly in step I; this assists the phase separation driven by both electrostatic interactions between KRKKRK and POMs, and the hydrophobic force of the segment from the C terminus of the peptide. Higher peptide ratios direct the full assembly for EuSiWMo–peptide as step II, forming bigger spherical assemblies that are comprised of isolated peptide-enwrapped POMs. Finally, the dispersed spherical assemblies grow and link together. It can be inferred that the assembled structure transition induces the large luminescence and I591/I614 variation of Eu3 + due to the altered microenvironment.
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Scheme 1. A schematic binding model and process between EuSiWMo and HPV16Ctb at different molar ratios.
We further used a POM cluster of EuW10 with lower surface charge to identify the influence of POM aggregation. The decay curves of EuW10 at a concentration of 50 and 120 mm illustrate that there is indeed a concentration-induced photophysical change and the longer lifetime contributes to the concentration increase (Figure S10 and Table S1 in the Supporting Information). We also compare ITC results for the interactions between HPV16Ctb and EuW10 at the two concentrations (Figure S11 in the Supporting Information). A single-step binding model matches the low-concentration system while a two-step binding fits well to the high concentration of this POM cluster. The thermodynamic data (Table S2 in the Supporting Information) confirm the crucial role of the initial aggregation of POM in the two-step assembly. To analyze the role of the intrinsic nature of the sequence of AAs in binding with EuSiWMo, several peptide analogues (Table 1) were designed as controls to understand the interaction between HPV16Ctb and POMs. The series of six polybasic residues (KRKKRK), the sequence length, and the substituted group at the C termini are found to strongly affect the assembly of peptide with POMs. In comparison with HPV16Ctb, both HPV18Ctb (Figure 1 d and Figure S12 in the Supporting Information) and sequence scrambled peptide HPV16Ctb-Scr (Figure S13 in the Supporting Information), possessing dispersive basic residues, exhibit obviously one-step assembly being consistent with step II when binding with EuSiWMo. This confirms the crucial action of the polybasic residue in the two-step process. When using HPV16Ctb-21, a longer SSTSTTG series connected to the N terminus of HPV16Ctb, a more complicated and stronger binding process is observed in ITC measurements (Figure S14 a in the Supporting Information). The result implies that the length increase at the terminal far from the polybasic residue also leads to varied self-assembly structures because of the additional intermolecular interactions and packing effects. In addition, the strong exothermicity observed in the step I process of HPV16Ctb to EuSiWMo suggests that, in addition to the involvement of electrostatic interactions, the hydrophobic residue Leu14 at the C terminus contributes to the binding steps. To confirm the impact of the hydrophobic group there, a synthesized peptide, HPV16Ctb-F, with an additional phenylalanine (F) at the C terminus of HPV16Ctb was used for comparison. The ITC isotherm for HPV16Ctb-F can also fit well to Chem. Eur. J. 2015, 21, 9028 – 9033
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a two-set binding model (Figure S14 b in the Supporting Information). But the obtained binding constants, being more than twice of K1 and almost identical of K2 of HPV16Ctb, respectively (Table S2 in the Supporting Information), indicate the enhanced effect of the extended hydrophobic group. In addition, a direct proof for the hydrophobic reaction is supplied by the HPV18Ctb–EuSiWMo interaction, as polar and hydrophilic C termini of HPV18Ctb result in a lower binding constant (Table S2 in the Supporting Information), and especially a one-step assembly with EuSiWMo (Figure S12 in the Supporting Information). Importantly, these results indicated that the step I process deals with hydrophobic interactions deriving from the C termini while there is no observable assistance for the step II process. In summary, a two-step assembly has been observed when a basic-AA-rich sequence peptide of HPV capsid protein L1 is introduced into a solution of Eu-containing POMs. The initial electrostatic interaction of POMs is shown to dissociate the assembly of peptide and then collect them to form strip-like assemblies under the assistance of hydrophobic effects between peptides. Increasing the ratio of peptide induces the transformation of the POMs and peptide self-assemblies into spherical structures with the additional help of hydrogen-bonding interactions. As a consequence, the present study proves that the basic sequence and the length, as well as other residues in the peptide all sense the interaction from POMs and self-adapt to form a synergistic assembly. The charge and concentration as well as the aggregation of POMs contribute greatly to the interaction with the peptide and the self-adaption-induced selfassembly. It should be noted, the highly charged POMs might bring new interaction forces when binding with peptides or proteins, which have not been taken into consideration in previous investigations. Therefore, the present study demonstrates both a meaningful preparation for bioinorganic materials and a strategy using POMs to modulate the assembly of peptides and even proteins, which could be extended to other proteins and/or viruses by using the peptides and POMs with similar properties. This strategy could be useful in the future treatment of virus.
Acknowledgements We greatly appreciate the financial support from the projects of NSFC (No. 91027027, 91227110, 21373101, 21003061 and 21221063), National 973 Program (2013CB834503) and the Innovation Program of the State Key Laboratory of Supramolecular Structure and Materials, Jilin University. Keywords: binding mechanisms · luminescence enhancement · peptides · polyoxometalates · self-assembly
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Received: March 30, 2015 Published online on May 26, 2015
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& Self-Assembly
The Two-Step Assemblies of Basic-Amino-Acid-Rich Peptide with a Highly Charged Polyoxometalate Teng Zhang, Hong-Wei Li, Yuqing Wu,* Yizhan Wang, and Lixin Wu*[a] Abstract: Two-step assembly of a peptide from HPV16 L1 with a highly charged europium-substituted polyoxometalate (POM) cluster, accompanying a great luminescence enhancement of the inorganic polyanions, is reported. The mechanism is discussed in detail by analyzing the thermodynamic parameters from isothermal titration calorimetry (ITC), time-resolved fluorescent and NMR spectra. By comparing the actions of the peptide analogues, a binding process and model are proposed accordingly. The driving forces in each binding step are clarified, and the initial POM aggregation, basic-sequence and hydrophobic C termini of peptide are revealed to contribute essentially to the two-step assembly. The present study demonstrates both a meaningful preparation for bioinorganic materials and a strategy using POMs to modulate the assembly of peptides and even proteins, which could be extended to other proteins and/or viruses by using peptides and POMs with similar properties.
Polyoxometalates (POMs), as a typical class of metal–oxygen anionic clusters,[1] have been found to possess activity in many biological systems such as antibacterial, antivirus, anticancer, and so forth.[2] Recently, POMs have been recognized to possess the capacity to induce cell apoptosis[3] and inhibit the aggregation of amyloid peptides.[4] Thus, clarification for the interaction of POMs with biomolecules is critical for understanding their biofunctions. The known investigations have demonstrated that the binding is mainly dependent on favorable electrostatic attraction and cluster size effects. However, there is little information for specific interactions in virus-related protein systems because in many cases general proteins, for example, human serum albumin (HSA) and bovine serum albumin (BSA), have been employed. In addition, few studies have dealt with the selective recognition of highly charged POMs to define binding site or peptidic segments which can affect the assembled structures relate to the function of proteins.[5] Therefore, elucidating the association mechanisms of POMs with representative peptides would be beneficial for the deter[a] T. Zhang, Dr. H.-W. Li, Prof. Dr. Y. Wu, Dr. Y. Wang, Prof. Dr. L. Wu State Key Laboratory of Supramolecular Structure and Materials Jilin University, No. 2699, Qianjin Street, Changchun, 130012 (China) E-mail: [email protected] [email protected] Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201501243. Chem. Eur. J. 2015, 21, 9028 – 9033
mination of binding sites to proteins and for controlling their assemblies. As a family of non-enveloped viruses human papillomavirus (HPVs) can easily infect epithelial cells in the skin and mucosa. Both epidemiologic and biochemical studies indicate that infection with certain high-risk types is directly associated with anogenital malignancies.[6] The interactions of several arginineand lysine-rich peptides of HPV-16 and HPV-18 capsid proteins, called nuclear localization signals (NLS), with the negatively charged cell surface receptor heparan/heparin have been reported to be involved in the infection of virus particles in host cells.[7] To analyze the binding-generated change of protein structure and to find a possible POM inhibitor, we herein investigated the highly charged POM-binding NLS peptides toward revealing the selective recognition to HPVs capsid protein for possible antiviral mechanism clarification in future work. Different from simple POMs with fewer charges used in previous studies,[8] a europium decatungstate (K13[Eu(SiW10MoO39)2]·28 H2O, EuSiWMo) that has a sandwich structure is used, accompanied by its analogue, Na9[EuW10O36]·32 H2O (EuW10) for comparison. Typical NLS peptides from high-risk PV types, HPV-16 and -18 capsid proteins (Table 1) were chosen. The binding site and interaction model between EuSiWMo and NLS peptides were used to evaluate the biological activity of the inorganic cluster. Interestingly, the synergistic assembly of the two components that induce a great enhancement of luminescence of the POM and a twostep assembly of the peptide is observed, being different from published results. The present study also opens an alternative method for the use of new fluorescent bioinorganic materials with increased biocompatibility. EuSiWMo (30 mm) in buffer solution shows luminescent peaks that are assigned to the transitions of 5D0 !7F1 (591 nm) and 5D0 !7F2 (614 nm) of Eu3 + .[9] Upon successive addition of HPV16Ctb, the luminescent intensity undergoes a dramatic enhancement (ca. 57-time; Figure 1 a). For a dilute POM solution (10 mm), a much higher enhancement (68-times) is achieved (Figure S1 in the Supporting Information). In contrast, similar but much lower expansion (8-times) was found for the binding of this POM with basic amino acids (AAs) electrostatically.[10] Therefore, enhancement can be well attributed to the stronger electrostatic combination, which expels water molecules from the secondary coordination sphere of Eu3 + , and leads to an increase in luminescence. A similar binding can be expected for the peptide and the enhanced luminescence can be explained as the synergistic performance of AAs in the peptides.
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Communication tion geometry around Eu3 + for both peptides, but different Peptide Amino acid (AA) sequence Description binding kinetics after this ratio. Apparently, the two peptides HPV16Ctb SSTSTTAKRKKRKL 492–505 of HPV16 L1 follow different routes when HPV16Ctb-F SSTSTTAKRKKRKL-F F added at C terminus of HPV16Ctb HPV16Ctb-21 SSTSTTG-SSTSTTAKRKKRKL SSTSTTG added at N terminus of HPV16Ctb the concentration is increased HPV16Ctb-Scr SKSRTKSRTKTAKL sequence scrambled HPV16Ctb above than the turning point. HPV18Ctb SSKPAKRVRVRARK 555–568 of HPV18 L1 The time-resolved fluorescence of EuSiWMo was performed before and after binding with HPV16Ctb to explain the intensity ratio changes of luminescence. The fitting to the lifetime (Figure S2 in the Supporting Information) shows a bi-exponential decay. The short phase can be attributed to the POM bearing coordinated water molecules,[11] while the longer phase should come from the peptide-induced assembly and change of chemical environments. A previous study indicates that an Eu3 + -containing POM, EuW10, shows a similar transition from a mono-exponential decay to a bi-exponential process after binding with histone H1 or BSA.[12, 13] As illustrated in Figure 2 and Table S1 in the Supporting Information, Figure 1. The luminescent spectra of EuSiWMo (30 mm) in MES/NaOH buffer solution (10 mm, pH 6.0) upon graduupon increasing the ratio of al addition of: a) HPV16Ctb, and c) HPV18Ctb, as representative of three repeated experiments. b) The averaged intensity changes at 591 nm of HPV16Ctb; inset: photographs of EuSiWMo (left) and HPV16Ctb/EuSiWMo (3:1; peptide, there is an obvious deright) mixture in quartz cuvettes. d) The averaged intensity ratios (I591/I614) of EuSiWMo versus the concentration of crease of the shorter-lifetime peptide. component (t1) and a concomitant increase of the longer-lifeThe fluorescence titration for POM displays a concentrationtime phase (t2), suggesting the tight influence of peptide for dependence of peptide while the saturation appears at 86 mm the chemical environment of POM cluster. Upon further addi(Figure 1 b). The luminescence can be visualized under UV light tion of HPV16Ctb, the longer luminescent decay time increases irradiation (right cell in inset), being much stronger than that dramatically. The ratio of longer lifetime segment reaches its of pure EuSiWMo (left in inset). A basic-AAs-rich peptide from maximum at component ratio 0.3 of the peptide to EuSiWMo another high-risk HPV, 18L1 (HPV18Ctb, Table 1) also shows en(Figure 2 b), indicating the contribution of the state with this hancement (Figure 1 c), indicating a possible general ability for lifetime to the luminescence. The lifetime changes are perfectly basic-AA-rich peptides to promote POM luminescence. In comconsistent with that occurring in the I591/I614 plot (Figure 1 d). Both should be attributed to the reduced coupling of water to parison, under identical condition, isolated peptide solutions POM when the amount of HPV16Ctb increases. do not show any apparent luminescence (data not shown). To clarify the binding process of peptides to POMs, isotherThe microenvironment of Eu3 + during binding HPV16Ctb mal titration calorimetry (ITC) measurement was conducted. and HPV18Ctb was assayed by means of fluorescent intensity Monitoring the time-dependent luminescence intensity of Euratio at I591/I614 (Figure 1 d). Dramatic changes are clearly seen SiWMo after the addition of HPV16Ctb indicates that a quick upon addition of very little peptide (peptides/EuSiWMo < 0.3). binding maximum can be reached within 3 min (Figure S3 in For the POM alone, the ratio I591/I614 is calculated to be 0.831, the Supporting Information), confirming the usefulness of ITC. whereas for the mixed systems the values increase to 1.397 Figure 3 a shows a calorimetric heat-flow trace and the correand 1.102 at a ratio of 0.3 for HPV16Ctb and HPV18Ctb to sponding titration curve obtained by adding HPV16Ctb into POM, respectively. Over this concentration range, the ratio rea solution of EuSiWMo. Differing from previously reported mains constant for HPV18Ctb, but for HPV16Ctb it first declines combinations of HPV16Ctb with heparin[7b] and that of and then reaches a stable state after molar ratios above 2.4. The turn at 0.3 implies the similar transformation of coordinaHPV18Ctb with EuSiWMo (Figure 3 b), a definite two-step bindTable 1. The sequences and origins of HPV L1 peptides used in this study.
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Figure 2. a) The molar ratio-dependent longer lifetimes (t2) of EuSiWMo, which reached maximum at a value of 1:3 for HPV16Ctb/EuSiWMo and then decreased. b) Plot of longer-lived component ratio of t2 versus the molar ratio of HPV16Ctb/EuSiWMo.
ing model can be well fitted to the titration plot of the integrated heat for HPV16Ctb and the POMs. The detailed thermodynamic parameters calculated from the Gibbs free energy change (DG) is summarized in Table S2 in the Supporting Information. Considering the contribution of enthalpy and entropy to the processes, in step I, the former mainly drives the strong binding of HPV16Ctb and EuSiWMo, while the latter is apparently favorable for step II, as well as the assembly of HPV18Ctb and POMs.[7b, 14] To track the binding kinetics, calorimetric titrations for HPV16Ctb and HPV18Ctb were performed at 5, 15 and 25 8C (Figure S4 in the Supporting Information). The slopes of the DH values versus temperature indicate the change of constant pressure heat capacity (DCp) for the interaction of peptide with POMs. It is known that the DCp is correlated with the binding-induced burial of surface area. A negative DCp value refers to a reduction of number for nonpolar surface-exposed residues, while a positive DCp suggests a reduction in the number of polar surface-exposed residues.[15] Here, the negative DCp emerging in step I of HPV16Ctb can be described in terms of the surface polarity increase due to its electrostatic and hydrophobic interactions with POMs. In contrast, the positive DCp values in step II of HPV16Ctb and the binding of HPV18Ctb can be ascribed to the surface polarity decrease involved in electrostatic and hydrogen-bonding interactions. The binding constant for step II of HPV16Ctb, calculated from the data in Table S2 in the Supporting Information, is at Chem. Eur. J. 2015, 21, 9028 – 9033
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Figure 3. Binding isotherms for the interaction of EuSiWMo with: a) HPV16Ctb, and b) HPV18Ctb. The calorimetric traces (top panels) are recorded by titrating EuSiWMo (20 mm) in a cell with HPV16Ctb or HPV18Ctb (450 mm, in syringe) at 25 8C. The bottom panels plot the integrated heat (squares) and the fitted lines (solid) of the bindings.
a magnitude of approximately 108 m¢1, being much (at least two orders) higher than many of the reported values.[11a, 16] Meanwhile, the binding constant for step I of this peptide is in the order of about 109 m¢1, being even tenfold higher than step II and that of HPV18Ctb. A reverse titration of EuSiWMo to the HPV16Ctb solution was conducted and a similar inflexion to that of step II was found (Figure S5 in the Supporting Information). At component ratio of 0.30 for EuSiWMo to HPV16Ctb, a binding state bearing an almost identical binding constant is found at the reciprocal ratio of HPV16Ctb to EuSiWMo (2.75) in the normal titration. The consistent transition in the two titration processes reveals that step II is in its thermodynamic equilibrium state. 1 H NMR spectra were used to determine the binding sites (Figure 4) and the chemical shifts of amide NH protons of
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Communication HPV16Ctb in the absence and presence of POM clusters were assigned based on the published results.[17] According to the reported explanations, the sensitivity of six cationic residues in the peptide to the addition of POMs should be the source of the electrostatic interactions.[18] The weakening-to-vanishing of peaks is attributed to NH (8.20–8.42 ppm) and the broadening of peaks deriving from guanidinium of arginine (7.15– 7.23 ppm) point to the binding sites in the peptide. Notably, a large change (0.031 ppm) of chemical shift at 8.05 ppm from leucine (Leu14), the hydrophobic residue at the C terminus, is observed. This result implies the hydrophobic interaction occurring between Leu14 with POMs upon the interaction of peptide, as supported by the negative DCp for step I of HPV16Ctb (Figure S4 in the Supporting Information). Beside electrostatic interactions, the hydrogen bonding inferred from ITC results can be supported by the chemical-shift change of amide protons of Ser4 at 8.39 ppm, which should come from the interaction between polar NH group and oxygen atoms of POM clusters. In addition, there was no obvious change for Thr6 at 8.12 ppm, Ala7 at 8.24 ppm, Thr3 and -5 at 8.30– 8.32 ppm,[17] illustrating either nonbinding between these sites and POMs or that the binding is too weak to be detected by NMR spectroscopy under the present conditions.
each assembly, indicating a hybrid composition. The morphology of pure HPV16Ctb solution shows that the peptide self-assembles into spheres with an average size of approximately 300 nm (Figure S8 in the Supporting Information). Hence, a complicated process may occur when mixing the two components together. The z-potentials present more detailed binding information for the synergistic self-assembly when using HPV16Ctb titrating EuSiWMo solution (Figure S9 in the Supporting Information). As expected, the interaction of the positively charged peptides with negatively charged EuSiWMo causes obvious changes of surface charge. The z-potential values of EuSiWMo increase gradually with the addition of peptide from negative, as that of POM cluster, to neutral and then to the positive threshold, similar to the peptide. These results further elucidate the electronic neutralization of the polyanionic cluster and the domination of the peptide on the surface of the final assemblies.
Figure 5. High resolution TEM images of EuSiWMo (30 mm) in the: a) absence, and presence of different molar ratios of HPV16Ctb to EuSiWMo at: b) 0.25:1 (insets: scaled-up views); c) 1:1; d) 1:1 scaled-up views.
Figure 4. 1H NMR spectra of 1.2 mm HPV16Ctb at molar ratios of HPV16Ctb/ EuSiWMo: a) 1:0, b) 1:0.01, c) 1: 0.02, d) 1: 0.05, and e) 1: 0.1 in solution (90 %H2O/10 %D2O). The spectral intensity at 7.10–7.25 ppm is multiplied by 2.
The TEM results demonstrate pure EuSiWMo (30 mm) existing in a small aggregate (~ 4 nm) comprised of 3–5 clusters (Figure 5 a). After the addition of HPV16Ctb with a molar ratio to EuSiWMo lower than 0.25:1, strip-like aggregates are found (Figure 5 b) in bearing with the high resolution lattice structure of POMs. When their ratio is increased to 1:1, spherical assemblies are observed (Figure 5 c and d). Further increasing the ratio to 3:1 (Figure S6 in the Supporting Information), causes the assemblies to associate while maintaining the spherical morphology. Energy dispersive X-ray (EDX) spectroscopy attached to TEM and SEM (Figure S7 in the Supporting Information) confirmed the presence of europium, silicon, tungsten, molybdenum, oxygen, nitrogen and carbon elements within Chem. Eur. J. 2015, 21, 9028 – 9033
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Based on the above results, a binding model and assembly procedure during the titration are proposed in Scheme 1. At the initial stage, the EuSiWMo in small aggregates associate with a small amount of HPV16Ctb through electrostatic and hydrophobic interaction, which breaks the peptide assembly; this leads to the formation of a small constitution unit for further self-assembly. The increase of peptide ratio results in the formation of strip-like co-assembly in step I; this assists the phase separation driven by both electrostatic interactions between KRKKRK and POMs, and the hydrophobic force of the segment from the C terminus of the peptide. Higher peptide ratios direct the full assembly for EuSiWMo–peptide as step II, forming bigger spherical assemblies that are comprised of isolated peptide-enwrapped POMs. Finally, the dispersed spherical assemblies grow and link together. It can be inferred that the assembled structure transition induces the large luminescence and I591/I614 variation of Eu3 + due to the altered microenvironment.
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Scheme 1. A schematic binding model and process between EuSiWMo and HPV16Ctb at different molar ratios.
We further used a POM cluster of EuW10 with lower surface charge to identify the influence of POM aggregation. The decay curves of EuW10 at a concentration of 50 and 120 mm illustrate that there is indeed a concentration-induced photophysical change and the longer lifetime contributes to the concentration increase (Figure S10 and Table S1 in the Supporting Information). We also compare ITC results for the interactions between HPV16Ctb and EuW10 at the two concentrations (Figure S11 in the Supporting Information). A single-step binding model matches the low-concentration system while a two-step binding fits well to the high concentration of this POM cluster. The thermodynamic data (Table S2 in the Supporting Information) confirm the crucial role of the initial aggregation of POM in the two-step assembly. To analyze the role of the intrinsic nature of the sequence of AAs in binding with EuSiWMo, several peptide analogues (Table 1) were designed as controls to understand the interaction between HPV16Ctb and POMs. The series of six polybasic residues (KRKKRK), the sequence length, and the substituted group at the C termini are found to strongly affect the assembly of peptide with POMs. In comparison with HPV16Ctb, both HPV18Ctb (Figure 1 d and Figure S12 in the Supporting Information) and sequence scrambled peptide HPV16Ctb-Scr (Figure S13 in the Supporting Information), possessing dispersive basic residues, exhibit obviously one-step assembly being consistent with step II when binding with EuSiWMo. This confirms the crucial action of the polybasic residue in the two-step process. When using HPV16Ctb-21, a longer SSTSTTG series connected to the N terminus of HPV16Ctb, a more complicated and stronger binding process is observed in ITC measurements (Figure S14 a in the Supporting Information). The result implies that the length increase at the terminal far from the polybasic residue also leads to varied self-assembly structures because of the additional intermolecular interactions and packing effects. In addition, the strong exothermicity observed in the step I process of HPV16Ctb to EuSiWMo suggests that, in addition to the involvement of electrostatic interactions, the hydrophobic residue Leu14 at the C terminus contributes to the binding steps. To confirm the impact of the hydrophobic group there, a synthesized peptide, HPV16Ctb-F, with an additional phenylalanine (F) at the C terminus of HPV16Ctb was used for comparison. The ITC isotherm for HPV16Ctb-F can also fit well to Chem. Eur. J. 2015, 21, 9028 – 9033
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a two-set binding model (Figure S14 b in the Supporting Information). But the obtained binding constants, being more than twice of K1 and almost identical of K2 of HPV16Ctb, respectively (Table S2 in the Supporting Information), indicate the enhanced effect of the extended hydrophobic group. In addition, a direct proof for the hydrophobic reaction is supplied by the HPV18Ctb–EuSiWMo interaction, as polar and hydrophilic C termini of HPV18Ctb result in a lower binding constant (Table S2 in the Supporting Information), and especially a one-step assembly with EuSiWMo (Figure S12 in the Supporting Information). Importantly, these results indicated that the step I process deals with hydrophobic interactions deriving from the C termini while there is no observable assistance for the step II process. In summary, a two-step assembly has been observed when a basic-AA-rich sequence peptide of HPV capsid protein L1 is introduced into a solution of Eu-containing POMs. The initial electrostatic interaction of POMs is shown to dissociate the assembly of peptide and then collect them to form strip-like assemblies under the assistance of hydrophobic effects between peptides. Increasing the ratio of peptide induces the transformation of the POMs and peptide self-assemblies into spherical structures with the additional help of hydrogen-bonding interactions. As a consequence, the present study proves that the basic sequence and the length, as well as other residues in the peptide all sense the interaction from POMs and self-adapt to form a synergistic assembly. The charge and concentration as well as the aggregation of POMs contribute greatly to the interaction with the peptide and the self-adaption-induced selfassembly. It should be noted, the highly charged POMs might bring new interaction forces when binding with peptides or proteins, which have not been taken into consideration in previous investigations. Therefore, the present study demonstrates both a meaningful preparation for bioinorganic materials and a strategy using POMs to modulate the assembly of peptides and even proteins, which could be extended to other proteins and/or viruses by using the peptides and POMs with similar properties. This strategy could be useful in the future treatment of virus.
Acknowledgements We greatly appreciate the financial support from the projects of NSFC (No. 91027027, 91227110, 21373101, 21003061 and 21221063), National 973 Program (2013CB834503) and the Innovation Program of the State Key Laboratory of Supramolecular Structure and Materials, Jilin University. Keywords: binding mechanisms · luminescence enhancement · peptides · polyoxometalates · self-assembly
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Received: March 30, 2015 Published online on May 26, 2015
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