Conformational Study of an Artificial Metal-Dependent Regulation Site for Use in Designer Proteins

This report describes the dimerisation of glutathione, and by extension, other cysteine-containing peptides or protein fragments, with a 5, 5’-disubstituted-2, 2’-bipyridine or 6, 6”-disubstituted-2, 2’:6’,2”-terpyridine unit. The resulting bipy-GS2 and terpy-GS2 were investigated as potential metal ion dependent switches in aqueous solution, and were found to predominantly adopt the transoïd conformation at physiological pH. Metal complexation with CuII and ZnII at this pH has been studied by UV/Vis, CD, NMR and ion-mobility mass spectrometry. ZnII titrations are consistent with the formation of a 1:1 ZnII:terpy-GS2 complex at pH 7.4, but bipy-GS2 was shown to form both 1:1 and 1:2 complexes with the former being predominant under dilute micromolar conditions. Formation constants for the resulting 1:1 complexes were determined to be log KM 6.86 (bipy-GS2) and 6.22 (terpy-GS2), consistent with a higher affinity for the unconstrained bipyridine, compared to the strained terpyridine. CuII coordination involves the initial formation of 1:1 complexes, followed by 1.5Cu:1bipy-GS2 and 2Cu:1terpy-GS2 complexes at micromolar concentrations. Binding constants for formation of the 1:1 complexes (log KM 12.5 (bipy-GS2); 8.04 and 7.14 (terpy-GS2)) indicate a higher affinity for CuII than ZnII. Finally, ion-mobility MS studies detected the free ligands in their protonated form, and were consistent with the formation of two different Cu adducts with different conformations in the gas-phase. We illustrate that the bipyridine and terpyridine dimerisation units can behave like conformational switches in response to Cu/Zn complexation, and propose that in future these can be employed in synthetic biology with larger peptide or protein fragments, to control large scale folding and related biological function.


Introduction
The primary, secondary, tertiary and quaternary structure of proteins, are all crucial in controlling biomolecular recognition events and biological function. In some cases small local structural changes can greatly impact the secondary, tertiary and even quaternary structure of a whole domain, and as a result can influence the biological activity or function. Regulation processes can arise from contact with other biomolecules, and can themselves sometimes be triggered in response to an external stimuli such as pH. [1] Substructure stabilisation or destabilisation, in response to an external stimulus or binding of an effector molecule, allows the protein to communicate molecular events over long distances, through a large variety of signal-response pathways. [2] Allostery is the generic term (terpy-GS 2 ), consistent with a higher affinity for the unconstrained bipyridine, compared to the strained terpyridine. Cu II coordination involves the initial formation of 1:1 complexes, followed by 1.5Cu:1bipy-GS 2 and 2Cu:1terpy-GS 2 complexes at micromolar concentrations. Binding constants for formation of the 1:1 complexes (log K M 12.5 (bipy-GS 2 ); 8.04 and 7.14 (terpy-GS 2 )) indicate a higher affinity for Cu II than Zn II . Finally, ion-mobility MS studies detected the free ligands in their protonated form, and were consistent with the formation of two different Cu adducts with different conformations in the gas-phase. We illustrate that the bipyridine and terpyridine dimerisation units can behave like conformational switches in response to Cu/ Zn complexation, and propose that in future these can be employed in synthetic biology with larger peptide or protein fragments, to control large scale folding and related biological function. used to describe a regulation site which is distinct from the active site in a biomolecule.
Chemists have invested significant effort into trying to model or engineer allosteric sites. [3] One attractive application is to introduce these into artificial biomolecular constructs for use in synthetic biology. [4] Metal complexation by bipyridine (bipy) and terpyridine (terpy) have previously been exploited as allosteric switches in organic solvents, [5,6] and have been inserted into a range of macromolecules including polymers [7] and DNA. [8] A number of reports describe their insertion into peptide sequences, [9][10][11] however, the majority focus on using them to achieve dimerisation on metal ion complexation. Only a small fraction takes advantage of a cisoïd-transoïd conformational transition on metal ion complexation, in an effort to achieve allosteric regulation. Kelly and co-workers prepared several short peptides with a bipy unit introduced directly into the peptide backbone. The peptide sequence and the pH were found to influence the conformational state of the bipy, as a result the free ligand was only able to adopt the transoïd conformation under alkaline conditions. At pH = 9.5 they demonstrated that Cu II coordination led to a structural reorganisation of the bipy linker to the cisoïd conformation, resulting in a secondary structure transition from random coil to β-sheet. However, the bpy linker could not be used as a switch at a physiologically relevant pH. [12] This highlights the need for a better understanding of the factors (c.a. protonation of polypyridine, substituent nature and positioning) which govern the conformational state of bipyridine and related polypyridine units, if they are to be exploited as potential artificial allosteric regulation sites.
The aim of this work was therefore to study the potential for using these polypyridine units as conformational metal ion dependent switches in designed artificial protein architectures. We therefore prepared and studied two small model compounds comprising a polypyridine switchable unit substituted at either end with a short peptide (rather than a complex protein fragment). It was proposed that metal ion coordination would constitute the triggering event, resulting in a conformational change from transoïd to cisoïd and subsequent realignment of the two peptide moieties. Ultimately our goal is to extend this work to the alignment of larger peptides and small protein fragments, however, the preparation of model compounds containing short peptides (3 amino acids) will allow the conformational change of the polypyridyl linker to be studied in greater detail.

Design and Synthesis
Various coupling techniques have been reported in the literature, however, in order for this approach to be widely applicable and for these polypyridyl units to be incorporated into structures based on natural protein motifs, we felt it was necessary to utilise native functionality for coupling. We therefore chose to take advantage of selective coupling to the sulfhydryl group of cysteine, the most nucleophilic naturally occurring amino acid. The model compounds reported here consist of polypyridyl linkers coupled through the Cys side chain of the naturally occurring tripeptide, glutathione (GSH).
Two different polypyridine units, differing in substitution pattern, were investigated. The conformation of the first, a 5,5'-disubstituted bipyridine (bipy) model, was proposed to be largely unaffected by the presence of metal ions, as upon transoïd to cisoïd rearrangement, the substituent at position 5 of the 2,2'-bipyridine experiences a 180°rotation around the 2,2'-inter ring linkage, relative to the substituent at position 5'. As these two substituents are located along the rotation axis the distance separating them remains unchanged (see Figure 1A). In our design, one needs to further account for the length and the flexibility of the dimethyl sulfide bridge linking the polypyridyl unit and the peptide backbone. The dimethyl sulfide bridge can freely rotate (see Figure 1C), and is therefore able to effectively compensate for the rotation of the 2pyridinyl. For these reasons 5,5'-disubstituted-2,2-bipyridine units can be considered allosteric ineffective. [13] The second model compound contains a 6,6''-disubstituted-2,2':6',2''-terpyridine (terpy) as the linker unit. In contrast to the bipy linker, a transoïd to cisoïd rearrangement of the terpy linker would alter the relative distance between the two peptide substituents. Methyl-methyl distances for cisoïd and transoïd conformations of both model compounds, were estimated based on reported structural information for analogous compounds (see Figure 1). [6,[14][15][16][17] 5,5Ј-Dibromomethyl-2,2Ј-bipyridine and 6,6ЈЈ-dibromomethyl-2,2Ј:6Ј,2ЈЈ-terpyridine were synthesized based on Figure 1. Schematic illustrating the impact of conformational transition in A) bipy-GS 2 and B) terpy-GS 2 on the inter-substituent distance and relative orientation. C) Illustration of the flexibility of the thioether linkage of (2-polypyridine) substituted at position 5-. Distances displayed are estimated based on reported structures for analogous compounds. [6,[14][15][16][17] previously reported procedures, [18] and were characterised by HR-MS, 1 H and 13 C NMR spectroscopy. The designed small model bipy-GS 2 and terpy-GS 2 allosteric regulation sites were synthesised by reacting each dibromomethyl linker with two equivalents of glutathione (GSH), in 50/50 mixture of acetonitrile and 100 mM aqueous Tris.HCl buffer pH 8.0. This method was adapted from previous reports. [9] The model compounds, bipy-GS 2 and terpy-GS 2 , were purified by reversed phase C18-HPLC and fully characterised by ESI-MS, 1 H, 13 C NMR, UV/Vis spectroscopy and analytical HPLC (see Figure  S1). The metal ion coordination chemistry and any associated conformational reorganisation of the model regulation sites, was studied by 1 H NMR (Zn II only), circular dichroism (CD), UV/Vis spectroscopy, and ion-mobility spectrometry (IMS) mass spectrometry (MS) over the millimolar to micromolar concentration range.

Circular Dichroism (CD)
The CD spectra of 350 μM solutions of bipy-GS 2 and terpy-GS 2 , recorded from 400 to 200 nm, did not display any notable signal. However, the addition of increasing concentrations of ZnCl 2 to the solution of bipy-GS 2 at pH 7.4 led to the appearance of new positive transitions centred at 220, 241, 310 and 320 nm, as well as a negative transition at 266 nm, with isosbestic points at 251 and 285 nm (Figure 2A). In contrast, addition of ZnCl 2 to terpy-GS 2 resulted in negative transitions at 228, 329, and 340 nm and positive transitions at 283 and 290 nm, with isosbestic points at 270 and 298 nm ( Figure 2B). A plot of molar ellipticity as a function of ZnCl 2 concentration reaches a plateau at ca. 0.9 (bipy-GS 2 ) and 1.0 equivalents (terpy-GS 2 ) of Zn II per model switch ( Figure 2).
Similarly, the addition of CuCl 2 to bipy-GS 2   clear isosbestic points at 265 and 292 nm. Further addition of CuCl 2 resulted in a gradual shift of the positive transitions toward 251 and 310 nm, and the negative transition toward 282 nm. All signals decreased in intensity, reaching a minimum on addition of 1.5 equivalents of CuCl 2 . No further spectral changes occur on addition of up to 3.0 equivalents CuCl 2 (see Figure 3A) The titration of increasing concentrations of CuCl 2 into a 350 μM solution of terpy-GS 2 at pH 7.4 resulted in the appearance of a negative transition centred at 215 nm. Two overlapping negative transitions at 335 and 347 nm appear on addition of more than 1.0 equivalent of CuCl 2 . A plot of molar ellipticity as a function of CuCl 2 concentration reaches a plateau for all transitions at ca. 2.0 equivalents of Cu II per terpy-GS 2 (see Figure 3B).
In all four cases, addition of excess EDTA (20 equiv. with respect to metal) resulted in CD spectra which were in good agreement with those of bipy-GS 2 and terpy-GS 2 recorded in the absence of metal ions. and terpy-GS 2 in aqueous solution is sensitive to the pH. The UV/Vis spectra of a 5 μM solution of bipy-GS 2 recorded between pH 6 and 10 display two transitions with λ max at 295 (ε 295 nm 19,600 m -1 cm -1 ) and 245 nm (ε 245 nm 15,200 m -1 cm -1 ), assigned as πǞπ* 1 and πǞπ* 2 transitions. [20] The analogous terpy-GS 2 spectra display a peak with λ max 297 nm (ε 297 nm 20,300 m -1 cm -1 ) attributed to πǞπ* 1 , however, the πǞπ* 2 transition which occurs around 221 nm, overlaps with that for the peptide bond ( Figure 4 and S2). Upon acidification by addition of concentrated HCl, these bands decrease in intensity whilst new bands appear at lower energy. A plot of absorbance as a function of pH allows for an approximation of the associated pK a values (see Figure S2).
A similar red-shift of the πǞπ* bands are observed upon Zn II and Cu II complexation, allowing an apparent binding constant to be determined [10] taking into account the competitive metal ion binding of the phosphate buffer employed in these experiments. [21] Aliquots of a stock solution of ZnCl 2 were titrated into a 5 μM solution of either bipy-GS 2 or terpy-GS 2 in 20 mM phosphate buffer pH 7.4. This resulted in the steady decrease in the absorbance at 295 (bipy-GS 2 ) and 297 nm (terpy-GS 2 ), and an increase in the absorbance at 308 and 320 nm (bipy-GS 2 ), and 330 and 340 nm (terpy-GS 2 ), respectively (see Figure 4A and B). A plot of the absorbance as a function of Zn II concentration indicates the formation of a 1:1 complex between Zn II and both the model ligands (see Figure 4E). The observation of an isosbestic point at 303 (bipy-GS 2 ) and 313  nm (terpy-GS 2 ) is consistent with the clean formation of the Zn II complex. The extinction coefficient at 320 nm for bipy-GS 2 and [Zn(bipy-GS 2 )X n ] m+ were determined to be 1,180 m -1 cm -1 and 23,000 m -1 cm -1 , respectively. The extinction coefficient at 340 nm for terpy-GS 2 and [Zn(terpy-GS 2 )X n ] m+ were estimated to be 800 m -1 cm -1 and 19,850 m -1 cm -1 , respectively. Formation constants, log K M , were calculated to be 6.86 Ϯ 0.04 for [Zn(bipy-GS 2 )X n ] m+ and 6.22 Ϯ 0.03 for [Zn(terpy-GS 2 )X n ] m+ , see Table 1.
The addition of CuCl 2 to solutions of bipy-GS 2 and terpy-GS 2 buffered at pH 7.4 was also accompanied by transitions between 400-900 nm. Aliquots of a stock solution of CuCl 2 titrated into a more concentrated 350 μM solution of bipy-GS 2 , resulted in an increase in the absorbance at 622 nm up to 1.5 equivalents of Cu II . Further addition of CuCl 2 led to only a small increase in the absorbance at 622 nm and an increase at 440 nm, consistent with addition of CuCl 2 to the blank buffered solution (see Figure 5A). An analogous titration of CuCl 2 into a 100 μM solution of terpy-GS 2 buffered at pH 7.4, resulted in an increase in the absorbance at 675 nm up to 2.0 equivalents of Cu II , see Figure 5B, which blue-shifted shifted slightly to 670 nm on addition of between 1.0 and 1.5 equivalents of Cu II . No further changes were observed upon addition of between 2.0 and 3.0 equivalents of CuCl 2 .

H NMR Spectroscopy
The 1 H NMR spectrum of a 5 mM solution of bipy-GS 2 in D 2 O at pD 1, displays two singlets at δ = 8.83 and 8.42 ppm in the aromatic region, integrating to two and four protons, respectively. In contrast, one singlet at δ = 8.59 ppm and two overlapping doublets at 8.04 and 8.00 (AB pattern), each integrating to 2 protons, are observed on raising the pD to 7.4 (see Figure S3). A titration of ZnCl 2 into a 5 mM solution of bipy-GS 2 Figure 6A). A plot of peak integration of the overlapping doublets (8.04 and 8.00 ppm) as a function of equivalence of ZnCl 2 indicates a 1:2 Zn:bipy-GS 2 ratio. Similarly a plot of peak integration for the resulting doublet for the Zn-bipy-GS 2 adduct at δ = 8.20 ppm, also plateaus at 0.5 equivalence ZnCl 2 consistent with a 1:2 Zn:bipy-GS 2 ratio (see Figure 6C). After 0.5 equivalents of ZnCl 2 have been added the peaks at δ = 8.59, 8.04 and 8.00 ppm appear to have been replaced with broad new peaks at δ = 8.42 and 8.20 ppm. Upon addition of between 0.5 and 1.0 equiv. ZnCl 2 these peaks sharpen into doublets and a broad peak attributed to H 6 appears at higher frequency , which were assigned using COSY and NOESY NMR (see Figures S4 and S5). The COSY spectrum displays cross-peaks between H 3 Ͻ-Ͼ H 4 and H 4a Ͻ-Ͼ H 5a (n.b. H 3a and H 4a are too close, so the cross-coupling overlaps with the diagonal peaks), see Figure S5A. In contrast, NOESY NMR recorded under similar conditions, displays an additional H 3a Ͻ-Ͼ H 3 inter-ring coupling (see Figure S5B).
On raising the pD to 7.4, three aromatic resonances are observed at 8.08 (1 H 4 , 2H 3 , 2H 3a ), 7.96 (2H 4a ) and 7.55 ppm (2H 5a ). A titration of ZnCl 2 into a 5 mM solution of terpy-GS 2 in D 2 O buffered at pD 7.4, resulted in the decrease in intensity of the peaks at δ = 8.08, 7.96 and 7.55 ppm, and the appearance of new broad peaks at δ = 8.31, 8.13 and 7.65 ppm. This is accompanied by a decrease in the intensity of the singlet at δ = 3.99 ppm assigned to the CH 2 -pyridinyl group. A plot of the peak integration for the singlet at δ = 3.99 ppm, as a function of ZnCl 2 concentration ( Figure 6B and D), is consistent with formation of a 1:1 complex between Zn II and terpy-GS 2 .
In both cases, addition of excess EDTA (20 equiv. with respect to ZnCl 2 ) resulted in 1 H NMR spectra which are in good agreement with those of bipy-GS 2 and terpy-GS 2 recorded in the absence of ZnCl 2 (see Figure 6A and 6B).

Ion Mobility Spectrometry (IMS) Mass Spectrometry (MS)
Ion mobility spectrometry (IMS) coupled to electrospray ionisation (ESI) mass spectrometry (MS), has been used to examine the model switches, bipy-GS 2 and terpy-GS 2 , in the absence and presence of CuCl 2 and ZnSO 4 . A single species is detected for the terpy-GS 2   However, the intensities of these two species is extremely different than for terpy-GS 2 , almost exclusively forming the [bipy-GS 2 -H+Cu] + species (5:95) (see Figure S6) + ). In the case of terpy-GS 2 this is the only species detected, however, two additional species are detected for bipy-GS 2 in the presence of ZnSO 4 (see Figure S6). One is consistent with a m/z of 851 and a DT of 6.02 ms. The second with m/z 855 (DT 4.09 ms), and with an isotopic distribution consistent with a +2 charge ion. However, these species were not subjected to further characterisation.

Zn II Coordination of Model Switches
Titration of Zn II into a 5 mM solution of terpy-GS 2 monitored by 1 H NMR, is consistent with a 1:1 binding ratio (see Figure 6B). Similar CD and UV/Vis titrations performed at micromolar concentrations, were also consistent with the formation of a 1:1 [Zn(terpy-GS 2 )X n ] m+ complex (where X can be an exogenous ligand such as a water molecule, hydroxide or chloride).
In contrast, the titration of Zn II into a 5 mM solution of bipy-GS 2 indicates the formation of a 2bipy-GS 2 :1Zn II complex, possibly followed by formation of a 1bipy-GS 2 :1Zn II complex. This is in stark contrast to the analogous titrations (CD, UV) recorded under more dilute (14 times and 1000 times respectively) and biologically relevant conditions, suggesting that the formation of a 2:1 complex with bipy-GS 2 only occurs at high concentrations, and that the 1:1 [Zn(bipy-GS 2 )X n ] m+ species dominates under more dilute conditions.
The relatively featureless CD spectra of the model switches, are altered dramatically upon coordination of Zn II . Titrations of bipy-GS 2 and terpy-GS 2 , display chiral induced CD signals relative to the bipy and terpy πǞπ* bands (250-400 nm range). The cotton effect induced upon Zn II addition is opposite for terpy-GS 2 and bipy-GS 2 (see Figure 2). This indicates that the lower energy transition for [Zn(terpy-GS 2 )X n ] m+ might arise from πǞπ* electronic absorptions involving molecular orbitals composed mainly from atomic orbitals from atoms composing the central pyridine ring, as previously suggested. [19] In contrast, the second lower energy transition (centred at 287 nm) for [Zn(terpy-GS 2 )X n ] m+ could involve orbitals comprising contributions mainly from the external pyridines, and is therefore more similar to the lower energetic absorption band for [Zn(bipy-GS 2 )X n ] m+ (see Figure 2). These observations are consistent with formation of a 1:1 complex with the Zn II coordinated to the intended polypyridine chelate of the model switches. Glutathione units might also contribute to the coordination sphere, but this currently remains unclear. [22]

Cu II Coordination of Model Switches
The shift in the bipy-GS 2 and terpy-GS 2 π Ǟ π* band (200-400 nm range) in the UV/Vis spectra upon addition of either Cu II or Zn II , is consistent with a ca. 1:1 ratio in all cases. However, CD spectra suggest that the complexation of Cu II is more complicated, and involves the formation of two different Cu II complexes with differing contributions to the metal-bound π Ǟ π* bands. This is not obvious in the UV/Vis spectra in this range, but is observed for Cu II complexation to both bipy-GS 2 and terpy-GS 2 by CD, due to exciton effects (see Figure 3). [23][24][25] The CD titration of Cu II into bipy-GS 2 , results in chiral induced signals relative to the bipy πǞπ* band (250-400 nm range) up to one equivalent consistent with the formation of a 1:1 complex involving coordination through the pyridine units. However, the CD titration indicates that this is followed by the formation of a 1.5Cu:1bipy-GS 2 complex, as a result of a reduction in these induced CD signals. In contrast, the analogous CD titration with terpy-GS 2 did not result in the formation of induced CD signals relative to the terpy πǞπ* band up to one equivalent of Cu II . However, it is consistent with the initial formation of a 1:1 complex, followed by a 2Cu:1terpy-GS 2 complex (see Figure 3B).
We hypothesise that on formation of the initial 1:1 complexes, the Cu II coordinates to the bipyridine or terpyridine ligand, and either exogenous water (or hydroxide) molecules or groups from the glutathione units (for example the N-or Ctermini, amino acid side chains or amide bonds [27] ). However, the formation of the 1.5Cu:1 bipy-GS 2 complex could involve the formation of a new intermolecular Cu II coordination site between two Cu(bipy-GS 2 ) complexes. Whereas on formation of the 2Cu:1terpy-GS 2 complex, the second Cu II could be coordinated exclusively by the glutathione units.

Binding Constants
The shift of the πǞπ* band was used to estimate the Cu II and Zn II binding constants to the model switches. Titrations were performed at low micromolar concentrations, and where necessary a competitor was introduced. Plots of the lower energy absorbance maxima for the resulting complexes πǞπ* transition [28] vs. metal ion concentration, were fit to a 1:1 model, as this shift corresponds to formation of the 1:1 polypyridine:metal complex (see Figure 4E). The binding constants reported in Table 1 are in good agreement with those reported previously for related ligands using similar methods. [10,11,[29][30][31][32] Our model compounds, bipy-GS 2 and terpy-GS 2 , display higher affinity for Cu II than for Zn II , consistent with previous reports; [29] and bipy-GS 2 displays a higher affinity for both Cu II and Zn II than terpy-GS 2 . The latter observation is consistent with lowering of the binding constant for terpy-GS 2 resulting from the strain introduced by substitutions at position 6-and 6''-, as previously reported for polypyridine amino-acid conjugates. [10] In the case of terpy-GS 2 , fitting the data for the two πǞπ* transitions as a function of Cu II concentration lead to different affinities, related by a factor of 10 (see Table 1). We postulate that these are due to the different Cu II coordination environments, where the two species contribute differently to the absorbance at 335 and 347 nm (fitted to obtain the formation constants).

UV/Vis Spectroscopy
Nakamato first studied the pH dependence of 2,2-bipyridine and 2,2':6',2''-terpyridine in water, and demonstrated that at low pH, free bipy and terpy display similar absorption profiles to those of the metal complexes, consistent with a cisoïd-conformation. However, the πǞπ* bands shift to higher energy on increasing the pH, and resembles those recorded in organic solvents, consistent with the transoïd-conformation. [19,29] It was hypothesised that deprotonation of the pyridinyl ring on increasing the pH, resulted in a conformational transition of the bipy and terpy from cisoïd-to transoïd-, and has been supported more recently by theoretical studies. [33][34][35] The equilibria are characterised by clear isosbestic points, and pK a values of 4.44 (bipy), 2.59 and 4.16 (terpy) were reported. [20] Similar pH titrations performed on our model compounds, bipy-GS 2 and terpy-GS 2 , are consistent with the reported protonation constants ( Figure S2) and the presence of a predominantly transoïd-conformation at physiological pH. These results importantly illustrate that introduction of short peptides into the design, does not alter the pH dependent behaviour of these polypyridine switching units as monitored in solution under dilute micromolar conditions by UV/Vis spectroscopy. Therefore, unlike Kelly and co-workers [12] our model switches adopt the transoïd conformation at neutral and physiologically relevant pH. This could be due to coupling through an amino acid side chain, rather than introduction into the peptide backbone. Notably the addition of Cu II and Zn II to our bipy-GS 2 and terpy-GS 2 model compounds at pH 7.4, lead to a shift of πǞπ* bands toward higher energy, consistent with a transoïdto cisoïd-conformational transition. Importantly these results indicate that metal ions can be used to control our model switches under biologically relevant conditions (under dilute conditions in aqueous solution and at a physiologically relevant pH).

H NMR Spectroscopy
The 1 H NMR spectrum of bipy-GS 2 recorded under acidic conditions is very different from that recorded at neutral pD. A single resonance, attributed to H 3 and H 4 of bipy-GS 2 , is observed at pD 1, however, an AB pattern where the two overlapping doublets are located at a lower chemical shift, is observed at pD 7.4. Addition of ZnCl 2 to a 5 mM solution of bipy-GS 2 at pD 7.4 results in new broad peaks which indicate a species in slow/intermediary exchange on the NMR time-scale. A plot of peak integration as a function of Zn II equivalence is consistent with the formation of a 2bipy-GS 2 :1Zn complex. This spectrum at 0.5 equivalents Zn II does not display any signal assigned to H 6 , most likely due to signal broadening as a result of the clash between the two bipyridine in the binary complex. However a broad resonance assigned to H 6 reappears on addition of more ZnCl 2 (between 0.5 and 1 equivalent) and sharpens in the presence of excess Zn II (5 and 10 equivalents), which may be consistent with conversion of the 2bipy-GS 2 :1Zn complex into a 1bipy-GS 2 :1Zn complex. [36,37] Theoretical studies suggest that even though monoprotonated bipyridine and bidentate metal complexes of bipyridine have energy minima with similar conformations (cisoïd), flexibility around the axial bond of bipyridine is much higher in the monoprotonated bipyridine compared to the metal complexes. [38] In fact, the difference in potential energy separating cisoïd and transoïd conformations has been reported to be comparable for the monoprotonated bipyridine and the free bipyridine. [33,34,38] This could account for the similarity of resonances assigned as H 3 and H 4 in spectra of bipy-GS 2 recorded www.zaac.wiley-vch.de at acidic and neutral pD, which in turn differ from those recorded for the Zn II complex (see Figure S3). NMR spectra of bipyridine or derivatives where H 3 and H 4 resonances overlap have previously been recorded in aqueous solution at both acidic [36,39] and physiological [36,40] pH, however, this is not exclusively the case. [39] Interpretation of bipyridine conformation based on NMR chemical shift can therefore lead to contradictory results. For example, theoretical studies suggest that H 3 are deshielded in the transoïd (cation free) bipyridine due to the close proximity with the nitrogen on the second ring. [13] Cation binding to bipyridine is expected to result in deshielding of aromatic resonances. In contrast, H 3 can display only a moderate deshielding, [39] or shielding [41] upon transoïd to cisoïd conformational transition, as deshielding effects on H 3 from the proximal nitrogen are lost. [36,42] Therefore an attractive method by which to assign the bipy-GS 2 conformation is by monitoring intra-ring coupling by NOESY NMR, [43] however, this is only possible for an asymmetric bipyridine for which H 3 peaks are inequivalent, and so cannot be applied to bipy-GS 2 .
In contrast, NOESY can be applied to terpy-GS 2 . At pD 1, the cross-peak observed between H 3 and H 3a in the NOESY spectrum is consistent with at least half of the terpyridine adopting a cisoïd conformation (see Figure S5B). The 1 H NMR spectrum of terpy-GS 2 recorded at pD 7.4 is different from that recorded at acidic pD, and is not suitable for determination of intra-ring coupling as the resonances for H 3 and H 3a overlap. Upon raising the pD from 1 to 7.4, all aromatic signals move to lower frequency, and proton signals relative to external pyridine (H 3a , H 4a and H 5a ) experience higher shielding than their counterpart (H 3 and H 4 ), consistent with a transition from mixed (cis-trans) conformation to a transoïd (transtrans) conformation. [44] Addition of ZnCl 2 lead to formation of broad resonances for a 1terpy-GS 2 :1Zn complex in slow/ intermediary exchange on the NMR timescale. Signals are generally broad and difficult to assign, but integration of the three signals indicates that the spectrum is different from that recorded for terpy-GS 2 both at acidic and neutral pD, and could be consistent with the cisoïd conformation of a terpyridine metal complex. [29,31]

Ion Mobility Spectrometry (IMS) Mass Spectrometry (MS)
The transition between cisoïd-and transoïd-polypyridine conformers is not the only conformational transition involved in our peptide conjugates of bipy or terpy. The thioether linkage and the peptide backbone will contribute to the overall global structure of the molecule and is likely to dominate when considering the molecules collisional cross-section (CCS). In an attempt to monitor the overall conformation of the model switches in the gas-phase, we have employed ion mobility spectrometry (IMS) couple to ESI-MS to study bipy-GS 2 and terpy-GS 2, in the absence and presence of Cu II and Zn II . In the absence of any metal ions these measurements found that both bipy-GS 2 and terpy-GS 2 were detected in the protonated form in the gas-phase (samples prepared at pH 6.7), which is associated with the cisoïd (bpy) and a mixed cis-trans confor-mation (terpy) in solution. Therefore, it is possible that any changes observed in the CCS can be attributed to changes in the orientation of the glutathione units, rather than a transoïdcisoïd conformational transition.
As expected bipy-GS 2 traverses the mobility T-Wave with a short drift time (DT) and is consistent with a smaller CCS when compared with the terpy-GS 2 model. Coordination of Zn II to bipy-GS 2 or terpy-GS 2 leads to the formation of a species consistent with [M -H+Zn] + with a longer drift time but similar CCS to the model switch in the absence of Zn II . The ion-mobility spectrum recorded for bipy-GS 2 in the presence of Zn II indicates the formation of at least three different species. We have not been able to assign the remaining two, but they may involve partial decomposition of the complex and cluster formation in the gas-phase.
The addition of Cu II resulted in the detection of two different species by IMS. One of these Cu adducts could be consistent with Cu I replacing the proton and has a near identical drift time and CCS to the model switch in the absence of metal ions. The second Cu adduct has been assigned as [M -H+Cu] + (consistent with Cu in the +2 oxidation state), is detected after a longer drift time and has a slightly larger CCS than the model switch in the absence of metal ions. Comparison of the CCS measurements suggests that there is potentially some structural difference, which we propose is due to complexation of the bipyridine/terpyridine and subsequent repositioning and potential coordination of the glutathione units. Formation of Cu II complexes in the gas-phase involving a mixture of bipyridine and peptide ligands, have previously been described. [45] Both the Cu adducts are formed in nearly equal amounts on coordinating to the terpy-GS 2 model switch (ca. 60:40), however, the second Cu adduct, [M -H+Cu] + , is almost exclusively formed on coordination to bipy-GS 2 (ca. 5:95). This would be consistent with formation of polypyridine-peptide complexes with different stability constants in the gas-phase, [45] similar to the formation of a more stable bipy-GS 2 complex with Cu II in aqueous solution. The different CCS for the various species formed (Cu/Zn) could be due to the different preferred metal ion coordination geometries. Similar metal ion conformational dependence in the gas-phase was described previously in an IMS study of the Gramicidin peptide. [46]

Conclusions
In conclusion, two model switches for the metal dependent spatial alignment of protein fragments in synthetic biology, have been prepared and studied in aqueous solution by UV/Vis, CD, NMR and IMS-MS. The model switches, bipy-GS 2 and terpy-GS 2 , contain a polypyridine linker (either based on 5,5'-disubstituted-2,2'-bipyridine or 6,6''-disubstituted-2,2':6',2''-terpyridine) coupled through the native Cys side chain of the tripeptide glutathione. This approach can therefore be extended to the coupling of larger natural protein fragments. Introduction of the peptide substituents was shown to have little effect on the polypyridine pH dependence, adopting the predominantly transoid conformation in aqueous solution at neutral pH.

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Variations to the polypyridine design resulted in different Cu II and Zn II coordination properties. Zn II coordination involves formation of a 1:1 complex with both bipy-GS 2 and terpy-GS 2 under biologically relevant conditions (micromolar concentrations and neutral pH). The coordination of Cu II was more complicated and appears to initially involve the formation of a 1:1 complex followed by a 1.5Cu:1bipy-GS 2 and 2Cu:1terpy-GS 2 complex. Cu II binding constants were found to be higher than those obtained for Zn II , and the sterically less strained bipy-GS 2 forms more stable complexes with both metal ions than terpy-GS 2 . Metal ion complexation was further investigated by IMS-MS, and changes to CCS upon metal ion coordination were found to be similar for both model switches.
Though polypyridine metal ion complexation has been extensively studied in the literature in organic solvents, this has not received the same kind of attention in water. This work therefore represents an important contribution towards their use in biologically relevant systems. Work is now on-going to investigate the use of these conformational switches with larger peptide fragments attached to the polypyridyl ligand, to evaluate if the metal ion dependent spatial rearrangement can be exploited to align these fragments in order to achieve enhanced biomolecular recognition.
HRES and ES-TOF MS were recorded with a Micromass LCT TOF spectrometer equipped with a 3000 V capillary voltage, and a cone voltage of 35 V. GC-MS were recorded with a Waters GCT Premier Micromass equipped with an Electronic Impact (EI) probe. 5,5'-Dimethyl-2,2'-bipyridine (0.389 g, 2.11 mmol), N-bromosuccinimide (0.756 g, 4.23 mmol) and azobisisobutyronitrile (10 mg, 0.06 mmol) were dissolved in dichloromethane (20 mL), refluxed using a 500 W halogen lamp, and the reaction progress monitored by TLC (SiO 2 , eluent: CH 2 Cl 2 /CH 3 OH (9/1)). After 4 hours, more Nbromosuccinimide (0.375 g, 2.10 mmol) was added and the reaction was refluxed for a further 2 hours. The mixture was allowed to cool to room temperature and the solution extracted with 0.1 m aqueous NaHCO 3 (5 ϫ 20 mL). The organic layer was dried with Na 2 SO 4 , filtered, and concentrated in vacuo. The solid was re-dissolved in 15 mL of a 50/50 mix CHCl 3 /CH 3

Synthesis of 2-Bromo-6-methylpyridine
2-Bromopicoline (10.8 g, 115 mmol) in hydrobromic acid (40 mL, 48 %) was cooled to -20°C in an ethanol bath. Bromine (14.4 mL, 280 mmol) was added dropwise, and the suspension was stirred for 90 min at -20°C. An aqueous solution of NaNO 2 (30 mL, 8.9 m, 268 mmol) was added dropwise, and the solution was allowed to warm to room temperature over 2 hours with stirring. The mixture was recooled to -20°C, and a cool NaOH aqueous solution (110 mL, 16.5 M, 1.81 mol) added slowly, while maintaining the temperature below -10°C. The mixture was allowed to warm to room temperature over the course of an more hour with continuous stirring. The mixture was then extracted with ethyl acetate and the organic layer dried with Na 2 SO 4 , filtered, and concentrated in vacuo. The dark oil was then purified by Kugelrohr distillation to yield a colourless oil (9.

Synthesis of 2-Tributylstannyl-6-methylpyridine
A solution of 2-bromo-6-methylpyridine (4.63 g, 27 mmol) in dry THF (20 mL) was cooled to -60°C, 27 mL of n-butyllithium in hexane (1.1 m, 30.1 mmol) added dropwise, and the solution was stirred for 2 hours at 0°C. Tributyltin chloride (8.9 mL, 32.8 mmol) was slowly added and the solution allowed to return to room temperature over 20 minutes with continuous stirring. Water (30 mL) was added into the reaction mixture, and phases were separated. The organic phase was washed with water (3 ϫ 30 mL), and the combined aqueous layers washed with ethyl ether (4 ϫ 30 mL). The combined organic phases were dried with Na 2 SO 4 , filtered, and concentrated in vacuo to afford a black oil (9.534 g, 92 %). The crude product was determined to be ca. 95 % pure by GC analysis and so used directly in the following synthesis

UV/Vis Spectroscopy
UV/Vis spectra were recorded in a 1 cm pathlength quartz cuvette at 298 K with a Shimadzu 1800 spectrometer. For the pH titration of model switches, aliquots of HCl, NaOH (terpy-GS 2 ) or KOH (bipy-GS 2 ) solutions of various concentrations (0.01, 0.1, 1 m) were added to cuvettes containing 3 mL of a 5 μM solution of the model switch. The solution was allowed to equilibrate for 10 min prior to recording the pH on a Jenway 3510 pH meter, and recording a UV/Vis spectrum.
For the metal titrations, aliquots of an aqueous 0.75 mM stock solution of either CuCl 2 or ZnCl 2 , were titrated into 3 mL of a 5 μM solution of model switches in 20 mM potassium phosphate buffer pH 7.4, and the spectra recorded after 3 min equilibration. Non-linear fitting were performed with Kaleidagraph software version 4.0. K app values were calculated by fitting data for the absorbance maximum of the metal complexes as a function of Cu II /Zn II concentration, to Equation (1) and (2) The constant b corresponds to the cuvette pathlength, [complex] corresponds to the concentration of 1:1 complex, [Ligand] the total bipy-GS 2 or terpy-GS 2 and [M] the total CuCl 2 /ZnCl 2 concentration at each point.
In order to ensure an accurate estimation, measurements were performed at concentrations close to the apparent dissociation constants, such that: The K M values were corrected by accounting for the contribution from phosphate metal ion binding, based on values reported in the literature, [21] see Equation (3): This is based on the assumption that the amount of free phosphate is equal to the total amount of phosphate in solution. K M and K PM corresponds to the estimated binding constant of the metal ion to the ligand of interest and the phosphate anion, respectively. When glycine was added as a competitor, K M was estimated using Equation (4), based on the reported Cu II binding constant for glycine. [47] The contribution from the phosphate anion was regarded as negligible, when 20 mM glycine was present. (4)

NMR Spectroscopy
All 1 H and 13 C NMR spectra were collected with either Bruker DRX500 (500 MHz 1 H and 125 MHz 13 C, T = 300 K), AVIII400