DOI: 10.1002/chem.201405581
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& Protein Folding | Hot Paper |
Induced Folding of Protein-Sized Foldameric b-Sandwich Models with Core b-Amino Acid Residues Gbor Olajos,[a] Anasztzia Hetnyi,[b] Edit Wber,[a] Lukcs J. Nmeth,[a] Zsolt Szakonyi,[c] Ferenc Flçp,[c] and Tams A. Martinek*[a]
Abstract: The mimicry of protein-sized b-sheet structures with unnatural peptidic sequences (foldamers) is a considerable challenge. In this work, the de novo designed betabellin14 b-sheet has been used as a template, and a!b residue mutations were carried out in the hydrophobic core (positions 12 and 19). b-Residues with diverse structural properties were utilized: Homologous b3-amino acids, (1R,2S)-2aminocyclopentanecarboxylic acid (ACPC), (1R,2S)-2-aminocyclohexanecarboxylic acid (ACHC), (1R,2S)-2-aminocyclohex3-enecarboxylic acid (ACEC), and (1S,2S,3R,5S)-2-amino-6,6dimethylbicyclo[3.1.1]heptane-3-carboxylic acid (ABHC). Six a/b-peptidic chains were constructed in both monomeric
and disulfide-linked dimeric forms. Structural studies based on circular dichroism spectroscopy, the analysis of NMR chemical shifts, and molecular dynamics simulations revealed that dimerization induced b-sheet formation in the 64-residue foldameric systems. Core replacement with (1R,2S)-ACHC was found to be unique among the b-amino acid building blocks studied because it was simultaneously able to maintain the interstrand hydrogen-bonding network and to fit sterically into the hydrophobic interior of the bsandwich. The novel b-sandwich model containing 25 % unnatural building blocks afforded protein-like thermal denaturation behavior.
Introduction
cyclic a/b-peptides as hairpin models.[11] Sheet-like self-organization has been observed for a/b-peptides at the air/water interface[12] and in fibrillous b-peptidic nanostructures.[13] The construction of water-soluble, sizable, and well-folded b-sheet mimetic a/b-peptides with b-amino acids in arbitrary positions, however, is a difficult task. Horne and co-workers pointed out that in-registry a!b3 replacement in the hydrophobic core of the two strands resulted in decreased structural stability because of the changed hydrogen-bond pattern and an altered side-chain display as compared with the natural b-sheet structure.[14] In alternative design strategies, in which the native side-chain orientations are maintained in a 16-residue disulfide-cyclized b-hairpin, two a-amino acid residues are substituted by a single b-amino acid (aa!b2 or b3) or special b2,3 residues are applied.[15] We have systematically probed the effects of in-registry b3-amino acid substitutions by using a water-soluble, biologically active, b-sheet-forming 33-mer peptide as the parent sequence.[16] The b3-analogues displayed decreased folding propensities, whereas a micellar environment could induce b-sheet formation in a very similar way to that of the parent a-peptide. Bioactivity was retained by this inducible b-sheet formation, and the tendency to aggregation was reduced. These findings suggested that controlled, inducible b-sheet folding may be a useful approach to the construction of bioactive a/b-peptidic b-sheet mimetics. In this work we investigated b-sheet inducibility in a b-sandwich-forming protein-sized system in which b-amino acid mutations had been carried out in the hydrophobic core. The folding was enhanced by the dimerization of the b-sheet units in this case. By using the 32-residue betabellin-14 as a templa-
There are a large number of known b-sheet-rich protein interfaces that are potential biological targets,[1] but the design of functional, water-soluble, stand-alone b-sheet structures, and especially those with protein-like structural features, is a great challenge.[2] De novo designed structures have been reported to form b-hairpins,[3] three-stranded b-sheets,[4] and b-sandwiches,[5] and protein epitope mimetic hairpins have been shown to display bioactive potencies.[6] For biological applications, however, it is desirable that the peptidic sequence contains unnatural building blocks to improve proteolytic stability.[7] Non-peptidic template and turn units have been combined with peptides to construct parallel and antiparallel sheet structures that display protein aggregation/inhibitory effects,[8] and aromatic oligoamides have been constructed to mimic bsheets.[9] Successes have also been achieved with homologated amino acids, which can be used to build short turns[10] and [a] G. Olajos, Dr. E. Wber, L. J. Nmeth, Prof. Dr. T. A. Martinek Institute of Pharmaceutical Analysis, SZTE-MTA Lendlet Foldamer Research Group University of Szeged, 6720 Szeged (Hungary) E-mail: [email protected] [b] Dr. A. Hetnyi Department of Medical Chemistry University of Szeged, 6720 Szeged (Hungary) [c] Dr. Z. Szakonyi, Prof. Dr. F. Flçp Institute of Pharmaceutical Chemistry University of Szeged, 6720 Szeged (Hungary) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201405581. Chem. Eur. J. 2015, 21, 1 – 9
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Full Paper te,[5c] we showed that the b-sheet content of the a/b-peptidic analogues can be increased by dimerization through the formation of disulfide bridges. We successfully constructed a foldameric sequence that displays protein-like behavior.
phobic core of the b-sandwich because these core substitutions may have a major effect on the folding process. These residues are in isolated positions so as to allow the bresidues to accommodate the local environment (Figure 1b). We previously found that b3-residues with large hydrophobic side-chains tend to attain a gauche backbone conformation in a b-sheet environment.[16] To test the effects of conformational constraints, we synthesized analogues by using cyclic b-amino acids. In accordance with the rules of the stereochemical patterning approach,[17] a cis backbone arrangement (with R and S configurations at the a- and b-carbon atoms, respectively) was applied to optimize the interstrand hydrogen-bond network. It has previously been demonstrated that a steric clash between bulky side-chains can exert a major influence on the secondary structure.[18] The steric fit within the hydrophobic core of the b-sandwich is also an important factor, and cyclic b-residues with various steric requirements were therefore selected for the study (Figure 1): (1R,2S)-2-Aminocyclopentanecarboxylic acid (ACPC), (1R,2S)-2-aminocyclohexanecarboxylic acid (ACHC), (1R,2S)-2-aminocyclohex-3-enecarboxylic acid (ACEC), and (1S,2S,3R,5S)-2-amino-6,6-dimethylbicyclo[3.1.1]heptane-3carboxylic acid (ABHC).
Results and Discussion Design approach Betabellin-14 consists of a 32-residue palindromic pattern of polar (p), nonpolar (n), end (e), and turn (t, r) residues (Figure 1a). When b-turns are induced by d-Lys-d-Ala (tt) and dAla-d-Lys (rr) segments, the sequence tends to fold into an am-
Induced folding as determined by circular dichroism (CD) spectroscopy The overall propensity of the betabellin-14 analogues to attain a specific secondary structure was monitored by using CD spectroscopy. In accordance with the literature, the parent sequence 1 displays a CD spectrum characteristic of a b-sheet, even in its monomeric form (Figure 2a), and dimerization through a disulfide bridge further increases the b-sheet content. The a/b-analogues 2–4 and 6 yielded U-shaped CD curves with an intense negative band at around 199 nm and a lower-intensity band at around 220 nm. Dimerization, however, resulted in changes in the CD responses; the negative band disappeared or became less dominant. A positive Cotton effect was observed for 5, whereas 11 exhibits inversion and a decrease in band intensity, which can be explained in terms of exciton coupling to the side-chain double bond. Similar anomalous CD behavior has previously been observed for helical bpeptide foldamers containing the ACEC residue.[19] Overall, these changes suggest the formation of ordered conformational states upon dimerization. A CD spectrum is the weighted sum of the component spectra of the secondary structure elements, and the curves observed are therefore very sensitive to the conformational ensemble and visual inspection may be misleading. To extract quantitative information, a convex constraint analysis deconvolution[20] was carried out simultaneously on all the spectra recorded in this study, with the exception of the spectra recorded for 5 and 11 containing the ACEC building blocks. This approach allowed the calculation of the pure component spectra and their weights without making use of any input basis spectrum or structural hypothesis. The normalized residual mean square deviation of the back-calculated spectra from the experimental curves averaged over 1–4, 6–10, and 12 was
Figure 1. a) Amino acid sequence and palindromic design (gray) of the betabellin-14 analogues studied. Residues are coded with the standard oneletter a-amino acid notation; lower case letters indicate d-a-amino acids. B) Schematic representation of the b-sandwich fold of the betabellin-14 monomer with the positions selected for the mutations highlighted in gray. Constituent strand segments are designated by the capital letters A–D.
phiphilic b-sheet.[5c] The formation of a b-sheet can be promoted by dimerization of the betabellin-14 chains through a disulfide bridge involving the residues Cys21. The intermolecular interactions enforced by the linkage enhance the hydrophobic interactions between the sheets, and the covalent betabellin14 dimers therefore display increased order. We chose this de novo designed system because it afforded a water-soluble and inducible b-sheet fold. To study the effects of the b-amino acid substitutions, we modified residues I12 and A19 in the hydro&
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Full Paper originally for betabellin-14 were able to push the a/b-sequences towards the b-sheet fold. Residue-level structure propensities by NMR analysis Chemical shift analysis[21] was carried out to gain a deeper insight into the propensity to form b-sheets at the amino acid level. Heteronuclear NMR spectra were recorded and the resonances assigned for the monomeric and dimeric sequences in natural abundance under optimized conditions (HEPES buffer, 500 mm, 298 K; see Figures S2–S13 in the Supporting Information). All the identified 1Ha, 1HN, 13Ca, 13Cb and 15N resonances (see Tables S1 and S2) were included in the analysis. The secondary structure propensity (SSP) score[22] was calculated by using the ssp software, which gave a semiquantitative measure of the residue-level secondary structure distribution along the chains. The resonances of the b-and d-a-residues were excluded from the calculations because of the lack of reference values. Although secondary-structure-independent neighborhood effects of the b- and d-a-residues on the magnetic environment of the adjacent l-a-residues cannot be ruled out, our earlier observations indicated no major systematic influence preventing an analysis of the trends in the secondary structure.[16] The set of negative SSP scores obtained for the monomeric parent sequence 1 is a good indicator of the b-sheet tendency in line with the CD results (Figure 3a). The largest b-sheet content was found in segment B. Disulfide formation in 7 led to decreases in the SSP scores along the chain (Figure 3b). A marked increase in the b-sheet content was observed for strand C containing the tethering Cys2 residues, which propagated to strands B and D. This indicates that hydrophobic stabilization within the core of the forced b-sandwich is effective. These residue-level results also confirmed betabellin-14 as a useful inducible b-sandwich model. For 2–6, the SSP score increases around the b-residues (positions 12 and 19), as reflected in the local disorder caused by these mutations and in support of the CD findings. The dimeric a/b-peptides 8–12 maintain the destructuring pattern of the b-residues, but the disulfide bridge increases the b-sheet content in segment C up to the values observed in segment B. This enhancement effect is side-chain-dependent. To study these dimerization-induced structural changes, the differences in the SSP scores were calculated (Figure 3c). This representation eliminates the influence of the reference chemical shifts and the data depict the dimerization-induced b-sheet enhancement along the sequences for the l-a-residues. For the b-turns of the d-a-residues, secondary-structure-type changes cannot be directly assigned to the values, but the DSSP scores can be compared with those of the parent sequences. It is clear from this analysis that dimerization-induced b-sheet enhancements are present for the a/bsequences except for the ABHC-containing sequence 12. Interestingly, the largest increases are observed in the proximity of the b-residues in position 19, which indicates the inherent ability of b-residues to fit into a b-sheet environment if extra stabilization is introduced. The best results were obtained with derivative 10, which displays similar DSSP scores in segment C to
Figure 2. a) Mean residue ellipticities (MRE) obtained for monomeric betabellin-14 (1) and the a/b-peptidic analogues 2–6, indicated in gray, gray dotted, gray dashed, black, black dotted, and black dashed lines, respectively. b) MRE curves obtained for dimeric betabellin-14 (7) and the a/b-peptidic analogues 8–12, indicated in gray, gray dotted, gray dashed, black, black dotted, and black dashed lines, respectively. c) Secondary structure contents calculated by using the convex constraint analysis algorithm for the CD curve deconvolution. b-sheet, b-turn, and disordered components are indicated in black, gray, and white, respectively.
3.94 %, which indicates an acceptably low fitting error. The protocol resulted in three-component spectra (see Figure S1 in the Supporting Information) that can be assigned to b-sheet, disordered, and a b-turn conformation of d-a-amino acid residues according to literature findings.[20] The corresponding coefficients allowed the contributions of the components to be estimated and hence the percentage of the secondary structure content was determined (Figure 2c). For the parent a-peptide sequences (1 and 7), the b-sheet content changed from 43 to 67 % upon dimerization, which shows the stabilization effects of the increased hydrophobic interactions in the disulfide-stabilized b-sandwich. The analysis revealed that the a/bpeptides are mainly disordered in the monomeric form, with the b-sheet content varying in the range 4–24 %. On the other hand, dimerization increases the b-sheet content to 34–47 % for 8–10. In contrast, the data for the sequence containing the bulky ABHC residues (12) indicate that the b-sheet content decreases after the formation of the disulfide bridge, which points to a steric clash. The b-turn contents for 1 and 7 were found to be 23 and 25 %, respectively, which are in line with the nine residues forming the three-turn structures (28 %). Sequences 9 and 10 showed the values closes to these (28 and 27 %, respectively), whereas 8 and 12 displayed marked deviations (7 % and 71 %, respectively). These findings support our hypothesis that, despite the destructuring effects of the b-residue mutations, b-sheet formation can be induced if the hydrophobic interactions are partially restored. In this case the stabilization effects of the forced b-sandwich formation proposed Chem. Eur. J. 2015, 21, 1 – 9
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Full Paper less efficient than in 7. These results suggest that side-chain bulkiness and the preferred arrangement of backbone torsions in the b-residues have profound effects on the b-sheet packing and thereby on the stabilization of the fold. The residual flexibility of this sensitive system and the signal overlaps in the 2D 1 H homonuclear spectra did not permit the assignment of diagnostic NOE interactions. Thermal denaturation Both CD and NMR responses represent ensemble averages that indicate the presence of secondary structure elements, but folded protein-like behavior requires a certain amount of rigidity. Cooperative thermal denaturation is a characteristic of well-folded proteins and is possible if the residual flexibility of the folded system is sufficiently low, the stabilization is driven by a well-packed hydrophobic core, and the hydrogen-bonding network is mostly complete.[23] Temperature-dependent CD measurements have revealed that dimeric betabellin-14 undergoes thermal denaturation with a melting temperature (Tm) of 58 8C.[5c] This phenomenon could be reproduced for 7, but in our hands, the use of sodium phosphate buffer at pH 6.5 shifted the Tm to 45 8C (Figure 4a,f). Nevertheless, this finding supports the view that 7 is stabilized by hydrophobic contacts between residues remote in the sequence, and that the thermal unfolding is cooperative due to the tightly packed hydrophobic cluster of the b-sandwich fold. This analysis was performed for all the dimeric a/b-analogues except for 11, for which the shape of the CD curve was affected by side-chain exciton coupling. We found that thermal denaturation behavior similar to that of 7 could only be observed for 10. An increase in b-sheet content was detected upon elevation of the temperature from 5 to 25 8C, which is due to cold denaturation.[24] Similar behavior was observed for sequences 8, 9, and 12, but the thermal unfolding phenomenon could not be captured. These findings suggest that the ACHC building block in 10 has the structural features (local conformational preferences and side-chain shape) that are required to maintain sufficient compactness and rigidity of the b-sandwich fold, which mimics proteins. On the other hand, the other tested b-residues were unable to reach this limit. Effects of b-residues on the b-sandwich packing
Figure 3. Residue-level secondary structure propensity (SSP) scores calculated on the basis of the 1Ha, 1HN, 13Ca, 13Cb and 15N NMR chemical shifts. a) SSP scores obtained for monomeric betabellin-14 (1) and the a/b-peptidic analogues 2–6, indicated in gray, green, orange, red, blue and black, respectively. b) SSP scores measured for dimeric betabellin-14 (7) and the a/b-peptidic analogues 8–12, indicated in gray, green, orange, red, blue and black, respectively. SSP scores for b- and d-a-residues are not given due to the lack of secondary-structure-dependent reference chemical shifts. Negative and positive scores predict b-sheet and a-helical conformations, respectively. c) SSP score differences including d-a-residues obtained upon dimerization. Difference values hold structural meaning only for the l-a residues.
To find an explanation for the side-chain-dependent folding behavior, molecular dynamics calculations were performed for the dimeric sequences 7–12. The modeling was based on the AMBER ff03/TIP3P force field combination, which has been validated for the modeling of b-peptidic sequences in water.[25] The initial b-sandwich geometry for the parent betabellin family was taken from the literature.[5b, 26] Preliminary calculations with various intersheet orientations in our set-up confirmed that the published geometry provided the lowest solvent-accessible surface area for the hydrophobic core. Therefore simulations were started from the proposed b-sandwich fold, and the conformational space was sampled for 150 ns. Because the a/b-analogues were expected to change their
those of the parent sequence. The residue-level b-sheet enhancements in the a/b-analogues, however, are mostly located in the tethered segment C, which suggests that the propagation of the stabilization effects to the neighboring strands is &
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Full Paper tive to 7, and 10 was predicted to be the most rigid sequence of the a/b-peptides. Comparison of the lowest-energy structures with that of 7 revealed that the b-residue mutations not only led to an increase in flexibility, but also to a change in the overall shape of the constituent b-sheets. This deviation from the compact bsandwich fold was lowest for 10 (Figure 5c and 6a), which yielded the most compact hydrophobic core (Figure 6b) and the greatest ability to maintain the hydrogen-bonding network (Figure 6c,d). It was possible for the ACHC residues to form all the local backbone hydrogen bonds in the low-energy conformations, and the steric packing of the b-sandwich interior was acceptable (see Figure S16 in the Supporting Information). These results are in accord with the experiments. It is clear that b3-residues (8) introduce an overall curvature into the bsheet, which leads to increased solvent accessibility (Figure 6b) of the core-mutated residues, disruption of the hydrogenbonding network (Figure 6), and increased flexibility. Interestingly, ACPC residues (9) yielded a degree of core shielding comparable to that of 10, but its ability to form hydrogen bonds in the b-sheet environment was the worst of the b-residues. ACEC residues (11) were able to produce low-energy structures and a level of core compactness similar to those of 10, but its fit in the hydrogen-bonding network was less successful, as indicated by the diminished number of hydrogen bonds around the b-residue. Sequence 12 mostly unfolded over the simulation trajectory due to the steric repulsion between the bulky side-chains. The lowest-energy structures retained the b-sheets only partially, which resulted in solvent-exposed hydrophobic residues and a missing hydrogen-bonding network on one side of the b-sandwich. These findings correlate well with the results of the CD and NMR analyses. We found that a reduced number of hydrogen bonds were formed around the b-residues, even if the size of the side-chain matched the replaced a-residues (see Table S3 in the Supporting Information). This can explain the reduced b-sheet tendency and lateral propagation of order upon b-
Figure 4. Temperature-dependent CD curves recorded for 7–10 and 12 depicted in panels (a)–(e), respectively. The color code for the temperature scale is given as an inset in panel (a). MRE values at 195 nm for 7 (triangles) and 10 (circles) (f)
overall conformations relative to the parent sequence and to display increased flexibility, the convergence of the simulations was monitored by the rate of conformational cluster formation (see Figure S14 in the Supporting Information).[27] We found that the number of clusters reached a plateau within the time span of the modeling, which is indicative of acceptable sampling of the conformational ensembles. The results obtained with sequence 7 were in close correlation with the experimental observations. Inspection of the lowest-energy structures of 7 confirmed the target b-sandwich fold with a compact hydrophobic core (Figure 5a). The number of clusters calculated over the trajectory was 82, the flexibility being due to the fraying terminals. For the a/b-analogues 8–12, the number of clusters was 735, 751, 192, 430, and 414, respecFigure 5. Overlay of the five lowest-energy structures obtained from molecular dynamics simulations for sequentively, which indicates a significes a) 7, b) 8, c) 10, and d) 12. Top- and side-views of the structures are displayed in the left and right panels, recant increase in flexibility relaspectively. The structures of sequences 9 and 11 are shown in Figure S15. Chem. Eur. J. 2015, 21, 1 – 9
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Full Paper induced dimerization enhanced b-sheet formation, which closely mimics the behavior of the parent betabellin-14 structure. The inducibility of the b-sheet fold with b-residues in the chain has been demonstrated previously, and these results strongly suggest that this phenomenon could be extended to the chemically tethered b-sandwich systems. We observed that the folding propensity and residual flexibility of the chains were dependent on the nature of the b-residues. The results supported both the fit into the local b-sheet conformational environment with high hydrogen-bonding engagement and the steric fit into the hydrophobic core as being essential for the maintenance of b-sheet content and rigidity so as to obtain protein-like behavior. We found that (1R,2S)-ACHC, selected in accord with the stereochemical patterning principle, met both these requirements simultaneously acceptably well, and was therefore unique among the b-amino acid building blocks studied. The b3-open chain and ABHC mutations disrupted the hydrophobic core, whereas ACPC and ACEC introduced a conformational mismatch relative to ACHC. We speculated that backbone dihedral preferences played crucial roles here, as has been observed for foldameric helices.[19, 28] With the help of ACHC residues in the core and the d-a-amino acids in the original design, we constructed a de novo protein-sized b-sandwich model containing 25 % unnatural building blocks that is predicted to have promising protease resistance properties.
Figure 6. a) Root-mean-square deviations (RMSD) of the lowest-energy structures of the parent b-sandwich fold of 7. b) Solvent exposure ratio for the core hydrophobic residues in strands B and C relative to those calculated for Gly-Xxx-Gly tripeptidic sequences averaged over the trajectory (see Table S3). c) Number of hydrogen bonds in the b-sheets averaged over the trajectory (the theoretical maximum is 36). d) Number of hydrogen bonds observed for the residues in positions 12 (gray) and 19 (black) averaged over the trajectory (the theoretical maximum is 8).
Experimental Section Peptide synthesis and purification
sheet dimerization. The overall stability of the b-sandwich fold, however, is influenced by the properties of the residue in a complex manner. The results of the simulations suggest that the local conformational fit into the b-sheet geometry and the compatibility with the hydrophobic core of the b-sandwich are equally important. For example, the b3-residues display a reduced state of compactness around the side-chains, but the backbone flexibility affords relatively good contacts with the hydrogen-bonding network. In contrast, ACPC sits well in the hydrophobic interior, whereas the local conformational preferences reduce the number of hydrogen bonds. The ABHC residues provide an extreme case in which the steric clash between the side-chains prevents the formation of the solventshielded b-sandwich core, thereby leading to unfolding, despite the fact that the backbone conformation is fixed and the same as that for ACHC. The fit into both the b-sheet strands and the hydrophobic core is best for ACHC among the b-residues. This results in considerable b-sheet content, inducibility of the structure, and protein-like thermal unfolding behavior.
All materials, except Fmoc-protected ACEC and ABHC, were commercially available. The synthesis and characterization of these building blocks have been described in the literature.[19, 29] Tentagel R RAM resin was used as the solid support and (7-azabenzotriazol1-yl)tetramethyluronium hexafluorophosphate (HATU) as the coupling reagent. Couplings were performed by microwave irradiation of a 3 equiv amino acid excess at 75 8C for 15 min for a-amino acids, and for 30 min for b-residues. Histidine and cysteine were coupled at 50 8C. Peptides were cleaved with TFA/water/d,l-dithiothreitol/triisopropylsilane (90:5:2.5:2.5), and then precipitated in ice-cold diethyl ether. The resin was washed with acetic acid and water, and subsequently filtered and lyophilized. Peptides were purified by RP-HPLC on a C4 column (Phenomenex Jupiter, 4.6 250 mm). The HPLC eluents were (A) 0.1 % TFA in water and (B) 0.1 % TFA and 80 % ACN in water, with a gradient from 25 to 55 % B over 60 min at a flow rate of 4 mL min1. Dimeric peptides were obtained by air oxidation of the purified peptides (5 mg mL1) in 20 % DMSO/water at 37 8C for 24 h.[30] The samples were diluted with water and purified analogously to the monomer peptides. Their purity was confirmed by analytical RP-HPLC and ESI-MS measurements.
Conclusion
Circular dichroism measurements
By using the de novo designed betabellin-14 b-sandwich structure as a template, we constructed six a/b-peptidic chains in both monomeric and dimeric forms to study the effects of bresidue mutations on the hydrophobic core. The mutated sequences displayed a reduced propensity to fold, but disulfide&
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CD measurements were performed with a Jasco J-815 CD spectrometer. The CD spectra were recorded by using a 1 mm thermally jacketed quartz cell from 250 to 190 nm at a scan speed of 100 nm min1 with 10 accumulations. Compounds were dissolved in pH 6.5 sodium phosphate buffer, and the peptide concentration
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Acknowledgements This work was supported by the Hungarian Academy of Sciences (Lendlet program LP-2011-009), Gedeon Richter Plc. (TP7017), and the Hungarian Research Foundation (OTKA K112442). Computations were carried out at the HPC Center of the University of Szeged (TAMOP-4.2.2.C-11/1/KONV-2012-0010).
NMR experiments NMR spectra were recorded on a Bruker Avance III 600 MHz spectrometer equipped with a 5 mm CP-TCI triple-resonance cryoprobe. Compounds were dissolved at a concentration of 500 and 250 mm for monomeric and dimeric peptides, respectively, in 20 mm [D18]HEPES buffer (90 % H2O, 10 % D2O) containing 0.02 % NaN3 at pH 6.5. 4,4-Dimethyl-4-silapentane-1-sulfonic acid was used as an external standard. 2D homonuclear TOCSY and NOESY, and 2D heteronuclear 15N and 13C HSQC experiments were performed to assign resonances. The NOESY mixing time was 225 ms and the number of scans was 32. TOCSY measurements were made with homonuclear Hartman–Hahn transfer by using the DIPSI2 sequence for mixing with a mixing time of 80 ms; the number of scans was 16. For all the 2D homonuclear spectra, 2K time domain points and 512 increments were applied. 13C HSQC experiments were performed under the same sample conditions, but the buffer was prepared in D2O. All spectra were acquired by using the excitation sculpting solvent suppression pulse scheme. Processing was carried out by using Topspin 3.1 (Bruker), a cosine-bell window function, single zero-filling, and automatic baseline correction. Spectra were analyzed by Sparky 3.114 (T. D. Goddard and D. G. Kneller, University of California, San Francisco).
Keywords: amino acids · peptidomimetics engineering · protein folding · protein structures
Molecular modeling The molecular structures of the peptides were generated by using Schrçdinger’s Maestro and CCG’s MOE. The folded b-sheets were aligned according to the facing reported for the betabellin structure.[26] Molecular dynamics simulations were performed by using the GROMACS molecular dynamics software with the AMBER ff03 force field extended to b-amino acids.[25] The peptides were solvated in a cubic box by using a TIP3P explicit solvent model. The net charges of the peptides were neutralized by adding Cl counter ions. The energies were minimized by means of the steepest descent algorithm. The system was then heated to 300 K during a 100 ps constant-volume simulation with a 1 fs time step. The pressure was next equilibrated to 1 atm during a 100 ps NPT simulation with a 2 fs time step. Simulations of 150 ns were performed for all sequences with a step size of 2 fs. Temperature coupling was carried out with the V-rescale algorithm and pressure coupling was performed with the Parinello–Rahman algorithm. The structures were clustered by using the gromoss method with a 0.1 nm cut-off for a-carbon RMSD.[32] The average solvent-accessible surface area (SASA) was calculated from the trajectory for each residue. The maximum allowed SASA values for the amino acids were calculated by using Gly-Xxx-Gly tripeptides as the host–guest system. Solvent exposure was calculated as the fraction of the folded and the maximum surface area of each residue. The presence of hydrogen bonds was determined by using a 0.35 nm cutoff radius and a 308 cut-off angle. www.chemeurj.org
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protein
[1] A. M. Watkins, P. S. Arora, ACS Chem. Biol. 2014, 9, 1747 – 1754. [2] a) P. S. P. Wang, J. B. Nguyen, A. Schepartz, J. Am. Chem. Soc. 2014, 136, 6810 – 6813; b) R. Guerois, M. Lpez de La Paz, Protein design : methods and applications, Humana Press, Totowa, N.J., 2006; c) S. H. Gellman, Curr. Opin. Chem. Biol. 1998, 2, 717 – 725; d) M. Ramrez-Alvarado, T. Kortemme, F. J. Blanco, L. Serrano, Bioorg. Med. Chem. 1999, 7, 93 – 103; e) K. H. Mayo, E. Ilyina, H. Park, Protein Sci. 1996, 5, 1301 – 1315; f) M. S. Searle, Biopolymers 2004, 76, 185 – 195; g) J. F. Espinosa, F. A. Syud, S. H. Gellman, Protein Sci. 2002, 11, 1492 – 1505; h) H. E. Stanger, S. H. Gellman, J. Am. Chem. Soc. 1998, 120, 4236 – 4237. [3] a) M. Ramrez-Alvarado, F. J. Blanco, L. Serrano, Nat. Struct. Mol. Biol. 1996, 3, 604 – 612; b) E. d. Alba, M. A. Jimnez, M. Rico, J. L. Nieto, Folding and Design 1996, 1, 133 – 144. [4] a) K. H. Mayo, J. Haseman, E. Ilyina, B. Gray, Biochim. Biophys. Acta 1998, 1425, 81 – 92; b) A. M. Fernndez-Escamilla, S. Ventura, L. Serrano, M. A. Jimnez, Protein Sci. 2006, 15, 2278 – 2289; c) T. Kortemme, M. RamrezAlvarado, L. Serrano, Science 1998, 281, 253 – 256; d) F. A. Syud, H. E. Stanger, H. S. Mortell, J. F. Espinosa, J. D. Fisk, C. G. Fry, S. H. Gellman, J. Mol. Biol. 2003, 326, 553 – 568; e) E. D. Alba, J. Santoro, M. Rico, M. A. Jimnez, Protein Sci. 1999, 8, 854 – 865; f) H. L. Schenck, S. H. Gellman, J. Am. Chem. Soc. 1998, 120, 4869 – 4870; g) G. J. Sharman, M. S. Searle, J. Am. Chem. Soc. 1998, 120, 5291 – 5300. [5] a) A. Lim, A. M. Makhov, M. J. Saderholm, J. D. Griffith, B. W. Erickson, Biochem. Biophys. Res. Commun. 1999, 264, 498 – 504; b) A. Lim, M. J. Saderholm, A. M. Makhov, M. Kroll, Y. Yan, L. Perera, J. D. Griffith, B. W. Erickson, Protein Sci. 1998, 7, 1545 – 1554; c) Y. Yan, B. W. Erickson, Protein Sci. 1994, 3, 1069 – 1073. [6] J. A. Robinson, Acc. Chem. Res. 2008, 41, 1278 – 1288. [7] a) L. M. Johnson, S. H. Gellman in Methods in Enzymology, Vol. 523 (Ed.: E. K. Amy), Academic Press, 2013, pp. 407 – 429; b) D. Seebach, M. Overhand, F. N. M. Kuhnle, B. Martinoni, L. Oberer, U. Hommel, H. Widmer, Helv. Chim. Acta 1996, 79, 913 – 941; c) L. M. Johnson, D. E. Mortenson, H. G. Yun, W. S. Horne, T. J. Ketas, M. Lu, J. P. Moore, S. H. Gellman, J. Am. Chem. Soc. 2012, 134, 7317 – 7320. [8] a) J. S. Nowick, Acc. Chem. Res. 2008, 41, 1319 – 1330; b) P. N. Cheng, C. Liu, M. Zhao, D. Eisenberg, J. S. Nowick, Nat. Chem. 2012, 4, 927 – 933. [9] a) L. Sebaoun, B. Kauffmann, T. Delclos, V. Maurizot, I. Huc, Org Lett. 2014, 16, 2326 – 2329; b) L. Sebaoun, V. Maurizot, T. Granier, B. Kauffmann, I. Huc, J Am Chem. Soc 2014, 136, 2168 – 2174. [10] a) Y. J. Chung, B. R. Huck, L. A. Christianson, H. E. Stanger, S. Krauthauser, D. R. Powell, S. H. Gellman, J. Am. Chem. Soc. 2000, 122, 3995 – 4004; b) Y. J. Chung, B. R. Huck, L. A. Christianson, H. E. Stanger, S. Krauthauser, D. R. Powell, S. H. Gellman, J. Am. Chem. Soc. 2001, 123, 5851 – 5851; c) D. Seebach, S. Abele, K. Gademann, B. Jaun, Angew. Chem. Int. Ed. 1999, 38, 1595 – 1597; Angew. Chem. 1999, 111, 1700 – 1703; d) C. R. Jones, M. K. N. Qureshi, F. R. Truscott, S. T. D. Hsu, A. J. Morrison, M. D. Smith, Angew. Chem. Int. Ed. 2008, 47, 7099 – 7102; Angew. Chem. 2008, 120, 7207 – 7210; e) M. K. N. Qureshi, M. D. Smith, Chem. Commun. 2006, 5006 – 5008. [11] E. Vass, U. Strijowski, K. Wollschl ger, I. M. Mndity, G. Szilvgyi, M. Jewgin´ski, K. Gaus, S. Royo, Z. Majer, N. Sewald, M. Hollsi, J. Pept. Sci. 2010, 16, 613 – 620. [12] S. Segman, M. R. Lee, V. Vaiser, S. H. Gellman, H. Rapaport, Angew. Chem. Int. Ed. 2010, 49, 716 – 719; Angew. Chem. 2010, 122, 728 – 731. [13] a) T. A. Martinek, A. Hetenyi, L. Fulop, I. M. Mandity, G. K. Toth, I. Dekany, F. Fulop, Angew. Chem. Int. Ed. 2006, 45, 2396 – 2400; Angew. Chem.
The SSP score was calculated by using the ssp software.[22] The refDB random coil reference set based on the chemical shifts of properly referenced known protein structures was used. The builtin reference set for cysteine residues was replaced for the dimer structures by the corresponding values of oxidized cysteine described in the literature.[31]
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[24] D. Chandler, Nature 2005, 437, 640 – 647. [25] L. J. Nmeth, Z. Hegeds, T. A. Martinek, J. Chem. Inf. Model. 2014, 54, 2776 – 2783. [26] H. Inouye, J. E. Bond, S. P. Deverin, A. Lim, C. E. Costello, D. A. Kirschner, Biophys. J. 2002, 83, 1716 – 1727. [27] L. J. Smith, X. Daura, W. F. van Gunsteren, Proteins: Struct. Funct. Bioinf. 2002, 48, 487 – 496. [28] E. F. Lee, J. D. Sadowsky, B. J. Smith, P. E. Czabotar, K. J. Peterson-Kaufman, P. M. Colman, S. H. Gellman, W. D. Fairlie, Angew. Chem. Int. Ed. 2009, 48, 4318 – 4322; Angew. Chem. 2009, 121, 4382 – 4386. [29] Z. Szakonyi, T. A. Martinek, R. Sillanp , F. Flçp, Tetrahedron: Asymmetry 2008, 19, 2296 – 2303. [30] J. P. Tam, C. R. Wu,W. Liu, J. W. Zhang, A highly selective and effective reagent for disulfide bond formation in peptide synthesis and protein folding, in Peptides: Chemistry and Biology (Eds.: N. Sewald, H.-D. Jakubke), ESCOM Science Publishers, Leiden, The Netherlands, 1992. [31] T. D. Goddard and D. G. Kneller, SPARKY 3, University of California, San Francisco. [32] H. Zhang, S. Neal, D. S. Wishart, J. Biomol. NMR 2003, 25, 173 – 195. [33] a) Schrçdinger Release 2014-2: Maestro, version 9.8, Schrçdinger, LLC, New York, NY, 2014.; b) Molecular Operating Environment (MOE), 2013.08; Chemical Computing Group Inc., 1010 Sherbooke St. West, Suite #910, Montreal, QC, Canada, H3A 2R7, 2015. [34] X. Daura, W. F. van Gunsteren, A. E. Mark, Proteins: Struct. Funct. Bioinf. 1999, 34, 269 – 280.
Received: October 9, 2014 Published online on && &&, 0000
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FULL PAPER & Protein Folding G. Olajos, A. Hetnyi, E. Wber, L. J. Nmeth, Z. Szakonyi, F. Flçp, T. A. Martinek* && – && Dissolving sandwiches: A water-soluble b-sandwich has been constructed by using cyclic b-amino acids in the hydrophobic core (see figure). The structural stability is highly dependent on the side-chain, and the destructuring effects
of the b-residues could be minimized by using (1R,2S)-2-aminocyclohexanecarboxylic acid. The b-sandwich displays protein-like thermal denaturation behavior.
Induced Folding of Protein-Sized Foldameric b-Sandwich Models with Core b-Amino Acid Residues
The b-sandwich structure…… is the target of many de novo engineered protein mimetics. In the foldameric approach, a-amino acids are replaced by unnatural building blocks, which have major effects on the secondary structure in residue-dependent way. In their Full Paper on page && ff., T. Martinek and co-workers carried out b-amino acid mutations in the hydrophobic core of a b-sandwich template by using a diverse set of building blocks. It was found that protein-like properties are maintained by using the (1R,2S)-2-aminocyclohexanecarboxyic acid building blocks.
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& Protein Folding | Hot Paper |
Induced Folding of Protein-Sized Foldameric b-Sandwich Models with Core b-Amino Acid Residues Gbor Olajos,[a] Anasztzia Hetnyi,[b] Edit Wber,[a] Lukcs J. Nmeth,[a] Zsolt Szakonyi,[c] Ferenc Flçp,[c] and Tams A. Martinek*[a]
Abstract: The mimicry of protein-sized b-sheet structures with unnatural peptidic sequences (foldamers) is a considerable challenge. In this work, the de novo designed betabellin14 b-sheet has been used as a template, and a!b residue mutations were carried out in the hydrophobic core (positions 12 and 19). b-Residues with diverse structural properties were utilized: Homologous b3-amino acids, (1R,2S)-2aminocyclopentanecarboxylic acid (ACPC), (1R,2S)-2-aminocyclohexanecarboxylic acid (ACHC), (1R,2S)-2-aminocyclohex3-enecarboxylic acid (ACEC), and (1S,2S,3R,5S)-2-amino-6,6dimethylbicyclo[3.1.1]heptane-3-carboxylic acid (ABHC). Six a/b-peptidic chains were constructed in both monomeric
and disulfide-linked dimeric forms. Structural studies based on circular dichroism spectroscopy, the analysis of NMR chemical shifts, and molecular dynamics simulations revealed that dimerization induced b-sheet formation in the 64-residue foldameric systems. Core replacement with (1R,2S)-ACHC was found to be unique among the b-amino acid building blocks studied because it was simultaneously able to maintain the interstrand hydrogen-bonding network and to fit sterically into the hydrophobic interior of the bsandwich. The novel b-sandwich model containing 25 % unnatural building blocks afforded protein-like thermal denaturation behavior.
Introduction
cyclic a/b-peptides as hairpin models.[11] Sheet-like self-organization has been observed for a/b-peptides at the air/water interface[12] and in fibrillous b-peptidic nanostructures.[13] The construction of water-soluble, sizable, and well-folded b-sheet mimetic a/b-peptides with b-amino acids in arbitrary positions, however, is a difficult task. Horne and co-workers pointed out that in-registry a!b3 replacement in the hydrophobic core of the two strands resulted in decreased structural stability because of the changed hydrogen-bond pattern and an altered side-chain display as compared with the natural b-sheet structure.[14] In alternative design strategies, in which the native side-chain orientations are maintained in a 16-residue disulfide-cyclized b-hairpin, two a-amino acid residues are substituted by a single b-amino acid (aa!b2 or b3) or special b2,3 residues are applied.[15] We have systematically probed the effects of in-registry b3-amino acid substitutions by using a water-soluble, biologically active, b-sheet-forming 33-mer peptide as the parent sequence.[16] The b3-analogues displayed decreased folding propensities, whereas a micellar environment could induce b-sheet formation in a very similar way to that of the parent a-peptide. Bioactivity was retained by this inducible b-sheet formation, and the tendency to aggregation was reduced. These findings suggested that controlled, inducible b-sheet folding may be a useful approach to the construction of bioactive a/b-peptidic b-sheet mimetics. In this work we investigated b-sheet inducibility in a b-sandwich-forming protein-sized system in which b-amino acid mutations had been carried out in the hydrophobic core. The folding was enhanced by the dimerization of the b-sheet units in this case. By using the 32-residue betabellin-14 as a templa-
There are a large number of known b-sheet-rich protein interfaces that are potential biological targets,[1] but the design of functional, water-soluble, stand-alone b-sheet structures, and especially those with protein-like structural features, is a great challenge.[2] De novo designed structures have been reported to form b-hairpins,[3] three-stranded b-sheets,[4] and b-sandwiches,[5] and protein epitope mimetic hairpins have been shown to display bioactive potencies.[6] For biological applications, however, it is desirable that the peptidic sequence contains unnatural building blocks to improve proteolytic stability.[7] Non-peptidic template and turn units have been combined with peptides to construct parallel and antiparallel sheet structures that display protein aggregation/inhibitory effects,[8] and aromatic oligoamides have been constructed to mimic bsheets.[9] Successes have also been achieved with homologated amino acids, which can be used to build short turns[10] and [a] G. Olajos, Dr. E. Wber, L. J. Nmeth, Prof. Dr. T. A. Martinek Institute of Pharmaceutical Analysis, SZTE-MTA Lendlet Foldamer Research Group University of Szeged, 6720 Szeged (Hungary) E-mail: [email protected] [b] Dr. A. Hetnyi Department of Medical Chemistry University of Szeged, 6720 Szeged (Hungary) [c] Dr. Z. Szakonyi, Prof. Dr. F. Flçp Institute of Pharmaceutical Chemistry University of Szeged, 6720 Szeged (Hungary) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201405581. Chem. Eur. J. 2015, 21, 1 – 9
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Full Paper te,[5c] we showed that the b-sheet content of the a/b-peptidic analogues can be increased by dimerization through the formation of disulfide bridges. We successfully constructed a foldameric sequence that displays protein-like behavior.
phobic core of the b-sandwich because these core substitutions may have a major effect on the folding process. These residues are in isolated positions so as to allow the bresidues to accommodate the local environment (Figure 1b). We previously found that b3-residues with large hydrophobic side-chains tend to attain a gauche backbone conformation in a b-sheet environment.[16] To test the effects of conformational constraints, we synthesized analogues by using cyclic b-amino acids. In accordance with the rules of the stereochemical patterning approach,[17] a cis backbone arrangement (with R and S configurations at the a- and b-carbon atoms, respectively) was applied to optimize the interstrand hydrogen-bond network. It has previously been demonstrated that a steric clash between bulky side-chains can exert a major influence on the secondary structure.[18] The steric fit within the hydrophobic core of the b-sandwich is also an important factor, and cyclic b-residues with various steric requirements were therefore selected for the study (Figure 1): (1R,2S)-2-Aminocyclopentanecarboxylic acid (ACPC), (1R,2S)-2-aminocyclohexanecarboxylic acid (ACHC), (1R,2S)-2-aminocyclohex-3-enecarboxylic acid (ACEC), and (1S,2S,3R,5S)-2-amino-6,6-dimethylbicyclo[3.1.1]heptane-3carboxylic acid (ABHC).
Results and Discussion Design approach Betabellin-14 consists of a 32-residue palindromic pattern of polar (p), nonpolar (n), end (e), and turn (t, r) residues (Figure 1a). When b-turns are induced by d-Lys-d-Ala (tt) and dAla-d-Lys (rr) segments, the sequence tends to fold into an am-
Induced folding as determined by circular dichroism (CD) spectroscopy The overall propensity of the betabellin-14 analogues to attain a specific secondary structure was monitored by using CD spectroscopy. In accordance with the literature, the parent sequence 1 displays a CD spectrum characteristic of a b-sheet, even in its monomeric form (Figure 2a), and dimerization through a disulfide bridge further increases the b-sheet content. The a/b-analogues 2–4 and 6 yielded U-shaped CD curves with an intense negative band at around 199 nm and a lower-intensity band at around 220 nm. Dimerization, however, resulted in changes in the CD responses; the negative band disappeared or became less dominant. A positive Cotton effect was observed for 5, whereas 11 exhibits inversion and a decrease in band intensity, which can be explained in terms of exciton coupling to the side-chain double bond. Similar anomalous CD behavior has previously been observed for helical bpeptide foldamers containing the ACEC residue.[19] Overall, these changes suggest the formation of ordered conformational states upon dimerization. A CD spectrum is the weighted sum of the component spectra of the secondary structure elements, and the curves observed are therefore very sensitive to the conformational ensemble and visual inspection may be misleading. To extract quantitative information, a convex constraint analysis deconvolution[20] was carried out simultaneously on all the spectra recorded in this study, with the exception of the spectra recorded for 5 and 11 containing the ACEC building blocks. This approach allowed the calculation of the pure component spectra and their weights without making use of any input basis spectrum or structural hypothesis. The normalized residual mean square deviation of the back-calculated spectra from the experimental curves averaged over 1–4, 6–10, and 12 was
Figure 1. a) Amino acid sequence and palindromic design (gray) of the betabellin-14 analogues studied. Residues are coded with the standard oneletter a-amino acid notation; lower case letters indicate d-a-amino acids. B) Schematic representation of the b-sandwich fold of the betabellin-14 monomer with the positions selected for the mutations highlighted in gray. Constituent strand segments are designated by the capital letters A–D.
phiphilic b-sheet.[5c] The formation of a b-sheet can be promoted by dimerization of the betabellin-14 chains through a disulfide bridge involving the residues Cys21. The intermolecular interactions enforced by the linkage enhance the hydrophobic interactions between the sheets, and the covalent betabellin14 dimers therefore display increased order. We chose this de novo designed system because it afforded a water-soluble and inducible b-sheet fold. To study the effects of the b-amino acid substitutions, we modified residues I12 and A19 in the hydro&
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Full Paper originally for betabellin-14 were able to push the a/b-sequences towards the b-sheet fold. Residue-level structure propensities by NMR analysis Chemical shift analysis[21] was carried out to gain a deeper insight into the propensity to form b-sheets at the amino acid level. Heteronuclear NMR spectra were recorded and the resonances assigned for the monomeric and dimeric sequences in natural abundance under optimized conditions (HEPES buffer, 500 mm, 298 K; see Figures S2–S13 in the Supporting Information). All the identified 1Ha, 1HN, 13Ca, 13Cb and 15N resonances (see Tables S1 and S2) were included in the analysis. The secondary structure propensity (SSP) score[22] was calculated by using the ssp software, which gave a semiquantitative measure of the residue-level secondary structure distribution along the chains. The resonances of the b-and d-a-residues were excluded from the calculations because of the lack of reference values. Although secondary-structure-independent neighborhood effects of the b- and d-a-residues on the magnetic environment of the adjacent l-a-residues cannot be ruled out, our earlier observations indicated no major systematic influence preventing an analysis of the trends in the secondary structure.[16] The set of negative SSP scores obtained for the monomeric parent sequence 1 is a good indicator of the b-sheet tendency in line with the CD results (Figure 3a). The largest b-sheet content was found in segment B. Disulfide formation in 7 led to decreases in the SSP scores along the chain (Figure 3b). A marked increase in the b-sheet content was observed for strand C containing the tethering Cys2 residues, which propagated to strands B and D. This indicates that hydrophobic stabilization within the core of the forced b-sandwich is effective. These residue-level results also confirmed betabellin-14 as a useful inducible b-sandwich model. For 2–6, the SSP score increases around the b-residues (positions 12 and 19), as reflected in the local disorder caused by these mutations and in support of the CD findings. The dimeric a/b-peptides 8–12 maintain the destructuring pattern of the b-residues, but the disulfide bridge increases the b-sheet content in segment C up to the values observed in segment B. This enhancement effect is side-chain-dependent. To study these dimerization-induced structural changes, the differences in the SSP scores were calculated (Figure 3c). This representation eliminates the influence of the reference chemical shifts and the data depict the dimerization-induced b-sheet enhancement along the sequences for the l-a-residues. For the b-turns of the d-a-residues, secondary-structure-type changes cannot be directly assigned to the values, but the DSSP scores can be compared with those of the parent sequences. It is clear from this analysis that dimerization-induced b-sheet enhancements are present for the a/bsequences except for the ABHC-containing sequence 12. Interestingly, the largest increases are observed in the proximity of the b-residues in position 19, which indicates the inherent ability of b-residues to fit into a b-sheet environment if extra stabilization is introduced. The best results were obtained with derivative 10, which displays similar DSSP scores in segment C to
Figure 2. a) Mean residue ellipticities (MRE) obtained for monomeric betabellin-14 (1) and the a/b-peptidic analogues 2–6, indicated in gray, gray dotted, gray dashed, black, black dotted, and black dashed lines, respectively. b) MRE curves obtained for dimeric betabellin-14 (7) and the a/b-peptidic analogues 8–12, indicated in gray, gray dotted, gray dashed, black, black dotted, and black dashed lines, respectively. c) Secondary structure contents calculated by using the convex constraint analysis algorithm for the CD curve deconvolution. b-sheet, b-turn, and disordered components are indicated in black, gray, and white, respectively.
3.94 %, which indicates an acceptably low fitting error. The protocol resulted in three-component spectra (see Figure S1 in the Supporting Information) that can be assigned to b-sheet, disordered, and a b-turn conformation of d-a-amino acid residues according to literature findings.[20] The corresponding coefficients allowed the contributions of the components to be estimated and hence the percentage of the secondary structure content was determined (Figure 2c). For the parent a-peptide sequences (1 and 7), the b-sheet content changed from 43 to 67 % upon dimerization, which shows the stabilization effects of the increased hydrophobic interactions in the disulfide-stabilized b-sandwich. The analysis revealed that the a/bpeptides are mainly disordered in the monomeric form, with the b-sheet content varying in the range 4–24 %. On the other hand, dimerization increases the b-sheet content to 34–47 % for 8–10. In contrast, the data for the sequence containing the bulky ABHC residues (12) indicate that the b-sheet content decreases after the formation of the disulfide bridge, which points to a steric clash. The b-turn contents for 1 and 7 were found to be 23 and 25 %, respectively, which are in line with the nine residues forming the three-turn structures (28 %). Sequences 9 and 10 showed the values closes to these (28 and 27 %, respectively), whereas 8 and 12 displayed marked deviations (7 % and 71 %, respectively). These findings support our hypothesis that, despite the destructuring effects of the b-residue mutations, b-sheet formation can be induced if the hydrophobic interactions are partially restored. In this case the stabilization effects of the forced b-sandwich formation proposed Chem. Eur. J. 2015, 21, 1 – 9
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Full Paper less efficient than in 7. These results suggest that side-chain bulkiness and the preferred arrangement of backbone torsions in the b-residues have profound effects on the b-sheet packing and thereby on the stabilization of the fold. The residual flexibility of this sensitive system and the signal overlaps in the 2D 1 H homonuclear spectra did not permit the assignment of diagnostic NOE interactions. Thermal denaturation Both CD and NMR responses represent ensemble averages that indicate the presence of secondary structure elements, but folded protein-like behavior requires a certain amount of rigidity. Cooperative thermal denaturation is a characteristic of well-folded proteins and is possible if the residual flexibility of the folded system is sufficiently low, the stabilization is driven by a well-packed hydrophobic core, and the hydrogen-bonding network is mostly complete.[23] Temperature-dependent CD measurements have revealed that dimeric betabellin-14 undergoes thermal denaturation with a melting temperature (Tm) of 58 8C.[5c] This phenomenon could be reproduced for 7, but in our hands, the use of sodium phosphate buffer at pH 6.5 shifted the Tm to 45 8C (Figure 4a,f). Nevertheless, this finding supports the view that 7 is stabilized by hydrophobic contacts between residues remote in the sequence, and that the thermal unfolding is cooperative due to the tightly packed hydrophobic cluster of the b-sandwich fold. This analysis was performed for all the dimeric a/b-analogues except for 11, for which the shape of the CD curve was affected by side-chain exciton coupling. We found that thermal denaturation behavior similar to that of 7 could only be observed for 10. An increase in b-sheet content was detected upon elevation of the temperature from 5 to 25 8C, which is due to cold denaturation.[24] Similar behavior was observed for sequences 8, 9, and 12, but the thermal unfolding phenomenon could not be captured. These findings suggest that the ACHC building block in 10 has the structural features (local conformational preferences and side-chain shape) that are required to maintain sufficient compactness and rigidity of the b-sandwich fold, which mimics proteins. On the other hand, the other tested b-residues were unable to reach this limit. Effects of b-residues on the b-sandwich packing
Figure 3. Residue-level secondary structure propensity (SSP) scores calculated on the basis of the 1Ha, 1HN, 13Ca, 13Cb and 15N NMR chemical shifts. a) SSP scores obtained for monomeric betabellin-14 (1) and the a/b-peptidic analogues 2–6, indicated in gray, green, orange, red, blue and black, respectively. b) SSP scores measured for dimeric betabellin-14 (7) and the a/b-peptidic analogues 8–12, indicated in gray, green, orange, red, blue and black, respectively. SSP scores for b- and d-a-residues are not given due to the lack of secondary-structure-dependent reference chemical shifts. Negative and positive scores predict b-sheet and a-helical conformations, respectively. c) SSP score differences including d-a-residues obtained upon dimerization. Difference values hold structural meaning only for the l-a residues.
To find an explanation for the side-chain-dependent folding behavior, molecular dynamics calculations were performed for the dimeric sequences 7–12. The modeling was based on the AMBER ff03/TIP3P force field combination, which has been validated for the modeling of b-peptidic sequences in water.[25] The initial b-sandwich geometry for the parent betabellin family was taken from the literature.[5b, 26] Preliminary calculations with various intersheet orientations in our set-up confirmed that the published geometry provided the lowest solvent-accessible surface area for the hydrophobic core. Therefore simulations were started from the proposed b-sandwich fold, and the conformational space was sampled for 150 ns. Because the a/b-analogues were expected to change their
those of the parent sequence. The residue-level b-sheet enhancements in the a/b-analogues, however, are mostly located in the tethered segment C, which suggests that the propagation of the stabilization effects to the neighboring strands is &
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Full Paper tive to 7, and 10 was predicted to be the most rigid sequence of the a/b-peptides. Comparison of the lowest-energy structures with that of 7 revealed that the b-residue mutations not only led to an increase in flexibility, but also to a change in the overall shape of the constituent b-sheets. This deviation from the compact bsandwich fold was lowest for 10 (Figure 5c and 6a), which yielded the most compact hydrophobic core (Figure 6b) and the greatest ability to maintain the hydrogen-bonding network (Figure 6c,d). It was possible for the ACHC residues to form all the local backbone hydrogen bonds in the low-energy conformations, and the steric packing of the b-sandwich interior was acceptable (see Figure S16 in the Supporting Information). These results are in accord with the experiments. It is clear that b3-residues (8) introduce an overall curvature into the bsheet, which leads to increased solvent accessibility (Figure 6b) of the core-mutated residues, disruption of the hydrogenbonding network (Figure 6), and increased flexibility. Interestingly, ACPC residues (9) yielded a degree of core shielding comparable to that of 10, but its ability to form hydrogen bonds in the b-sheet environment was the worst of the b-residues. ACEC residues (11) were able to produce low-energy structures and a level of core compactness similar to those of 10, but its fit in the hydrogen-bonding network was less successful, as indicated by the diminished number of hydrogen bonds around the b-residue. Sequence 12 mostly unfolded over the simulation trajectory due to the steric repulsion between the bulky side-chains. The lowest-energy structures retained the b-sheets only partially, which resulted in solvent-exposed hydrophobic residues and a missing hydrogen-bonding network on one side of the b-sandwich. These findings correlate well with the results of the CD and NMR analyses. We found that a reduced number of hydrogen bonds were formed around the b-residues, even if the size of the side-chain matched the replaced a-residues (see Table S3 in the Supporting Information). This can explain the reduced b-sheet tendency and lateral propagation of order upon b-
Figure 4. Temperature-dependent CD curves recorded for 7–10 and 12 depicted in panels (a)–(e), respectively. The color code for the temperature scale is given as an inset in panel (a). MRE values at 195 nm for 7 (triangles) and 10 (circles) (f)
overall conformations relative to the parent sequence and to display increased flexibility, the convergence of the simulations was monitored by the rate of conformational cluster formation (see Figure S14 in the Supporting Information).[27] We found that the number of clusters reached a plateau within the time span of the modeling, which is indicative of acceptable sampling of the conformational ensembles. The results obtained with sequence 7 were in close correlation with the experimental observations. Inspection of the lowest-energy structures of 7 confirmed the target b-sandwich fold with a compact hydrophobic core (Figure 5a). The number of clusters calculated over the trajectory was 82, the flexibility being due to the fraying terminals. For the a/b-analogues 8–12, the number of clusters was 735, 751, 192, 430, and 414, respecFigure 5. Overlay of the five lowest-energy structures obtained from molecular dynamics simulations for sequentively, which indicates a significes a) 7, b) 8, c) 10, and d) 12. Top- and side-views of the structures are displayed in the left and right panels, recant increase in flexibility relaspectively. The structures of sequences 9 and 11 are shown in Figure S15. Chem. Eur. J. 2015, 21, 1 – 9
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Full Paper induced dimerization enhanced b-sheet formation, which closely mimics the behavior of the parent betabellin-14 structure. The inducibility of the b-sheet fold with b-residues in the chain has been demonstrated previously, and these results strongly suggest that this phenomenon could be extended to the chemically tethered b-sandwich systems. We observed that the folding propensity and residual flexibility of the chains were dependent on the nature of the b-residues. The results supported both the fit into the local b-sheet conformational environment with high hydrogen-bonding engagement and the steric fit into the hydrophobic core as being essential for the maintenance of b-sheet content and rigidity so as to obtain protein-like behavior. We found that (1R,2S)-ACHC, selected in accord with the stereochemical patterning principle, met both these requirements simultaneously acceptably well, and was therefore unique among the b-amino acid building blocks studied. The b3-open chain and ABHC mutations disrupted the hydrophobic core, whereas ACPC and ACEC introduced a conformational mismatch relative to ACHC. We speculated that backbone dihedral preferences played crucial roles here, as has been observed for foldameric helices.[19, 28] With the help of ACHC residues in the core and the d-a-amino acids in the original design, we constructed a de novo protein-sized b-sandwich model containing 25 % unnatural building blocks that is predicted to have promising protease resistance properties.
Figure 6. a) Root-mean-square deviations (RMSD) of the lowest-energy structures of the parent b-sandwich fold of 7. b) Solvent exposure ratio for the core hydrophobic residues in strands B and C relative to those calculated for Gly-Xxx-Gly tripeptidic sequences averaged over the trajectory (see Table S3). c) Number of hydrogen bonds in the b-sheets averaged over the trajectory (the theoretical maximum is 36). d) Number of hydrogen bonds observed for the residues in positions 12 (gray) and 19 (black) averaged over the trajectory (the theoretical maximum is 8).
Experimental Section Peptide synthesis and purification
sheet dimerization. The overall stability of the b-sandwich fold, however, is influenced by the properties of the residue in a complex manner. The results of the simulations suggest that the local conformational fit into the b-sheet geometry and the compatibility with the hydrophobic core of the b-sandwich are equally important. For example, the b3-residues display a reduced state of compactness around the side-chains, but the backbone flexibility affords relatively good contacts with the hydrogen-bonding network. In contrast, ACPC sits well in the hydrophobic interior, whereas the local conformational preferences reduce the number of hydrogen bonds. The ABHC residues provide an extreme case in which the steric clash between the side-chains prevents the formation of the solventshielded b-sandwich core, thereby leading to unfolding, despite the fact that the backbone conformation is fixed and the same as that for ACHC. The fit into both the b-sheet strands and the hydrophobic core is best for ACHC among the b-residues. This results in considerable b-sheet content, inducibility of the structure, and protein-like thermal unfolding behavior.
All materials, except Fmoc-protected ACEC and ABHC, were commercially available. The synthesis and characterization of these building blocks have been described in the literature.[19, 29] Tentagel R RAM resin was used as the solid support and (7-azabenzotriazol1-yl)tetramethyluronium hexafluorophosphate (HATU) as the coupling reagent. Couplings were performed by microwave irradiation of a 3 equiv amino acid excess at 75 8C for 15 min for a-amino acids, and for 30 min for b-residues. Histidine and cysteine were coupled at 50 8C. Peptides were cleaved with TFA/water/d,l-dithiothreitol/triisopropylsilane (90:5:2.5:2.5), and then precipitated in ice-cold diethyl ether. The resin was washed with acetic acid and water, and subsequently filtered and lyophilized. Peptides were purified by RP-HPLC on a C4 column (Phenomenex Jupiter, 4.6 250 mm). The HPLC eluents were (A) 0.1 % TFA in water and (B) 0.1 % TFA and 80 % ACN in water, with a gradient from 25 to 55 % B over 60 min at a flow rate of 4 mL min1. Dimeric peptides were obtained by air oxidation of the purified peptides (5 mg mL1) in 20 % DMSO/water at 37 8C for 24 h.[30] The samples were diluted with water and purified analogously to the monomer peptides. Their purity was confirmed by analytical RP-HPLC and ESI-MS measurements.
Conclusion
Circular dichroism measurements
By using the de novo designed betabellin-14 b-sandwich structure as a template, we constructed six a/b-peptidic chains in both monomeric and dimeric forms to study the effects of bresidue mutations on the hydrophobic core. The mutated sequences displayed a reduced propensity to fold, but disulfide&
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CD measurements were performed with a Jasco J-815 CD spectrometer. The CD spectra were recorded by using a 1 mm thermally jacketed quartz cell from 250 to 190 nm at a scan speed of 100 nm min1 with 10 accumulations. Compounds were dissolved in pH 6.5 sodium phosphate buffer, and the peptide concentration
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Full Paper was 100 and 50 mm for monomeric and dimeric peptides, respectively. For thermal control, a Julabo water thermostat was used with a 10 min equilibration time for each temperature. The solvent baseline was subtracted. Convex constraint analysis deconvolution of the CD curves was performed by using the CCA + program.[20]
Acknowledgements This work was supported by the Hungarian Academy of Sciences (Lendlet program LP-2011-009), Gedeon Richter Plc. (TP7017), and the Hungarian Research Foundation (OTKA K112442). Computations were carried out at the HPC Center of the University of Szeged (TAMOP-4.2.2.C-11/1/KONV-2012-0010).
NMR experiments NMR spectra were recorded on a Bruker Avance III 600 MHz spectrometer equipped with a 5 mm CP-TCI triple-resonance cryoprobe. Compounds were dissolved at a concentration of 500 and 250 mm for monomeric and dimeric peptides, respectively, in 20 mm [D18]HEPES buffer (90 % H2O, 10 % D2O) containing 0.02 % NaN3 at pH 6.5. 4,4-Dimethyl-4-silapentane-1-sulfonic acid was used as an external standard. 2D homonuclear TOCSY and NOESY, and 2D heteronuclear 15N and 13C HSQC experiments were performed to assign resonances. The NOESY mixing time was 225 ms and the number of scans was 32. TOCSY measurements were made with homonuclear Hartman–Hahn transfer by using the DIPSI2 sequence for mixing with a mixing time of 80 ms; the number of scans was 16. For all the 2D homonuclear spectra, 2K time domain points and 512 increments were applied. 13C HSQC experiments were performed under the same sample conditions, but the buffer was prepared in D2O. All spectra were acquired by using the excitation sculpting solvent suppression pulse scheme. Processing was carried out by using Topspin 3.1 (Bruker), a cosine-bell window function, single zero-filling, and automatic baseline correction. Spectra were analyzed by Sparky 3.114 (T. D. Goddard and D. G. Kneller, University of California, San Francisco).
Keywords: amino acids · peptidomimetics engineering · protein folding · protein structures
Molecular modeling The molecular structures of the peptides were generated by using Schrçdinger’s Maestro and CCG’s MOE. The folded b-sheets were aligned according to the facing reported for the betabellin structure.[26] Molecular dynamics simulations were performed by using the GROMACS molecular dynamics software with the AMBER ff03 force field extended to b-amino acids.[25] The peptides were solvated in a cubic box by using a TIP3P explicit solvent model. The net charges of the peptides were neutralized by adding Cl counter ions. The energies were minimized by means of the steepest descent algorithm. The system was then heated to 300 K during a 100 ps constant-volume simulation with a 1 fs time step. The pressure was next equilibrated to 1 atm during a 100 ps NPT simulation with a 2 fs time step. Simulations of 150 ns were performed for all sequences with a step size of 2 fs. Temperature coupling was carried out with the V-rescale algorithm and pressure coupling was performed with the Parinello–Rahman algorithm. The structures were clustered by using the gromoss method with a 0.1 nm cut-off for a-carbon RMSD.[32] The average solvent-accessible surface area (SASA) was calculated from the trajectory for each residue. The maximum allowed SASA values for the amino acids were calculated by using Gly-Xxx-Gly tripeptides as the host–guest system. Solvent exposure was calculated as the fraction of the folded and the maximum surface area of each residue. The presence of hydrogen bonds was determined by using a 0.35 nm cutoff radius and a 308 cut-off angle. www.chemeurj.org
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The SSP score was calculated by using the ssp software.[22] The refDB random coil reference set based on the chemical shifts of properly referenced known protein structures was used. The builtin reference set for cysteine residues was replaced for the dimer structures by the corresponding values of oxidized cysteine described in the literature.[31]
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Received: October 9, 2014 Published online on && &&, 0000
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FULL PAPER & Protein Folding G. Olajos, A. Hetnyi, E. Wber, L. J. Nmeth, Z. Szakonyi, F. Flçp, T. A. Martinek* && – && Dissolving sandwiches: A water-soluble b-sandwich has been constructed by using cyclic b-amino acids in the hydrophobic core (see figure). The structural stability is highly dependent on the side-chain, and the destructuring effects
of the b-residues could be minimized by using (1R,2S)-2-aminocyclohexanecarboxylic acid. The b-sandwich displays protein-like thermal denaturation behavior.
Induced Folding of Protein-Sized Foldameric b-Sandwich Models with Core b-Amino Acid Residues
The b-sandwich structure…… is the target of many de novo engineered protein mimetics. In the foldameric approach, a-amino acids are replaced by unnatural building blocks, which have major effects on the secondary structure in residue-dependent way. In their Full Paper on page && ff., T. Martinek and co-workers carried out b-amino acid mutations in the hydrophobic core of a b-sandwich template by using a diverse set of building blocks. It was found that protein-like properties are maintained by using the (1R,2S)-2-aminocyclohexanecarboxyic acid building blocks.
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