Helix Stabilization of Poly(ethylene glycol)–Peptide Conjugates

ARTICLE pubs.acs.org/Biomac

Helix Stabilization of Poly(ethylene glycol) Peptide Conjugates Amit Jain and Henry S. Ashbaugh* Tulane University, Department of Chemical and Biomolecular Engineering, 300 Lindy Boggs Center, New Orleans, Louisiana 70118, United States

bS Supporting Information ABSTRACT: Hybrid polymer peptide conjugates offer the potential for incorporating biological function into synthetic materials. The secondary structure of short helical peptides, however, frequently becomes less stable when expressed independent of longer protein sequences or covalently linked with a conformationally disordered synthetic polymer. Recently, new amphipathic peptide poly(ethylene glycol) conjugates were introduced (Shu, J., et al. Biomacromolecules 2008, 9, 2011), which displayed enhanced peptide helicity upon polymer functionalization while retaining tertiary coiled-coil associations. We report here a molecular simulation study of peptide helix stabilization by conjugation with poly(ethylene glycol). The polymer oxygens are shown to favorably interact with the cationic lysine side chains, providing an alternate binding site that protects against disruption of the peptide hydrogenbonds that stabilize the helical conformation. When the peptide lysine charges are neutralized or poly(ethylene glycol) is conjugated with polyalanine, the polymer exhibits a negligible effect on the secondary structure. We also observe the interactions of poly(ethylene glycol) with the amphipathic peptide lysines tends to segregate the polymer away from the nonpolar face of the helix, suggesting no disruption of the interactions that drive tertiary contacts between helicies.

’ INTRODUCTION R-Helices are a ubiquitous secondary structural element in folded proteins that have been exploited for potential drugs1 3 and as components of synthetic materials that incorporate biological activity.4 9 A significant problem in the development of peptide-based drugs is that when short, therapeutically active peptide sequences are cut from a longer folded protein sequence, the peptides tend to be unstructured as a result of competition for hydrogen-bonds with water and lost cooperativity,10 making the peptide vulnerable to protease digestion.11,12 The protease resistance of short, helical peptides can be enhanced, however, if their conformation is stabilized.13 One strategy for helix stabilization is to cross covalently-link side-chain residues separated by one or two helical turns.14 These cross-links introduce conformational constraints that reduce the entropic penalty for forming a helix. Covalent cross-links may be formed, for example, by lactam bridges,15 17 disulfide bonds,18,19 or through hydrocarbon staples.13,20 Alternatively, cationic and anionic amino acid pairs separated by one turn may form a salt bridge to stabilize the helix through attractive Coulombic forces,21 23 or metal ion ligating side chains can be introduced to form cross-links through divalent ion complexation.24,25 Working on hybrid synthetic/biological materials, Xu and coworkers recently designed new helix-bundle forming polymer peptide conjugates.6 Specifically, they covalently attached short poly(ethylene glycol) (PEG) chains to de novo designed amphipathic peptides that self-organize into triple helix coiled-coil bundles. The PEG was attached through a cysteine on the hydrophilic side of the helix. Surprisingly, the polymer stabilized the R-helical secondary structure as evidenced by postconjugation circular dichorism spectra. The helicity of the peptide increased r 2011 American Chemical Society

with increasing molecular weight of the polymer, attaining maximum secondary structure stabilization for PEG side chains 2000 Da in mass and greater. Additionally, the PEG did not disrupt the peptide tertiary structure. Klok and coworkers also observed PEG stabilization of amphipathic helicies at low peptide concentrations.7 Different from Xu’s peptides, the helicity of Klok’s polymer peptide conjugates decreased relative to the unconjugated peptide with increasing concentration, and tertiary aggregation was diminished. The hybrid peptides synthesized by Klok were conjugated with PEG at the N-terminus, whereas those prepared by Xu were modified toward the middle of the sequence, suggesting a sequence position dependence of polymer conjugation on helix stability. Independent of the PEG functionalization position, however, the secondary structures of the conjugated peptides are more resilient to increasing temperature than their unconjugated parents.6,7,26 On the basis of the experimental observations, a question follows: What role does the disordered PEG chain play in stabilizing the structure of Xu’s R-helical peptides? Whereas it was postulated that PEG can provide osmotic stress to stabilize protein structure27 and a microhydrophobic environment to stabilize intrapeptide hydrogen bonds, Xu noted that because “...we do not know the spatial arrangement of the conjugated PEG chains relative to the peptide...” the mechanism for helical stabilization is unclear.6 To gain insight into the stabilization mechanism, we have performed molecular dynamics simulations of polymer peptide conjugate helix formation. Our simulations Received: April 13, 2011 Revised: June 3, 2011 Published: June 09, 2011 2729

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Table 1. Peptide Polymer Naming Conventions Used in the Present Study sequence identifier 1CW

description Ac-EVEALEKKVAALESKVQALEKKVEALEHGWDGRCONH2 the original peptide sequence investigated by Xu and coworkers.

1CW0

sequence 1CW with the cationic lysine charges neutralized to

A33

Ac-A33-CONH2

zero. a polyalanine sequence the same length as 1CW A2K

Ac-A6K2A6KA5K2A11-CONH2 a mutant of the polyalanine sequence with lysines placed at the same positions as in 1CW.

A2R

Ac-A6R2A6RA5R2A11-CONH2 a mutant of the polyalanine sequence with arginine’s placed at the same positions as the lysines in 1CW.

peptide- the suffix -xEO indicates conjugation of the peptide with an xEO

ethylene oxide chain x monomers in length. To affect conjugation position 14 is mutated to a cystine to attach the oligomer through the sulfur side chain.

focus on the peptide 1CW studied by the Xu group. This peptide was chosen because experimentally it showed the most significant helical stabilization after PEG conjugation that persisted to higher peptide concentrations, unlike the sequences studied by Klok and coworkers. In addition to 1CW, we also simulated a series of polymer peptide conjugates of polyalanine and polyalanine mutants to identify those amino acid side chains that play the largest role in moderating peptide interactions with PEG to reinforce the secondary structure.

’ SIMULATION METHODS Molecular dynamics28 simulations were performed using AMBER 8.29 The peptides were modeled using the AMBER 2003 force field.30 Charges on acidic and basic side chains were set to their values at neutral pH. Partial charges for the ethylene oxide monomers of PEG and the maleimide end unit were obtained from ab initio Gaussian0331 calculations using the RESP algorithm.32 Lennard-Jones, bond length, bond angle, and dihedral contributions to the potential for PEG and maleimide were modeled with generalized AMBER force field.33 Water was modeled using the generalized Born implicit solvent model.34 Replica exchange molecular dynamics35 (REMD) simulations were performed to evaluate the helix melting curves. Sixteen replicas were simulated at temperatures distributed exponentially over the range 260 to 700 K. To equilibrate the system, the polymer peptide conjugates were initially simulated for 10 ps at each temperature, followed by 15 ns of simulation with exchanges between neighboring temperatures following a Metropolis acceptance criterion.28 Subsequent to equilibration, an additional 50 ns of REMD simulation was performed to evaluate thermodynamic and structural averages. A time step of 2 fs was used to integrate the equations of motion. The SHAKE algorithm36 was used to constrain bond lengths between the heavy atoms and hydrogens. Andersen’s thermostat37 with a coupling constant of 0.1 ps was used to maintain the temperature of each replica. Postsimulation analysis of the average

Figure 1. Helix melting curves of 1CW and its conjugates with poly(ethylene glycol). The symbols defined in the Figure legend indicate results for 1CW, 1CW-20EO, and 1CW 40EO. The error bars denote one standard deviation.

helical content of the peptides was evaluated based on intrachain hydrogen-bonding using the DSSP method.38 To begin, we simulated Xu’s peptide designated 1CW (AcEVEALEKKVAALESKVQALEKKVEALEHGWDGR-CONH2), a 33 amino acid sequence.6 For the polymer peptide conjugates, the serine at position 14 (bolded in the sequence above) was mutated to cysteine to serve as the polymer functionalization site. The PEG chains were end-capped with maleimide, which reacts with cysteine to form a carbon sulfur bond that anchors the polymer to the peptide. The peptide was conjugated with PEG chains 20 and 40 ethylene oxide units long, corresponding to molecular weights of 880 and 1760 Da, respectively. Below we denote the unconjugated and conjugated peptides as 1CW, 1CW-20EO, and 1CW 40EO, respectively, indicating the number of polymer monomers. In addition to 1CW, we also simulated four alternative peptides: 1CW with the charges on the lysine side chains neutralized (denoted 1CW0), a 33 amino acid polyalanine peptide (Ac-A33-CONH2, denoted A33), a mutant of A33 with lysines placed at the same sequence positions as in 1CW (AcA6K2A6KA5K2A11-CONH2, denoted A2K), and a mutant of A33 with arginines placed at the same sequence positions as the lysines in 1CW (Ac-A6R2A6RA5R2A11-CONH2, denoted A2R). As for 1CW, the fourteenth position of A33, A2K, and A2R were mutated to cysteine for PEG conjugation. Polymer conjugates of these alanine-based peptides are indicated using the -xEO notation for 1CW (e.g., A33 40EO denotes A33 conjugated with a 40-mer PEG chain). The sequence naming nomenclature used in the Article is defined in Table 1 for the reader’s convenience. The implicit treatment of water given by the generalized Born model34 neglects specific solvent-mediated interactions between water and the protein to gain computational speed. Helix melting curves, however, are less cooperative and can be drawn out to higher temperatures using the generalized Born model for water.39 To validate the conclusions drawn from our simulations of 1CW using the generalized Born implicit solvent model, we have performed preliminary REMD simulations in an explicit representation of water using the TIP3P model.40 These results are reported in the Supporting Information supplement. We note, however, that simulated helix41 and hairpin42 melting curves 2730

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Figure 2. Fractional helicities for individual amino acids at 296 K for 1CW and 1CW 40EO along the peptide backbone. The symbols defined in the Figure legend indicate results for 1CW and 1CW 40EO. The error bars denote one standard deviation. The pink bars highlight the lysine positions. The serine (S14) mutated to a cysteine to attach the PEG chain is highlighted by the red box. Stretches 1 and 2 indicate those sequence segments most stabilized by PEG.

in explicit water can be drawn out to higher temperatures than experiment as well as a result of deficiencies in both the water and protein force fields. To assess the impact of PEG hydridization on peptide helix stabilization, then we supplement our implicit solvent simulations by comparison with an explicit treatment of water to determine if the stabilization mechanism is affected by the solvent model used.

’ RESULTS AND DISCUSSION The helix melting curves as determined from simulations of 1CW and its polymer conjugates are shown in Figure 1. These melting curves show that PEG conjugation progressively increases the peptide helicity with increasing polymer chain length over the entire temperature range. At 296 K, the simulated helicity increases from 41% for 1CW to 54% for 1CW 40EO, in reasonable quantitative agreement with the experimentally observed infinite dilution helicites of 44 and 60% for 1CW and 1CW conjugated with a 2000 Da PEG chain (∼45 ethylene oxide units in length), respectively, at 298 K and pH 8.6 In difference to the peptide backbone, the PEG chain is amorphous, adopting a panoply of conformations. (See the simulation snapshot in the left-hand inset of Figure 1.) The simulated helix melting curves are stretched out to higher temperatures than experiment, which is potentially attributable to the implicit treatment of the solvent.39 Simulations of 1CW and 1CW 40EO in explicit water show a similar postconjugation helical enhancement. (See Figure S1 of the Supporting Information). Given the agreement between our simulations and experiment, the question follows: How does the disordered PEG chain stabilize the helical conformation of peptide 1CW? The helical enhancement of 1CW by the 40-mer PEG chain is not uniform along the peptide backbone but is largely confined to two stretches from the valine at position 2 to the lysine at position 8 and the leucine at position 12 to the alanine at position 18 (Figure 2). Examining the amino acid content of these two enhanced stretches, we observe a mix of hydrophobic (alanine, leucine, and valine), polar (glutamine), anionic (glutamate), and

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Figure 3. Amino acid/poly(ethylene glycol) oxygen distance probability distribution function at 296 K for 1CW 40EO. Part a compares results for representative hydrophobic (V2), polar (Q17), anionic (E3), and cationic (K7) amino acids, each of which sit within either stretch 1 or 2 (Figure 2). Part b compares results for all of the lysines of 1CW (K7, K8, K15, K21, and K22). Figure symbols are defined in the Figure legend. The probability distribution function, P(r), describes the probability of observing a PEG oxygen a distance, r, away from the center-ofmass of a given amino acid side chain.

cationic (lysine) amino acids. The strongest potential interaction between the PEG chain and these amino acids is between lysine’s cationic amine and the electronegative oxygen of PEG. This interaction can be directly observed in the amino acid side-chain center-of-mass/PEG oxygen distance probability distribution functions (pdfs). Representative hydrophobic, polar, anionic, and cationic amino acid pdfs from the two enhanced helicity stretches are reported in Figure 3a. Whereas the pdfs for valine, glutamine, and glutamate exhibit broad, distant peaks at separations of ∼15 Å, a sharp, distinctive peak is observed for lysine 7 at ∼4 Å indicating intimate contact with the PEG oxygens. A similar contact peak is observed for all lysines down the sequence length (Figure 3b). This peak is strongest for those lysines within the two enhanced helicity stretches (K7, K8, and K15) and weaker for those outside those regions (K21 and K22). The lysines at positions 21 and 22 are located at the bend in the helix structure where the helix effective performs a u-turn, as observed in simulation snapshots. (See, for example, the left-hand inset of Figures 1 and 4.) This turn is also observed for the unmodified 1CW sequence, suggesting that this local conformation is robust and unperturbed by PEG. From simulations performed in explicit water at 293 K, the lysine pdfs exhibit a similarly strong PEG contact interaction peak, suggesting that this feature is not an artifact of the implicit generalized Born solvent model’s treatment of electrostatic interactions. (See Figure S2 of the Supporting Information.) In addition, the u-turn near lysines 21 and 22 is also observed in our simulations in explicit water. (See Figure S3 of the Supporting Information). 2731

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Figure 4. Molecular simulation snapshots of 1CW 40EO and 1CW0 40EO at 296 K. The peptide backbone and PEG chain are indicated by the cyan ribbon and gray licorice, respectively. (a) Positions of the lysine residues relative to the PEG side chain of 1CW 40EO indicated by the van der Waals surface. The lysine carbons are highlighted in green, and the protonated lysine nitrogens that interact most strongly with PEG are highlighted in blue. (b) Positions of the hydrophobic leucine and valine residues of 1CW 40EO that drive helix aggregation relative to the PEG side chain. The leucine and valine residues are indicated by the yellow and red van der Waals surfaces, respectively. (c) Positions of the lysine residues relative to the PEG side chain of 1CW0 40EO indicated by the green van der Waals surface. The lysine carbons are highlighted in green and the neutralized lysine nitrogens are highlighted in blue.

The intimate contact between the peptide lysines and PEG can be directly visualized from instantaneous simulation configurations. As can be seen in Figure 4a, the PEG chain is coiled around four of the lysines (K7, K8, K15, and K21) in this snapshot with the protonated amines in direct contact with the polymer backbone, enjoying attractive interactions with the PEG oxygens. These contacts appear similar to the amorphous structures between lithium cations and PEG oligomers.43 Only one of the lysines is not in contact with the polymer (K22). The stronger pdf peak for lysine 22 relative to lysine 21 (Figure 3b) indicates that PEG and the lysine are periodically able to interact as the polymer explores conformational space. Snapshots from our simulations in explicit water show similar lysine contact patterns with PEG. (See Figure S3a of the Supporting Information.) The strong interactions between the peptide lysines and PEG largely confine the polymer to the polar side of the amiphipathic helix (Figure 4b). The leucine and valine side chains that drive helix association are segregated to the hydrophobic side of the helix away from polymer. We may then anticipate that because PEG segregates toward the polar-side of the helix, tertiary helical bundle formation through hydrophobic interactions will be largely unaffected by the polymer. This conclusion is borne out from experimental analytical ultracentrifugation assays of 1CW and 1CW polymer conjugate self-assembly.6 On the basis of the strong interactions observed between PEG and the lysines, we propose the PEG oxygens act as an alternative interaction site for lysine rather than the carbonyl oxygens along the peptide backbone. The lysine interactions with PEG thereby allow enhanced carbonyl oxygen hydrogen-bonding with the amide hydrogens along the peptide backbone to stabilize the helix. To test this hypothesis, we have performed simulations in which the positive charge on the quaternary amines for all lysines of 1CW have been neutralized and reduced to zero (1CW0). The helix melting curves for 1CW0, 1CW0 20EO, and 1CW0 40EO are reported in Figure 5. To begin, the helicity of 1CW0 is significantly greater than that of 1CW (Figure 1), indicating the lysine charge does disrupt peptide hydrogen-bonding. More importantly, the melting curves of the 1CW0 polymer conjugates are practically identical with that of 1CW0. It is observed from molecular snapshots of 1CW0 40EO that the

Figure 5. Effect of poly(ethylene glycol) conjugation on helix melting curve of 1CW with the charge of each lysine reduced from +1 to 0. The symbols defined in the Figure legend indicate results for 1CW0, 1CW0 20EO, and 1CW0 40EO. The error bars denote one standard deviation.

intimate contact between the lysine side chains and PEG is lost after lysine neutralization (Figure 4c), which is further confirmed by the loss of the lysine/PEG oxygen contact peak in the pdfs (results not shown). To test further the PEG/lysine interaction mechanism, we consider PEG conjugation with two additional peptide sequences: polyalanine with the same sequence length as 1CW (A33) and a lysine mutant of A33 with lysines placed at the same positions as 1CW (A2K). The helix melting curves of A33 and A33 40EO are essentially indistinguishable (Figure 6a). Without any cationic groups to interact with, the polymer plays little role in the conformation of A33, similar to 1CW0 (Figure 5). Incorporation of lysines into the A33 sequence reintroduces PEG stabilization, as observed in the melting curves for A2K and A2K 40EO (Figure 6b). Specifically, PEG functionalization raises the helicity of A2K 40EO relative to A2K by ∼10% at 296 K, comparable to the stabilization observed for 1CW 40EO relative to 1CW (Figure 1). Moreover, the melting curve of A2K is reduced relative to A33, similar to the helical disruption observed 2732

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Figure 6. Effect of poly(ethylene glycol) conjugation on helix melting for polyalanine and polyalanine mutants. Parts a c report results for A33, A2K, and A2R, respectively. The symbols defined in the Figure legend indicate results for the peptides and their conjugates with the 40-mer PEG chain. The error bars denote one standard deviation.

for 1CW relative to 1CW0 when the lysine charges are turned on and consistent with the lower helical propensity of lysine relative to alanine observed experimentally.44 The helix stabilization moderated by PEG is not limited to sequences incorporating lysine. Similar to lysine, arginine is cationic and connected to the peptide backbone through a flexible side chain. To test the potential for PEG to enhance peptide helicity through arginine/PEG interactions, we mutated the lysines of A2K to arginine to form the sequence A2R. The helix melting curves of A2R and A2R 40EO obtained from simulation are shown in Figure 6c. As hypothesized, PEG stabilizes the helical conformation of A2R, suggesting the same interactions are at play as in the case of the lysine containing sequences examined above. Interestingly, the extent of stabilization of A2R by PEG is comparatively greater than that for A2K. Integrating the pdfs (not reported here) between the PEG oxygens and the cationic side chains of A2K and A2R out to 6 Å, corresponding to the first minimum in the distribution (e.g., Figure 3b), we find 7.1 and 8.2 ether oxygens in the primary interaction shells of lysine and arginine, respectively. We conclude that the polymer interacts slightly more strongly with the arginine side chains than with lysine, providing greater helical stabilization for A2R over A2K.

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’ CONCLUSIONS To understand the role of PEG in stabilizing the helical conformation of polymer peptide conjugates, we performed extensive molecular simulations of peptide 1CW and a series of polyalanine-based sequences. Our simulations found that the oxygens of the conjugated PEG chain favorably interact with the cationic lysine side chains in a manner reminiscent of ammonium cation binding to PEG45 48 and crown ethers.49,50 This binding proceeds through electrostatic and hydrogen-bonding interactions between the ammonium and ether oxygens, and can be reduced by increasing the degree of substitution of the ammonium hydrogens. The PEG oxygens thereby provide an alternative interaction site for lysine to the peptide backbone carbonyl oxygens. As a result, the carbonyl oxygens neighboring a lysine more readily enjoy hydrogen-bonding with the peptide backbone amide group, thereby stabilizing the R-helix. Similar to 1CW, we believe that preferential PEG interactions with lysine also play a role in the helix stabilization observed by Klok and coworkers7 for their polymer peptide conjugates at low peptide concentrations. In addition, we found that the multiple interactions of PEG with the 1CW lysines largely confine the polymer to the polar side of the helix. This segregation of the PEG away from the hydrophobic valine and leucine side chains is consistent with the experimental observation that polymer conjugation does not disrupt the formation of a tertiary 1CW helical bundle. On the basis of our results for polyalanine mutants, we believe that helix stabilization by PEG is not limited to sequences containing lysine but also applies to sequences containing arginine, which is chemically similar to lysine. Indeed, experimentally, the guanidinium cation forms complexes with crown ethers, and arginine displays a greater affinity for crown ether complexation than lysine,50 52 consistent with our observation that the sequence A2R is stabilized to a greater extent than A2K after PEG hybridization. Previous simulation and experimental studies of glycosylated proteins have found that the pendant glycans can stabilize the protein fold by modification of local sequence conformational preferences that destabilize the unfolded state.53,54 Our results for the neutralized 1CW0 and polyalanine sequences find no stabilization after conjugation with PEG. This suggests steric modification of the peptide conformational preferences by the polymer does not play a role in stabilizing the helices examined here. Whereas we have identified a potentially important mechanism in the stabilization of lysine (arginine)-rich helical peptides, open questions remain, including: What is the dependence of the peptide helicity on the position of PEG conjugation?6,26 How does PEG interact with lysine in the assembled trimer bundle where lysine and glutamate residues on opposing peptide chains can also form salt bridges?55 What is the role of the synthetic polymer’s chemistry (e.g., hydrophilic versus hydrophobic) on helix stabilization/destabilization?26 ’ ASSOCIATED CONTENT

bS

Supporting Information. Results from our simulations of peptide 1CW in explicit TIP3P water including helix melting curves, PEG-side chain probability distribution functions, and simulation snapshots are presented in this supplement. This material is available free of charge via the Internet at http://pubs.acs.org.

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’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected].

’ ACKNOWLEDGMENT We gratefully acknowledge support from the National Science Foundation (EPS-0701491) for financial support. We acknowledge computational support from the Louisiana Optical Network Initiative (www.loni.org) where simulations were performed on Poseidon, a 128 node Dell Cluster housed at the University of New Orleans. ’ REFERENCES (1) Drahl, C. Chem. Eng. News 2008, 86, 18–23. (2) Wilson, A. J. Chem. Soc. Rev. 2009, 38, 3289–3300. (3) Dathe, M.; Wieprecht, T. Biochim. Biophys. Acta 1999, 1462 71–87. (4) Nowak, A. P.; Breedveld, V.; Pakstis, L.; Ozbas, B.; Pine, D. J.; Pochan, D.; Deming, T. J. Nature 2002, 417, 424–428. (5) Petka, W. A.; Harden, J. L.; McGrath, K. P.; Wirtz, D.; Tirrell, D. A. Science 1998, 281, 389–392. (6) Shu, J. S.; Tan, C.; DeGrado, W. F.; Xu, T. Biomacromolecules 2008, 9, 2111–2117. (7) Vandermeulen, G. W. M.; Tziatzios, C.; Klok, H. A. Macromolecules 2003, 36, 4107–4114. (8) Vandermeulen, G. W. M.; Tziatzios, C.; Duncan, R.; Klok, H. A. Macromolecules 2005, 38, 761–769. (9) B€orner, H. G.; Schlaad, H. Soft Matter 2007, 3, 394–408. (10) Lifson, S.; Roig, A. J. Chem. Phys. 1961, 34, 1963–1974. (11) Walensky, L. D.; Kung, A. L.; Escher, I.; Malia, T. J.; Barbuto, S.; Wright, R. D.; Wagner, G.; Verdine, G. L.; Korsmeyer, S. J. Science 2004, 305, 1466–1470. (12) Zhang, H.; Zhao, Q.; Bhattacharya, S.; Waheed, A. A.; Tong, X.; Hong, A.; Heck, S.; Curreli, F.; Goger, M.; Cowburn, D.; Freed, E. O.; Debnath, A. K. J. Mol. Biol. 2008, 378, 565–580. (13) Schafmeister, C. E.; Po, J.; Verdine, G. L. J. Am. Chem. Soc. 2000, 122, 5891–5892. (14) Andrews, M. J. I.; Tabor, A. B. Tetrahedron 1999, 55 11711–11743. (15) Osapay, G.; Taylor, J. W. J. Am. Chem. Soc. 1990, 112 6046–6051. (16) Houston, M. E.; Campbell, A. P.; Lix, B.; Kay, C. M.; Sykes, B. D.; Hodges, R. S. Biochemistry 1996, 35, 10041–10050. (17) Taylor, J. W. Biopolymers 2002, 66, 49–75. (18) Jackson, D. Y.; King, D. S.; Chmielewski, J.; Singh, S.; Schultz, P. G. J. Am. Chem. Soc. 1991, 113, 9391–9392. (19) Pellegrini, M.; Royo, M.; Chorev, M.; Mierke, D. F. J. Peptide Res. 1997, 49, 404–414. (20) Blackwell, H. E.; Grubbs, R. H. Angew. Chem., Int. Ed. 1998, 37, 3281–3283. (21) Marquesee, S.; Baldwin, R. L. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 8898–8902. (22) Marqusee, S.; Robbins, V. H.; Baldwin, R. L. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 5286–5290. (23) Mayne, L.; Englander, S. W.; Qiu, R.; Yang, J.; Gong, Y.; Spek, E. J.; Kallenbach, N. R. J. Am. Chem. Soc. 1998, 120, 10643–10645. (24) Ruan, F.; Chen, Y.; Hopkins, P. B. J. Am. Chem. Soc. 1990, 112, 9403–9404. (25) Ma, M. T.; Hong, H. N.; Scully, C. C. G.; Appleton, T. G.; Fairlie, D. P. J. Am. Chem. Soc. 2009, 131, 4505–4512. (26) Shu, J. S.; Huang, Y. J.; Tan, C.; Presley, A. D.; Chang, J.; Xu, T. Biomacromolecules 2010, 11, 1443–1452. (27) Stanley, C. B.; Strey, H. H. Biophys. J. 2008, 94, 4427–4434.

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