Mechanical Reinforcement of Proteins with Polymer Conjugation

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Mechanical Reinforcement of Proteins with Polymer Conjugation Elizabeth P. DeBenedictis, Elham Hamed, and Sinan Keten ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.5b06917 • Publication Date (Web): 19 Dec 2015 Downloaded from http://pubs.acs.org on December 21, 2015

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Mechanical Reinforcement of Proteins with Polymer Conjugation Elizabeth P. DeBenedictis†, Elham Hamed†, and Sinan Keten* Department of Civil and Environmental Engineering and Mechanical Engineering, Northwestern University, Evanston, Illinois 60208, United States *

Corresponding author: [email protected]

†

Elizabeth DeBenedictis and Elham Hamed equally contributed to this work

ABSTRACT Conjugating poly ethylene glycol (PEG) to peptides, also known as PEGylation, is proven to increase the thermodynamical stability of peptides, and has been successfully applied to prolong the lifetime of peptide-based vaccines and therapeutic agents. While it is known that protein structure and function can be altered by mechanical stress, whether PEGylation can reinforce proteins against mechanical unfolding remains to be ascertained. Here, we illustrate that PEGylation prolongs the lifetime of α-helices subject to constant stress. PEGylation is found to increase the unfolding time through two mechanisms. We see that: (1) the unfolding rate of a helical segment is decreased through prolonged plateau regimes where the peptide helical content remains constant, and (2) the proportion of refolding to unfolding is increased, primarily by shielding water molecules from replacing forcibly exposed backbone hydrogen bonds near the conjugation site. Our findings demonstrate the feasibility of improving peptide mechanical stability with polymer conjugation. This provides a basis for future studies on optimizing conjugation location and chemistry to build custom biomolecules with unforeseen mechanical functions and stability. KEYWORDS: Coiled coils, Polymer conjugation, Atomistic simulation, Mechanical stability, Drug delivery

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Many proteins are subject to mechanical stress in vivo, have load-bearing and loadsensing functions, and their structure is sensitive to external forces. The force-response of α-helical protein domains and coiled coils is of particular significance given their mechanical role in diverse biological systems. Serving as the safety belt of cells,1 vimentin provides resistance to large deformations of cells2, 3 and absorbs large amounts of energy through unfolding and releasing sacrificial hidden length.4 Protein unfolding also helps maintain both extensibility and permeability in fibrin, which is relevant for blood clotting, wound healing, and thrombosis.5 Molecular Dynamics (MD) has proven useful in observing mechanical unfolding mechanisms of proteins, and how their stability depends on factors such as structure and topology, amino acid distribution, direction and rate of applied forces, and orientation and number of backbone hydrogen bonds.6,

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Macroscale mechanical properties of peptide biomaterials are chiefly governed by these secondary structures as shown previously for explaining the superior mechanical properties of spider silk.7 A strategy for further improving stability of biomaterials may be to shield the load-bearing H-bonds from surrounding water molecules. It was recently shown that exposing the force-resisting domains to solvent by selectively deleting peptide segments jeopardizes the stability of small proteins.8 Yet, synthetic strategies focusing on solvent shielding through addition of side-conjugated materials to improve mechanical stability of proteins have not been explored.

Improving protein stability is highly relevant to tissue engineering, immunology, biosensing, biomaterials, and drug delivery, and may be achieved by attaching polymer chains to the side-groups of proteins. Conjugation of α-helices with polymer chains, particularly poly ethylene glycol (PEG), or so-called PEGylation, has emerged as a highly effective strategy to enhance peptide stability under elevated temperatures or pressures.9-15 PEGylation was also shown to lengthen peptide degradation time in drug delivery applications,16, 17 enhance thermal stability of coiled coils without harming the structural configuration,12,

18-20

and decrease immune response in therapeutic

applications.21 It is worth mentioning that polymer conjugation does not necessarily always stabilize proteins. In fact, the stabilization effect of conjugated polymers on

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proteins depends upon several factors, including the size and sequence of protein, the type, molecular weight, conjugation site, and grafting density of polymers, and the environmental and boundary conditions. Although numerous studies have shown that polymer-conjugated proteins have a higher stability than native proteins, cases of destabilization upon polymer conjugation have also been reported.22-26 In majority of the latter studies, the destabilization effect is due entropic repulsion arising from excluded volume interactions of polymer chains. This may occur, for example, due to confinement of a large number of polymer monomers near the peptide surface, or due to steric interactions of polymer chains with the surface to which the polymer-conjugated protein is tethered.

Understanding and improving upon the stabilization effects induced by PEGylation requires knowledge of polymer chain conformations on the helix surface. MD has shown the stabilization effect of PEG on α-helices is due to (1) formation of a protecting polymer shell around the peptide that protects the helix backbone hydrogen bonds from solvent attacks and (2) interactions between peptide side chain groups and the conjugated polymer chains.19, 20 MD simulations have also shown similar stabilization mechanisms upon PEGylation of other proteins.25, 27-29

While it is now established that polymer conjugation can improve the ability of small proteins to resist thermal and chemical denaturation, the impact of PEGylation on mechanical stability of peptides has not been fully explored. This investigation into protein materials is of particular significance as the temperature-induced and stretched unfolding pathways of α-helical domains have been shown to be different: helices undergo fraying and fragmentation and unfold much earlier at high temperatures compared to forced stretching, while the unfolding process is more cooperative under the applied forces.30 Uncovering the mechanical response of polymer-conjugated proteins is a necessary first step in creating engineered protein materials with tunable resistance to stress.

To address this, here we study how α-helices unfold under constant mechanical stress and

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determine if PEGylation is an effective strategy for reinforcing small helical proteins against mechanical insults. In order to assess the impact of PEGylation on the helix unfolding process, constant tensile forces are applied to both peptide termini to accelerate unfolding, both in the absence and presence of an attached PEG chain (Figure 1). Using this model, we quantify the correlation between the force magnitude and helix unfolding time for native and PEGylated peptides. Additionally, we investigate the reinforcement mechanisms the PEG chain imparts during the unfolding process.

RESULTS AND DISCUSSION Here we present results for a model system of a small α−helical peptide subject to a constant tensile force applied at its termini. The unfolding time (τunfolding), defined here as the time elapsed to reach a completely unfolded state under a constant applied force (F), is taken as the metric for mechanical stability. In this study, we investigate how PEGylation of a peptide influences its τunfolding and therefore stability, and the molecular mechanisms through which this occurs. The values of τunfolding for all simulation trials at all force levels are listed in Table S.1 of the Supporting Information for the native peptide and PEGylated peptide. The average unfolding times measured from multiple trials are presented in Figure 2, showing that the helix τunfolding is greater for PEGylated peptides compared to native helices for all forces studied here, ranging over an order of magnitude from 52 pN to 695 pN. This enhanced unfolding resistance against mechanical forces is observed over all forces and trials, where the most significant increase in helix unfolding time upon conjugation is observed for the lowest force. The unfolding time is related to the off-rate, , through =1/τunfolding. The off-rate in mechanical unfolding experiments of proteins can be obtained according to the classical Bell’s model:31 =

∆

,

(1)

where is a natural vibration frequency, and and denote, respectively, the energy barrier and the distance between the equilibrated and transition state. Combining these, the relationship between applied force and unfolding time follows a logarithmic relation of the form !

'

= − " ∆# $ % + " ∆# − $.

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(2)

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Numerous studies have noted deviations from a single energy barrier model, where the slope of the curve may vary depending on the magnitude of force applied.32 This trend is also observed in our system for both native and PEGylated helices. Three regimes, marked by different slopes, can be distinguished for force-induced unfolding response of the α-helix: small, intermediate, and large force regimes (Figure 2). The results in each regime are plotted with best-fitted lines, following Bell’s model. The estimations for energy barriers and distances to the transition state for each force regime are provided in Table 1, to qualitatively describe the differences in the stability of two systems. The results show that the PEG stabilization effect is more pronounced for the small force regime than the other two regimes. This is evident from the energy barrier and transition distance differences in different regimes (Table 1) as well as the increase in unfolding time upon PEGylation. For example, the average τunfolding increases by 84% upon PEG conjugation for the smallest force tested (F=52 pN), but the corresponding increase is ~14% for the largest force applied (F=695 pN). This data suggests that the unfolding time increase at large forces can be considered as a lower limit of the stabilization effect, and low force unfolding, thermal denaturing, or experiments under equilibrium conditions should exhibit a greater reinforcement effect if the trend of increasing slope holds for lower forces than tested here. Diminishing reinforcement effects with increasing force are expected, as the unfolding time scale becomes comparable to the time required for the PEG and surrounding water to adopt new configurations in response to protein unfolding. This relationship between the PEG-induced reinforcement effect and applied force calls for a deeper understanding of the mechanisms of PEG-protein interactions, which will be discussed next.

To clarify how PEGylation slows the unfolding process, we first investigate how the presence of PEG changes the lifetime of helix backbone H-bonds during the unfolding process. The secondary structure of an α-helix is stabilized primarily by backbone Hbonds between carbonyl (C=O) group of the amino acid i and amino (N-H) group of the amino acid i+4, known as i→i+4 H-bonds, which break as the helix unfolds. We measure the occupancy of these hydrogen bonds to indirectly observe how unfolding progresses throughout the helix length during the course of the trial. The occupancy of these

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backbone H-bonds during the helix unfolding process can be seen in Figure 3a for the smallest tested force F=52 pN. Here, occupancy is quantified as the fraction of the total time a specific H-bond remains intact and thus it represents the time-averaged occupancy of H-bonds, rather than their instantaneous occupancy. The conjugated PEG chain clearly increases the helix backbone H-bond occupancy, particularly for the residues closer to the PEG conjugation site (residue 14). For the majority of trials, the unfolding starts at the helix C terminus (residue 29), due to the unhelical configuration of residues 27, 28, and 29 in the native structure of peptide. Then, the helix N terminus (residue 1) unfolds, and finally the unfolding propagates toward the helix middle section. This unfolding path can also be recognized in Figure 3a, where the occupancy of H-bonds is generally lower for the residues located closer to helix C terminus compared to N terminus, since the Cterminal region of the peptide is less helical to begin with. The results of Figure 3a show that the unfolding process at the two ends is minimally affected by the presence of conjugated PEG chain (Figure 3a). However, the progression of unfolding slows down more for PEGylated helices than native helices nearing the helix core, where the PEG chain is conjugated. Among all residues, the largest increase in H-bond occupancy upon PEG conjugation occurs for center residues 14, 15, and 16, where the time-averaged Hbond occupancy of PEGylated helices is roughly twice that of native helices.

These results clearly illustrate that unfolding progresses with backbone H-bonds breaking, which is followed by the exposed polar groups forming H-bonds with the surrounding water molecules. Unfolding of helix segments depends on the probability of unfolding and refolding events that are governed by the relative free energies of the two states. We hypothesize that the relative stability of the unfolded state should depend on the accessibility of the backbone to surrounding water, where water-backbone H-bonds would lower the free energy of this state. The central question is whether PEGylation changes the dynamics of this transition by modulating the solvent accessible surface area. To answer this, we measured the time elapsed between the irreversible breakage of helixbackbone H-bonds and formation of new backbone-water H-bonds for the central residues (12-16) located at the helix core (Figure 3b). For all the residues analyzed, PEGylation increases the time required for water molecules to access the backbone and

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form H-bonds. Thus, we conclude that the excluded volume of the PEG chain near the surface of the peptide effectively blocks water molecules. This reduces the probability of permanently replacing the backbone H-bonds, and thus prolongs the onset of subsequent unfolding events. The process of replacement of helix backbone H-bonds with backbonewater H-bonds is depicted in Figure 3c.

The analyses so far indicate that PEG prolongs the lifetime of backbone H-bonds and thus the helical peptide lifetime. Next, we aim to determine if this effect arises from PEGylation impacting the sequence of unfolding events the peptide experiences, or by simply slowing the progression of unfolding events. Apart from continuous unfolding events where individual H-bonds sequentially break, we see that unfolding may occur in bursts where several H-bonds may break concertedly, as previously observed.33 Here we define these unfolding bursts when the helical content suddenly decreases by more than 3 residues. Likewise, refolding of unfolded segments is also possible, marked here by increases in helical content larger than a single residue, and may also exhibit cooperative transitions involving multiple residues. Additionally, plateau regions where the unfolding process seemingly comes to a halt over prolonged periods can also be observed. Plateau periods are classified as periods of time without significant refolding or unfolding, with no more than 1 residue change in helicity during a time period of 0.8 ns for the lowest (52 pN) force level. Examples of refolding and unfolding burst events are illustrated in Figure 4. Although examples of these events can be found over the range of forces, this analysis focuses on the lowest force, F=52 pN. Here, the PEGylated peptide spends 58% of the unfolding time in a plateau region on average (standard deviation s = 8.4%), whereas the native peptide spends 45.7% of the time in a plateau (s = 10.5%), suggesting the likelihood of experiencing a plateau state is higher for PEGylated peptides. As the refolding and unfolding burst events are largely coupled – the helix must unfold before it can refold – we quantify the proportion of backward motion to rapid forward motion, R, as the total helicity gained from refolding over the total helicity lost through unfolding bursts. R values of 3.02 (s = 1.33) and 2.11 (s = 0.55) are obtained for PEGylated peptides and native peptides, respectively, indicating PEGylation increases the amount of refolding events relative to unfolding bursts. These results, altogether, show that the

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stability resulting from PEGylation works through two mechanisms: (1) through increased resistance to unfolding by increasing the time the peptide spends in plateau periods, and (2) through an increased proportion of refolding events to unfolding bursts. This indicates that the presence of a PEG side chain does not merely slow the progression of unfolding events, but influences the sequence of events that occur and the proportion of these events relative to one another.

Observing now, the impact of PEGylation on unfolding events across all three force regimes identified in Figure 2, we see the percentage of the total unfolding time the peptide spends in an unfolding burst is consistently higher for native peptides than PEGylated peptides. Plateau events are only seen occurring in forces below 209 pN, and for all low and intermediate forces, the PEGylated peptide spends a larger percentage of the total unfolding time in a plateau region. Refolding events are dependent on having an unfolded segment to refold as they most commonly directly follow unfolding bursts, unlike unfolding bursts and plateaus, which can occur at any time. As such, there is no consistent trend for whether the PEGylated or native peptide experiences a larger portion of the unfolding time in a refolding burst. This data suggests that in the large-force regime, PEG stabilizes the peptide through prevention of unfolding bursts, but as the force decreases, more refolding events and increased plateaus contribute more appreciably to the PEG stabilization effects.

Now that the events dictating mechanical reinforcement by PEGylation are revealed, we investigate how the dynamic conformations of PEG near the helix contribute to stabilization. The conformation of the PEG chain on the helix surface depends on both PEG interactions with the surface and the solvent.34 Because the shapes (mushroom, pancake, etc.) predicted by common polymer models are within the error for current macromolecular sizing techniques, such as small-angle neutron scattering and smallangle X-ray scattering, the distribution of conformations is difficult to determine experimentally. Additionally, dynamics of the chain require an extremely high spatiotemporal resolution that is currently not available. This further underlines the significance of MD as a means to relate PEG conformations to stabilization mechanisms.

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To better identify how PEG conformations impact peptide stability, here, we present heat maps showing the location of the PEG chain over the course of 3 trials of force 52 pN (Figures 5b). We note that the unfolding times in these three trials show broad variations even though the applied force is identical - due to the relatively short timescale of simulations compared to experiments. Here, we make use of the random configurations sampled in each trial to assess whether the differences in the unfolding times between the trials can be linked to PEG conformations. Parameters quantifying potential shielding effects of the PEG chain are of particular interest, such as proximity and the degree to which it surrounds the helix surface. Heat maps in Figure 5b characterize the conformation of PEG chain projected onto the XY plane at each time frame, where the Zaxis represents the helix longitudinal axis. For each trial, the heat map is normalized by the trial length to allow a quantitative comparison, where the normalized densities 0 and 1 correspond to areas least and most frequently populated by the PEG chain, respectively. A number of visual distinctions can be made between the three trials: trials 1 and 3 have a more complete ring of high intensity better centered around the peptide, while trial 2 has an area near the top surface with lower intensity, breaking the ring. These density maps imply that the peptides with the largest unfolding time also experience a more idealized version of the pancake conformation model, while trials with less proximal surface coverage exhibit weaker stabilization effects. To better quantify the position of the PEG chain, we define two parameters: average radial distance “ravg” and coverage angle “θiqr” (Figure 5a). The radial distance measures, for each time step, the average distance of each of the PEG monomers from the peptide center of mass in the XY-plane (Figure 5a). The coverage angle is calculated by projecting the coordinates of the PEG chain onto the XY plane, and for each time step, the amount of the peptide surface that is surrounded by the PEG chain is calculated as an angle (Figure 5a), where 360 degrees indicates the PEG chain forms a full circle around the peptide. As this angle varies greatly over the course of the trial, the interquartile range is used to represent the middle 50th percentile of PEG coverage angles. Use of a coverage angle calculation per frame distinguishes between the PEG chain winding over a large surface of the peptide over the course of the trajectory versus winding over a large surface at any given time. Figure 5b shows that the longer unfolding times generally correspond to a smaller distance between each PEG monomer

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and the peptide center of mass, consistent with the observation of increased concentricity for longer trials. Additionally, at each time step, the coverage angles are generally higher for longer-lasting trials, corroborating the theory that an ideal pancake model of PEG chain wrapped onto the helix surface contributes to an increased stability of peptide.

Having seen that PEG occupancy relative to the peptide influences unfolding time, we aim to better understand where on the helix surface the PEG chain is likely to interact. The interaction between the PEG chain and varying peptide residues is quantified by inspecting the number of PEG monomers within a 5Å cutoff distance of the most surfaceexposed carbon in each residue during the unfolding process (Figure 5c). These are taken as the percentage of the total PEG monomers within the cutoff distance, or pair counts, to observe whether certain residues have more favorable interactions with the PEG chain than others. Figure 5c illustrates the percentage of PEG monomers within a 5Å cutoff near each particular residue. Because the PEG chain is conjugated at residue 14 (CYS), the seemingly high affinity to this residue is due to the constant presence of PEG. We note that hydrophobic residues make up 48% of the residues of the peptide, and on average experience 48% of the pair counts. Charged residues, however, only make up 17% of the residues in the peptide but make up 27% of the pair counts, whereas neutral residues account for 25% of the pair counts yet make up 35% of the peptide residues. We observe residues LYS and ALA have the strongest interactions with the PEG chain. The cationic LYS side chain attracts PEG oxygens, which provides a preferred site for PEG monomers on the helix surface19,

20

and contributes to PEG stabilization effects.

Comparing this with Figure 3b, we notice that residues 14 and 16 experience the smallest increase in time delay (in backbone Hbond formation), yet residues 13 and 16 see the smallest percentage of PEG interactions in this region of the helix. This may hint that adjacent residues affect PEG interaction, as residue 16 does not see as much shielding effects from nearby residues compared to residue 13. Local maxima in pair counts away from the conjugation site occur where the residue is flanked by hydrophobic or charged residues. The results indicate there is opportunity for optimizing sequence distribution in protein design to modulate the reinforcement effect arising from PEG affinity to particular residues.

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As it is shown that PEG proximity correlates to a longer unfolding time, we investigate the relationship between the PEG chain conformations around the peptide and specific unfolding events. We quantify the coverage angle and average distance to peptide center of mass for unfolding events separately (Figure 6). Although the absolute values of the PEG distance and angle vary between trials, the difference for various modes within each trial is consistent: Figure 6 shows that for the smallest force tested, F=52 pN, events that maintain or regain helicity occur when the PEG chain is proximal and surrounds the peptide. Here, the PEG chain is on average 1.3 A closer during refolding than unfolding bursts, and 2.0 A closer during plateaus than during unfolding bursts. During refolding events, the PEG chain covers an average of 22.6° more around the peptide compared to unfolding, and 28.7° more during plateau events compared to unfolding. It can be concluded that PEG chain proximity to the peptide and coverage of the peptide surface are critical in facilitating protein stabilization events, such as maintaining helicity during a plateau period or refolding the unfolded helix segments. The smaller difference between refolding and unfolding burst values follows that refolding typically occurs directly after an unfolding burst, which are both short events during which the PEG chain cannot travel a significant distance. Plateau events in this force range last 0.8–1 ns, during which the PEG chain can gradually move closer to the peptide. Because PEG conformation can be tied to specific unfolding events, the proportion of which dictates overall unfolding time, methods to control PEG conformation such as controlling sequence distribution, conjugation site, or molecular weight are promising avenues for improving protein stability.

The mechanical stabilization effect imparted on peptides through polymer conjugation is shown to become more significant as the force range decreases and thus the PEG chain has more time to move into a favorable conformation in the peptide vicinity. Despite statistical variation in low force trials, we exploit this to shed light on mechanistic principles of reinforcement, and how these are intertwined with PEG conformation. Knowing this, the probability of refolding can be increased by selecting appropriate residue sequences, tuning the molecular weight and conjugation site, among other

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methods. The relationship between PEG location and unfolding time is seen through the heat map analysis, where trials that require a longer time to unfold have a more proximal PEG chain and also exhibit more winding of the PEG chain around the peptide. Furthermore, it is shown that unfolding trials with a larger refolding to unfolding burst ratio have longer lifetimes, and that the PEG chain tends to be closer and covering a larger angle during refolding events. Together, these results indicate that control over the position and conformation of the PEG chain can have implications for the unfolding time and overall mechanical stability of peptides.

CONCLUSION By investigating how α-helical peptides side-conjugated with a PEG chain respond to mechanical stresses ranging over an order of magnitude, we found convincing evidence that small proteins can be mechanically reinforced against unfolding by polymer conjugation. This is confirmed by our results showing that the time required to fully unfold a PEGylated α-helix is consistently larger than for its native counterpart for constant tensile forces ranging over an order of magnitude.

We show that the unfolding of a protein can be viewed as a sequence of events, including unfolding bursts, refolding, and plateau periods. A high proportion of refolding events to unfolding bursts correlates to an extended lifetime of helices that is stochastically regulated by the proximity of the PEG chain to the protein surface and its surface coverage. Shielding of the water molecules by the nearby PEG monomers is found to promote refolding. With this understanding, efforts to design mechanically stable proteins can be directed to ensure the polymer maintains an optimum position in relation to the protein. Our analysis correlating peptide sequence to the proximity of the PEG chain to the peptide surface suggests that both hydrophobic and charged residues partake in creating highly affinity sites for the polymer. Sequence distribution and also the use of random block copolymers as a conjugation strategy may help enhance the effects observed in this study. Additionally, although our study focuses on the reinforcement effect of PEGylation on the mechanical response of α-helical peptides, similar trends are likely to be observed in proteins with other secondary structures, such as β-sheets.

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Conformational and thermodynamical stabilization of β-sheets via PEGylation has been reported before,27,

35, 36

and this offers a promising prospect for reinforcing the

mechanical response of PEGylated β-sheets under external loading. Such features can be readily studied by our computational framework in the future.

Our results unravel the mechanisms underlying the mechanical reinforcement of proteins upon polymer conjugation. The methodology used here can be extended to various peptide-polymer conjugates, and also to correlate conjugation location and reinforcement effects. Our study indicates that central locations that provide maximum surface coverage may be sufficient, but conjugation at multiple locations may conceivably help stabilize larger proteins in a variety of solvent environments. The finding that polymer conjugation can modulate the folding resistance of proteins against applied forces opens up possibilities for future studies on force resistance response of protein-based block copolymers, hydrogels and other de novo systems that will build upon our findings to design stable protein systems with tunable mechanical properties and tailored functionalities.

MATERIALS AND METHODS A single helical strand of a 3-helix coiled coil structure (PDBID “1coi” in the protein data bank37) was used as the model peptide in our study (Figure 1a). The structure and thermodynamical behavior of this helix bundle has been well characterized experimentally11,

12, 37

and computationally.38 A maleimide-capped PEG chain with 40

monomers (molecular weight of ~ 1763 Da) was covalently attached to the side of helix. We refer to this PEG chain as PEG40. Following ref. 12, residue serine at position 14 of the helix was mutated to cysteine to facilitate the PEG conjugation via formation of a carbon-sulfur bond. The schematic of helix−PEG conjugate is shown in Figure 1b. All-atomistic simulations were performed using NAMD.39 The helix-PEG conjugates were solvated in an explicit water solvent using the TIP3P model.40 Periodic boundary conditions were applied in the three dimensions, and an NPT ensemble with constant pressure of 1 atm and constant temperature of 300 K was used. Bonded interactions of

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peptide and PEG were modeled using the CHARMM force field.41 The standard LennardJones potential was used to model the long-range non-bonded interactions and the particle mesh Ewald technique was employed for electrostatics.

In order to assess the helix stability under mechanical loading, constant tensile forces were applied to both peptide termini in the direction of helix axis (Figure 1). Forces with different magnitudes, including 0.75 kcal/mol/Å, and 1, 2, 3, ..., 10 kcal/mol/Å (~ 52, 70, 139, 209, 278, 348, 417, 487, 556, 626, and 695 pN), were employed to investigate the correlation between helix unfolding behavior and the magnitude of applied force. For each force, both a native and PEGylated peptide were compared. Each helix was pulled until it reached complete unfolding and was repeated for 5 samples beginning with the same initial configuration but different random seeds for initial velocities. The only exceptions were (1) the cases under forces 209, 278, and 348 pN, which were repeated for 10 samples to increase the number of sampling points and thus the accuracy of data without a significant increase in the computational cost, and (2) the cases under the smallest force, 52 pN, which were run for 4 samples, due to the high computational cost of these long, all-atomistic simulations. Table S.1 of the Supporting Information lists the number of samples at each force level. Total simulation time for all forces and trials for both native and PEGylated helices was ~ 2.1 µs. Peptide helical content (helicity) was quantified at all times during the trajectory using the STRIDE algorithm implemented in VMD.42 Using the helicity-time data, helix unfolding time was measured as the time when peptide helical content reached zero and stayed zero for at least 10 ps.

Conflict of Interest: The authors declare no competing financial interest. Supporting Information Available: Additional Table S1 and its caption. This material is available free of charge via the Internet at http://pubs.acs.org.

Acknowledgements: The authors acknowledge funding from the Office of Naval Research (Grant No. N00014-13-1-0760). E.P.D. gratefully acknowledges support from the Ryan Fellowship and the Northwestern University International Institute for

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Nanotechnology. The authors thank Ertugrul Alemdar for his help in formatting the figures.

Author Contributions: S.K. conceived and designed the research with E.H and E.P.D. E.H. set up the model systems, and E.P.D. and E.H. performed the simulations and calculations. All authors analyzed the results and wrote the manuscript together.

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13. Top, A.; Zhong, S.; Yan, C.; Roberts, C. J.; Pochan, D. J.; Kiick, K. L. Controlling Assembly of Helical Polypeptides via PEGylation Strategies. Soft Matter 2011, 7, 9758-9766. 14. Vandermeulen, G. W. M.; Tziatzios, C.; Duncan, R.; Klok, H. A. PEG-Based Hybrid Block Copolymers Containing Alpha-Helical Coiled Coil Peptide Sequences: Control of Self-Assembly and Preliminary Biological Evaluation. Macromolecules 2005, 38, 761-769. 15. Vandermeulen, G. W. M.; Tziatzios, C.; Klok, H. A. Reversible Self-Organization of Poly(Ethylene Glycol)-Based Hybrid Block Copolymers Mediated by a De Novo Four-Stranded Alpha-Helical Coiled Coil Motif. Macromolecules 2003, 36, 41074114. 16. Harris, J. M.; Chess, R. B. Effect of Pegylation on Pharmaceuticals. Nat. Rev. Drug Discovery 2003, 2, 214-221. 17. Veronese, F. M.; Pasut, G. PEGylation, Successful Approach to Drug Delivery. Drug Discovery Today 2005, 10, 1451-1458. 18. Hamed, E.; Ma, D.; Keten, S. Effect of Polymer Conjugation Site on Stability and Self-Assembly of Coiled Coils. Bionanosci. 2015, 5, 140-149. 19. Hamed, E.; Xu, T.; Keten, S. Poly(Ethylene Glycol) Conjugation Stabilizes the Secondary Structure of α-Helices by Reducing Peptide Solvent Accessible Surface Area. Biomacromolecules 2013, 14, 4053-4060. 20. Jain, A.; Ashbaugh, H. S. Helix Stabilization of Poly(Ethylene Glycol)-Peptide Conjugates. Biomacromolecules 2011, 12, 2729-2734. 21. Hermeling, S.; Crommelin, D. J. A.; Schellekens, H.; Jiskoot, W. StructureImmunogenicity Relationships of Therapeutic Proteins. Pharm. Res. 2004, 21, 897903. 22. Carmichael, S. P.; Shell, M. S. Entropic (De)Stabilization of Surface-Bound Peptides Conjugated with Polymers. J. Chem. Phys. 2015, 143, 243103. 23. Couet, J.; Biesalski, M. Polymer-Wrapped Peptide Nanotubes: Peptide-Grafted Polymer Mass Impacts Length and Diameter. Small 2008, 4, 1008-1016.

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24. Dong, H.; Lund, R.; Xu, T. Micelle Stabilization via Entropic Repulsion: Balance of Force Directionality and Geometric Packing of Subunit. Biomacromolecules 2015, 16, 743-747. 25. Yang, C.; Lu, D.; Liu, Z. How PEGylation Enhances the Stability and Potency of Insulin: A Molecular Dynamics Simulation. Biochemistry 2011, 50, 2585-2593. 26. Ruiz, L.; Keten, S. Directing the Self-Assembly of Supra-Biomolecular Nanotubes Using Entropic Forces. Soft Matter 2014, 10, 851-861. 27. Chao, S.-H.; Matthews, S. S.; Paxman, R.; Aksimentiev, A.; Gruebele, M.; Price, J. L. Two Structural Scenarios for Protein Stabilization by PEG. J. Phys. Chem. B 2014, 118, 8388-8395. 28. Maullu, C.; Raimondo, D.; Caboi, F.; Giorgetti, A.; Sergi, M.; Valentini, M.; Tonon, G.; Tramontano, A. Site-Directed Enzymatic PEGylation of the Human Granulocyte Colony-Stimulating Factor. FEBS J. 2009, 276, 6741-6750. 29. Xue, Y.; O'Mara, M. L.; Surawski, P. P.; Trau, M.; Mark, A. E. Effect of Poly(Ethylene Glycol) (PEG) Spacers on the Conformational Properties of Small Peptides: A Molecular Dynamics Study. Langmuir 2011, 27, 296-303. 30. Paci, E.; Karplus, M. Unfolding Proteins by External Forces and High Temperatures: The Importance of Topology and Energetics. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 6521-6526. 31. Bell, G. I. Models for the Specific Adhesion of Cells to Cells. Science 1978, 200, 618-627 32. Sun, L.; Noel, J. K.; Sulkowska, J. I.; Levine, H.; Onuchic, J. N. Connecting Thermal and Mechanical Protein (Un)Folding Landscapes. Biophys. J. 2014, 107, 2941-2952. 33. Ackbarow, T.; Chen, X.; Keten, S.; Buehler, M. J. Hierarchies, Multiple Energy Barriers, and Robustness Govern the Fracture Mechanics of α-Helical and β-Sheet Protein Domains. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 16410-16415. 34. Pai, S. S.; Hammouda, B.; Hong, K.; Pozzo, D. C.; Przybycien, T. M.; D., T. R. The Conformation of the Poly(Ethylene Glycol) Chain in Mmno-PEGylated Lysozyme and Mono-PEGylated Human Growth Hormone. Bioconjugate Chem. 2011, 22, 2317-2323.

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35. Meg, W.; Guo, X.; Qin, M.; Pan, H.; Cao, Y.; Wang, W. Mechanistic Insights into the Stabilization of SrcSH3 by PEGylation. Langmuir 2012, 28, 16133-16140. 36. Pandey, B. K.; Smith, M. S.; Torgerson, C.; Lawrence, P. B.; Matthews, S. S.; Watkins, E.; Groves, M. L.; Prigozhin, M. B.; Price, J. L. Impact of Site-Specific PEGylation on the Conformational Stability and Folding Rate of the Pin WW Domain Depends Strongly on PEG Oligomer Length. Bioconjugate Chem. 2013, 24, 796-802. 37. Ogihara, N. L.; Weiss, M. S.; Degrado, W. F.; Eisenberg, D. The Crystal Structure of the Designed Trimeric Coiled Coil Coil-V(a)L(d): Implications for Engineering Crystals and Supramolecular Assemblies. Protein Sci. 1997, 6, 80-88. 38. Hamed, E.; Keten, S. Hierarchical Cascades of Instability Govern the Mechanics of Coiled Coils: Helix Unfolding Precedes Coil Unzipping. Biophys. J. 2014, 107, 477484. 39. Phillips, J. C.; Braun, R.; Wang, W.; Gumbart, J.; Tajkhorshid, E.; Villa, E.; Chipot, C.; Skeel, R. D.; Kale, L.; Schulten, K. Scalable Molecular Dynamics with NAMD. J. Comput. Chem. 2005, 26, 1781-1802. 40. Jorgensen, W. L.; Chandrasekhar, J.; Madura, J. D.; Impey, R. W.; Klein, M. L. Comparison of Simple Potential Functions for Simulating Liquid Water. J. Chem. Phys. 1983, 79, 926-935. 41. MacKerell, J., A. D.; Bashford, D.; Bellott, M.; Dunbrack, J., R. L.; Evanseck, J. D.; Field, M. J.; Fischer, S.; Gao, J.; Guo, H.; Ha, S.; Joseph-McCarthy, D.; Kuchnir, L.; Kuczera, K.; Lau, F. T. K.; Mattos, C.; Michnick, S.; Ngo, T.; Nguyen, D. T.; Prodhom, B.; Reiher, I., W. E. et al. All-Atom Empirical Potential for Molecular Modeling and Dynamics Studies of Proteins. J. Phys. Chem. B 1998, 102, 35863616. 42. Humphrey, W.; Dalke, A.; Schulten, K. VMD: Visual Molecular Dynamics. J. Mol. Graphics Modell. 1996, 14, 33-38.

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Tables

Table 1. Energy barrier (Eb) and transition distance (xb) for three force regimes: small, intermediate, and large. The errors denote standard deviations. Force regime Small-force regime Intermediate-force regime Large-force regime

Eb (kcal/mol)

xb (Å)

Peptide

Peptide-PEG40

Peptide

Peptide-PEG40

10.12±0.35

10.68±0.27

2.38±0.68

2.65±0.57

7.5±0.1

7.61±0.05

0.64±0.03

0.63±0.04

5.38±0.31

5.61±0.20

0.15±0.01

0.16±0.01

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Figures Figure 1

(a)

F

(b)

F

F

F

Figure 1. Schematics of (a) helix and (b) helix-PEG conjugate. Mechanical stability of both systems is investigated by applying tensile forces of varying magnitudes at the two helix termini.

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Figure 2

Figure 2. Logarithm of average helix unfolding time, τavg, versus the applied force, F. Depending on the force magnitude, three regimes of small forces (solid best fitted lines), intermediate forces (dashed best fitted lines), and large forces (dotted best fitted lines) can be distinguished. The inset indicates error bars corresponding to standard deviations for small forces (see Table S.1 of the Supporting Information for values of standard deviation for all forces). For all the forces tested, there is a statistically significant increase in unfolding time of the PEGylated peptides compared to the native peptides, suggesting that PEG conjugation reinforces helices against force-induced unfolding. As the applied force becomes smaller, the PEG stabilization effect becomes more noticeable.

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Figure 3

Figure 3. (a) Occupancy of helix backbone i→i+4 hydrogen bonds (H-bonds) broken during the unfolding process shown for different residues of the peptide. These results show that PEG conjugation increases the lifetime of backbone H-bonds and this effect is more noticeable closer to the PEG conjugation site (residue 14). (b) A schematic showing that first helix backbone H-bonds (black bonds) break and then, with a slight time lag, new H-bonds form between backbone and water (red bonds).

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Figure 4

Figure 4. Snapshots of (a) unfolding bursts and (b) refolding events. The unfolding burst shown has the largest decrease in the peptide helicity in Trial 4, F=70 pN. Snapshots are taken at 0.02 ns intervals. The refolding event shown has the largest helicity increase in Trial 1, F=70 pN. Snapshots are taken at 0.012 ns intervals. It can be seen that during the unfolding burst, the PEG chain is isolated to one side of the peptide, imparting minimal shielding effects. During the refolding event, however, the PEG chain wraps around the surface of the peptide, potentially blocking interactions with water.

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Figure 5

Figure 5. (a) Schematics showing the definition of the parameters radial distance, r, and coverage angle,θ, used in characterization of PEG conformations. (b) Heat maps showing the PEG chain occupancy during unfolding simulations of helix-PEG conjugate under the applied load F=52 pN for Trial 1, Trial 2, and Trial 3. The peptide boundary is shown by the red dashed circle. Here, longer unfolding times are marked by an increase in PEG density near the peptide, and a more complete occupancy ring centered around the peptide.

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Figure 6

Figure 6. The (a) coverage angle, θiqr, of the PEG chain around the peptide and (b) average distance of PEG chain from the peptide center of mass, ravg, displayed separately for unfolding bursts, refolding events, and plateau periods for F=52 pN. It can be seen that events that regain or maintain helicity occur when the PEG chain surrounds a larger portion of the peptide, and also when the PEG chain is closer to the peptide surface. This difference is more pronounced between plateau periods and unfolding bursts.

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Figure 7

Figure 7. (a) The time difference between the irreversible breakage of backbone H-bonds and formation of backbone-water H-bonds, ∆t, for the residues located in the helix middle section. The data shows that the time difference is longer for PEGylated peptide than native peptide, confirming the solvent shielding effects of PEG chain. (c) Profile of pair counts showing the total number of pair counts near each residue averaged for Trial 1, 2 and 3 of force F=52 pN. Peaks in the number of pair counts often occur in hydrophobic patches, and, in the cases of Residues 18, 22 and 25, correspond to an increased time delay for the PEGylated peptide. Neutral residues experience a lower percentage of pair counts relative to the proportion of the peptide they make up, whereas charged residues see a higher proportion of pair counts, which can be exploited in designing mechanically reinforced proteins to maximize surface interactions. The one letter residue names represent: A - Alanine, C - Cysteine, E - Glutamic Acid, G Glycine, H - Histidine, K - Lysine, L - Leucine, Q - Glutamine, and V - Valine.

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For table of contents use only:

Mechanical Reinforcement of Proteins with Polymer Conjugation Elizabeth P. DeBenedictis†, Elham Hamed†, and Sinan Keten*

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Mechanical Reinforcement of Proteins with Polymer Conjugation

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