Sequence Directionality Dramatically Affects LCST Behavior of Elastin

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Sequence directionality dramatically affects LCST behavior of elastin-like polypeptides Nan K Li, Stefan Roberts, Felipe Garcia Quiroz, Ashutosh Chilkoti, and Yaroslava G Yingling Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00099 • Publication Date (Web): 17 Apr 2018 Downloaded from http://pubs.acs.org on April 18, 2018

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Sequence directionality dramatically affects LCST behavior of elastin-like polypeptides Nan K. Li†, Stefan Roberts‡, Felipe Garcia Quiroz$, Ashutosh Chilkoti‡, and Yaroslava G. Yingling†* †Department of Materials Science and Engineering, North Carolina State University, 911 Partners Way, Raleigh, NC 27695, USA. ‡ Department of Biomedical Engineering, PO Box 90281, Duke University, Durham, North Carolina 27708, USA $ Howard Hughes Medical Institute, Laboratory of Mammalian Cell Biology & Development, The Rockefeller University, New York, New York 10065, USA

ABSTRACT

Elastin-like polypeptides (ELP) exhibit an inverse temperature transition or lower critical solution temperature (LCST) transition phase behavior in aqueous solutions. In this paper, the thermal responsive properties of the canonical ELP, poly(VPGVG), and its reverse sequence poly(VGPVG) were investigated by turbidity measurements of the cloud point behavior, circular dichroism (CD) measurements, and all-atom molecular dynamics (MD) simulations to gain a molecular understanding of mechanism that controls hysteretic phase behavior. It was shown

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experimentally that both poly(VPGVG) and poly(VGPVG) undergo a transition from soluble to insoluble in aqueous solution upon heating above the transition temperature (Tt). However, poly(VPGVG) resolubilizes upon cooling below its Tt, whereas the reverse sequence, poly(VGPVG), remains aggregated despite significant undercooling below the Tt. The results from MD simulations indicated that a change in sequence order results in significant differences in the dynamics of the specific residues, especially valines, which lead to extensive changes in the conformations of VPGVG and VGPVG pentamers and, consequently, dissimilar propensities for secondary structure formation and overall structure of polypeptides. These changes affected the relative hydrophilicities of polypeptides above Tt, where poly(VGPVG) is more hydrophilic than poly(VPGVG) with more extended conformation and larger surface area, which led to formation of strong interchain hydrogen bonds responsible for stabilization of the aggregated phase and the observed thermal hysteresis for poly(VGPVG).

KEYWORDS. Molecular dynamics simulations, hysteresis, ELP, VPGVG, VGPVG, LCST, sequence order, syntax


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Introduction Elastin-like polypeptides (ELP) are a class of synthetic polypeptides derived from elastin, which undergo a transition from soluble to insoluble in aqueous solution at a lower critical solution temperature (LCST).1 ELPs, at a given concentration in aqueous solution, are soluble below their LCST cloud point, or transition temperature (Tt), and undergo hydrophobic collapse and form insoluble aggregates at temperatures greater than the Tt.2,

3

ELPs are characterized by their

temperature responsiveness that can be tuned at the sequence level and their biocompatibility, which make them interesting as a class of synthetic biopolymers for a variety of applications including drug delivery engineering scaffolds

4-6

11, 12

, protein purification7-9, responsive nanoparticles10, and tissue

. The most widely studied ELP is poly(VPGVG) whose LCST

behavior as a function of concentration13-15 and ionic strength16 in aqueous solutions has been extensively characterized. This pentapeptide motif was also generalized into the VPGXG repeat unit by Urry, where the fourth residue (X) in the pentapeptide repeat is often termed the “guest’ residue that can be substituted with other amino acids except proline.14, 17 ELPs that exhibit LCST behavior may also display a hysteresis loop in their temperaturedependent turbidimetry profiles. For example, it has been long known that poly(VPAVG) displays significant thermal hysteresis in its heating-cooling cycle,18,

19

so that considerable

undercooling below the Tt is needed to resolubilize poly(VPAVG). We also recently investigated the LCST phase behavior of a large library of polypeptides composed of LCST motifs, which uncovered additional forms of hysteretic phase behaviors and large differences in thermal hysteresis for polypeptides with very subtle changes in amino acid sequence20. While this thermal hysteresis behavior opens exciting possibilities for the design of materials with new


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properties, the molecular determinants of how amino acid sequence governs hysteretic phase behavior remain poorly understood. The amino acid composition of an ELP is not the only sequence dependent factor that can affect its LCST behavior. Changes in amino acid order are likely to disrupt native secondary structure propensities of well-folded polypeptides, which can have a major effect on their function. However, for intrinsically disordered ELPs that exist in dynamic and flexible conformations, the rationalization of how changes in amino acid order influence their functions and phase behaviors are far from obvious. To provide insights into how the amino acid order controls LCST behavior of ELPs, we examined a unique pair of polypeptides20, poly(VPGVG) and poly(VGPVG), whose sequences are essentially mirror images or “reverse” motifs and hence identical if read in the opposite directions, N- to C-terminus or vice versa. A comparison of the LCST phase behavior of poly(VPGVG) and poly(VGPVG) allows us to examine the dependence between the arrangement of amino acids and LCST phase behavior with no change in the overall composition. The thermal behavior of aqueous solutions of these two polymers were examined using turbidity measurements and all-atom molecular dynamics (MD) simulations. Although the turbidity measurements can effectively describe the macroscopic “cloud point” behavior of an ELP, changes in the molecular properties associated with sequence reversal are less easy to characterize experimentally. In our previous study, the effect of temperature on the structure, dynamics and association of (VPGVG)18 in aqueous solution was investigated using atomistic MD simulations. And from that study we concluded that the LCST phase behavior of poly(VPGVG) is a collective phenomenon that originates from the correlated gradual changes in single polypeptide structure and the abrupt change in properties of hydration water around the peptide and is a result of a competition between peptide−peptide and peptide−water


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interactions.21 We hence carried out MD simulations to reveal how sequence reversal impacts the thermal behaviors of ELPs (VPGVG)18 and (VGPVG)18. Moreover, we attempt to also elucidate the mechanism underlying the hysteretic behavior observed in the LCST phase transition of (VGPVG)18. Materials and Methods Molecular Dynamics Simulations In this study, we employed the Amber 1122 and 1223 programs to run MD simulations. The ff99SB force field was employed for peptides and water was modeled using the TIP3P model24. The force field chosen here was demonstrated to be able to reproduce the inverse temperature transition properties of poly(VPGVG) in our previous studies.21, 25 The non-bonded interactions were truncated at 9 Å cutoff with a 0.00001 tolerance for Ewald convergence and the long-range electrostatic interactions were taken into account by Particle Mesh Ewald (PME) summation26. Simulations of a single poly(VPGVG) and poly(VGPVG) peptides in water were performed at ten different temperatures between 290 K and 350 K, which span the temperature range relevant to the experimental study of LCST phase transitions. The initial structures for the models of ELPs were built based on dihedral angles for each residue in Urry’s β-spiral model27, which was proposed based on pioneering work in the development of ELP by chemically synthesizing poly(VPGVG) by Urry’s group and widely used as the initial structure by previous MD studies on ELPs.21, 25, 28, 29 The initial structures were then solvated in explicit water with the closest distance between any polypeptide atom and the edge of the periodic box to be at least 8 Å to ensure that the polypeptide did not cross periodic boundaries and interact with its own image. However, the initial structure was completely distorted at the very early stage of MD trajectory. The simulation box for (VPGVG)18 contained 6856 water molecules with a size of 5.9 nm 7.7


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nm 5.0 nm in the equilibrium state and the simulation box for (VGPVG)18 contained around 8651 water molecules with a size of 6.9 nm 8.7 nm

5.7 nm in the equilibrium state. The

equilibrium box size varied slightly at different temperatures. The system was then equilibrated in eight stages, including several energy minimization, heating and MD run cycles. The equilibration protocol was the same as described in our previous study21. The system temperature was maintained using the Berendsen thermostat.30 The SHAKE algorithm was used to constrain the position of the hydrogen atoms.31 The production simulations were performed for at least 70 ns with a 2 fs time step. With a sampling rate of one frame per 1 ps (every 500 time steps) of the MD trajectories, we would expect that not only enough samples were collected, but also the simulation efficiency was enhanced. To ensure the convergence of all simulations, we calculated the time autocorrelation function for in-plane and out-of-plane backbone rotations, from which the relaxation time of the peptide backbone was estimated to be less than 25 ns (Figure S5, S6). The statistical and clustering analyses were carried out on MD trajectories for the last 40 ns using in-house scripts along with the tool suite accompanying Amber12.0. The interaction energies were calculated using the molecular mechanics energy function in NAMD 2.732. The hydrogen bond analyses were performed using an angle cut-off of 30 and a distance cutoff of 3.5 Å. To identify the most probable peptide structures from the MD simulations trajectory, the hierarchical RMSD-based clustering algorithm33 was used. Specifically, the last 40 ns of a trajectory from the single-peptide MD simulations were clustered to produce three structural clusters using the pairwise RMSD between frames as a metric and comparing the carbon alpha atoms with a critical distance of 12 Å. Representative snapshots of the ELP structures from the most populated clusters at 290 K and 350 K are shown in Figures S3 and S4. The lowest-energy


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representative single polypeptide structures from these clusters were then taken to be the initial structures in the simulations of the interaction between two polypeptides. The two selected single polypeptide structures were then placed side-by-side with a 32.3~32.4 Å distance between the centers of mass of the polypeptides and a 7 Å distance between the two closest surfaces. The structures were then solvated in explicit solvent with the closest distance between any solute atom and the edge of the periodic box specified as 12 Å and simulated at 350 K. The simulation box contained 10493 water molecules with the size of 9.2 nm 7.9 nm

5.8 nm for double-

poly(VPGVG) system and 9738 water molecules with size of 6.6 nm 9.4 nm 6.4 nm for double-poly(VGPVG) system. Equilibration protocols and MD simulations were the same as described for single-peptide simulations21. The production steps of the simulations were carried out at 350 K for more than 15 ns with a 2 fs time step and performed three times. Experimental protocol Recombinant synthesis and characterization of polypeptides: We synthesized genes encoding polymers (VPGVG)40, (VPGVG)80, (VGPVG)40 and (VGPVG)80 using recursive direction ligation by plasmid reconstruction and expressed them in E. coli BL21(DL3, Edgebio) as previously described34,35. Polymers of VPGVG were purified by inverse transition cycling (ITC)36. Polymers of VGPVG were purified from the insoluble fraction after sonication and centrifugation using 6 M guanidinium chloride followed by dialysis into water and centrifugation to remove insoluble proteins. Polymers of VGPVG, which remain soluble upon dialysis, were further purified by cycles of ITC using NaCl instead of temperature to trigger the phase transition37. The LCST phase behaviors of the purified polymers were studied by temperaturedependent turbidimetry using a Cary UV-vis spectrophotometer (Agilent) at a wavelength of 350 nm and heating/cooling rates of 1 C/min, except where otherwise specified. CD experiments


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were carried out using an Aviv Model 202 instrument and 1 mm quartz cells (Hellma USA). Scans were carried out in H2O with a polymer concentration of 10 µM. Polymers were scanned in triplicate from 190 -260 nm in 1 nm steps with a 1 s averaging time. Data points with a dynode voltage above 500 V were removed from analysis.


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Figure 1: Experimental comparison of LCST phase behavior of poly(VPGVG) and poly(VGPVG). (a,b) Temperature dependent turbidimetry for (VPGVG)40/80 and (VGPVG)40/80 (50 µM, PBS). Polymers with identical composition but sequencereversed orientation (reverse sequence) have different LCST phase behaviors. (VPGVG)40 and (VPGVG)80 —canonical ELP sequences—shows reversible phase behavior, but the reverse sequence, poly(VGPVG), shows irreversible aggregation as a function of solution temperature. (c) The reverse sequence VGPVG has a slightly higher transition temperature across all measured concentrations despite the identical amino acid composition. (d) CD measurements of the soluble polymers (10 µM at 15 C in H2O) reveal a significant difference in their secondary structure. Poly(VPGVG) has a negative “shelf” at 218 nm, indicating the presence of beta turns 35, 36, and this peak is absent in the CD spectrum of the reverse sequence, poly(VGPVG). Both polymers otherwise appear highly disordered. Results Experimental results Turbidity measurements for poly(VPGVG) and poly(VGPVG) reveal sharp differences in their LCST phase behavior (Figures 1a,b and Figures S1a,b). For both the 40-mer and 80-mer lengths,


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the two polymers have a similar Tt upon heating; however, poly(VPGVG) is fully reversible whereas the reverse sequence poly(VGPVG) remains aggregated despite significant undercooling below its Tt. Above its Tt, poly(VPGVG)40 and poly(VPGVG)80 aggregates also show signs of sedimentation (the optical density drops gradually as the coacervates settle below the UV light path). This sedimentation is not observed for poly(VGPVG) polymers on the time scale of our experiments, indicating that the aggregates are either smaller or less dense than those


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of poly(VPGVG). The irreversible aggregation observed for poly(VGPVG) is also independent of both concentration (Fig. S2), and heating and cooling rates, with a nearly identical Tt and complete irreversibility observed for heating and cooling rates ranging from 0.5– 5

C/min (Fig.

S1c-f). Interestingly, the Tt for poly(VPGVG) at both 40 and 80-mers tends to be slightly higher than that for poly(VGPVG), despite identical amino acid composition (Fig. 1c). This difference in transition temperatures, consistent for all concentrations and for both polymer chain lengths, is


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likely due to differences in polypeptide conformation. CD measurements for both polymers reveal slightly different molar ellipticity peaks (Fig. 1d and Fig. S1g). Poly(VPGVG) appears largely disordered with a slight shelf at 218 nm which has historically been attributed to unstable Pro-Gly beta turns in the polymer

38, 39

. Poly(VGPVG), which has a reversed Gly-Pro sequence,

is also largely disordered but does not exhibit a peak at 218 nm in its CD spectrum, indicating a


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slight shift in the secondary structure likely produced from disruption of the Pro-Gly beta-turn structure.

Figure 2. Temperature dependence of the properties of a single (VPGVG)18 and (VGPVG)18 ELP chain: (a) radius of gyration (Rg), (b) npw, the number of peptide-water hydrogen bonds, (c) solvent accessible surface area (SASA, (d) peptide-water interaction energy. Error bars represent the standard deviation. Structural properties and water-peptide interactions To explain the differences observed in the experiments, we examined dynamics of forward sequence (VPGVG)18 and reverse sequence (VGPVG)18 in water as a function of temperature using MD simulations. We chose 18 repeats because the forward sequence (VPGVG)18 has been


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investigated systematically and has been shown to exhibit inverse transition thermal behaviors.21 The evolution of single chain Rg for (VPGVG)18 and (VGPVG)18 show that both peptides adopt a collapsed state over a temperature range between 290 K and 350 K (Fig. 2, S3, S4).21 However, while the Rg of (VPGVG)18 gradually decreases, the Rg for the reverse sequence (VGPVG)18 stays approximately constant as the temperature increases. The comparison of the number of peptide-peptide (npp) and water mediated peptide-water-peptide (npwp) intrachain hydrogen bonds between the two polymers (Figure S7a) reveals that for (VPGVG)18, npwp monotonically decreases while npp increases as a function of temperature. However, for (VGPVG)18 system all observables vary only slightly upon heating. The temperature induced changes in the solvent accessible surface area (SASA) and number of water-peptide hydrogen bonds (npw) from the MD simulations (Fig 1b.c) indicated that for (VPGVG)18, SASA and npw decrease gradually upon heating until a sudden drop at around 330 K, which we previously connected to the Tt21. In contrast, for (VGPVG)18, the dependence of SASA and npw on the temperature is gradual with no abrupt change (Fig. 1 b,c). At high temperatures (VGPVG)18 has a greater solvent accessible surface area (SASA) and forms more hydrogen bonds with water molecules than (VPGVG)18, which indicate that the reversal of the amino acid order makes (VPGVG)18 more hydrophilic at elevated temperatures than (VGPVG)18. In our previous studies, we identified that the number of water molecules in the first hydration shell is an important parameter for characterization of the phase behavior, which is defined as the number of water molecules within a 2.23 Å distance of any atom on the polypeptide21, 25. This distance is taken as the first minima from the radial distribution function (RDF) between the oxygen of water and an atom on the polypeptide.21 Below 330 K, the number of first layer hydration water molecules around (VPGVG)18 and (VGPVG)18 is very similar (Fig. S7b). Above


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330 K, however, the differences between the two polymers become more significant, as (VPGVG)18 exhibits a sharp decrease which is correlated with a similar decrease in the SASA, unlike (VGPVG)18. The interaction energy between polypeptides and water provides information for the hydrophobicity change of a single peptide with temperature. As shown on Figure 2d, polypeptide-water interactions become energetically more unfavorable as the temperature increases. Moreover, at high temperatures the difference in the interaction energies between polypeptides indicates that the surface hydrophobicity of a single molecule of (VGPVG)18 is less than that of (VPGVG)18. Overall, reversing the amino acid order appears to cause minor structural and energetic changes in ELPs at low temperatures. However, the poly(VGPVG) is larger (higher Rg) and more hydrophilic (larger SASA, npw, and lower peptide-water interaction energy) than poly(VPGVG) at high temperatures, although their overall amino acid compositions are identical. Dihedral angles, secondary structures and role of individual residues. To explore the structural differences associated with different temperature-responsive behaviors of (VPGVG)18 and (VGPVG)18 in aqueous solution, we calculated the torsion angles of each amino acid (Figure 3 and S8). The high intensity region in the Ramachandran plot indicates the most favorable, low-energy torsion angle pairs. In our previous study, we discussed the features of torsion angles in VPGVG pentamer at low and high temperatures and compared our simulation results with multiple experimental studies in details.21 Overall, our simulations show that for (VPGVG)18, the change in dihedral angle as a function of temperature agrees well with the experimental observations.

40, 41, 42

Here, we found that structural dynamics of backward

sequence, (VGPVG)18 showed similarities but also significant differences to the forward


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sequence, (VPGVG)18, especially for valine residues (Figure 3). Val1 in VPGVG is preceded by proline and shows torsion angle distribution that is distinctive and different from the other valines in both VPGVG and VGPVG pentamer. The ψ angle of Val1 in VPGVG only populates around 150° and φ is around −130° or −75°, whereas Val4’ torsion angles are distributed between four regions where ψ is around 150° or −2° and φ is around −130° or −90°. However, Val1 and Val4 in VGPVG occupy similar conformational spaces in the left side of the Ramachandran plots. The ψ angle display the bimodal distribution with (ψ ~150° or −5°, respectively) while ϕ ranges from ~−45° to ~−170° with one peak around −126° for a4_1, a4_2, and d4_1, and −107.7° for d4_2. The torsion angles of Val4 residues in VGPVG also clustered at


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the left-handed alpha helical conformational space marked as d4_3 (52°, 30.3°). Glycine and proline are usually observed to have very different ϕ, ψ distribution outlines than the other amino acids, since glycine is conformationally less constrained and proline is conformationally more constrained. 43 In VPGVG, the Pro2 and Gly3 primarily adopt the torsion angle pairs (φ = −60°, ψ = 120°), (φ = −75°, ψ = 150°) as the i+1 and i+2 residues of a type-II βturn structure. Gly2 in VGPVG is also a pre-proline residue; however, Gly2 does not have a side chain which allows high flexibility as well as the torsion angles which are normally not allowed for other amino acid residues. Moreover, the most populated region for Gly3 in VPGVG is missing in the plots for Gly2 in VGPVG, indicating missing turn structures in the segment

Figure 3. (a-d) Ramachandran plot (ϕ, ψ distributions) for Val1 and Val4 in (VPGVG)18 and (VGPVG)18, respectively, at 350 K, colored by intensities. The most populated regions on the Ramachandran plots are labelled. (e-f) Representative structures for (e) G(VPGVG)V segment and (f) G(VGPVG)V segment at 350 K built with the dihedral angles obtained from the highest intensity points in Ramachandran plots (Figure 3 and Figure S8).


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VGPV. The most populated angles adopted by Gly2 in VGPVG correspond to the high intensity regions of the Ramachandran plot labeled by c3_1 (−76.5°, 150.6°), c3_2 (70°, −141.1°) and c4_1 (98.27°, −140.0°) as shown in Figure S8. The Gly5 in VGPVG are scattered over various conformational spaces of the Ramachandran plot, which may adopt various secondary structural motifs such as Helix, Turn and β-strand. Interestingly, the Ramachandran plot pattern for Gly5 in poly(VGPVG) at 350 K is similar to Gly5 in poly(VPGVG) at 290 K; the Ramachandran plot pattern for Gly5 in poly(VGPVG) at 290K is similar to Gly5 in poly(VPGVG) at 350 K. Gly5 residue in poly(VGPVG) at 290 K and 350 K occupy two areas with relatively high number of occurrences, such as e3_1 (−80.76°,135°) and e4_2 (−107.7°, −2.0°), respectively. In contrast to glycine amino acids, ψ angle for Pro3 in VGPVG varies in a wider range from −62° to 173° which indicates a larger flexibility of proline residues in VGPVG than that in VPGVG, where φ is fixed around −65° due to its backbone stereochemistry. To indicate the difference in the most probable conformation we build the representative structures for pentamers of VPGVG and VGPVG based on the most populated torsion angles obtained from Ramachandran plots, as shown in Figure 3e and f. The VGPVG segment has a more stretched out conformation than VPGVG at 350K (Fig. 3 e,f), which can explain the observed differences in radius of gyration. The secondary structure motifs were determined by a common structure recognition algorithm, the DSSP method, which is based on H-bonding patterns44. In the DSSP algorithm, the residues bracketed by the hydrogen bond between residue i and residue i+n (n=3, 4, 5) are considered to adopt a turn structure. The helical structure includes residues from α- , 3-10 and π helix; the βstrand structure includes residues from β-bridges and extended strands involved in parallel and anti- parallel β-sheets. The secondary structural propensities of each single polypeptide at various temperatures are listed in Table S2. Distinct changes were observed due to the reversing


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of the amino acid order in the secondary structural propensities, which is in agreement with the experimental observations (Figure 1). In general, there is a larger propensity for helix structures in the reverse sequence, (VGPVG)18 chain than in the forward sequence (VPGVG)18 at all temperatures, which is consistent with the observations from the Ramachandran plot. In addition, (VPGVG)18 adopts more turn structures than (VGPVG)18 for most groups at different temperatures. The propensity for β-strands are very close for the two polypeptides at low temperature; however, there is a smaller propensity for β-strands for (VGPVG)18 chain at high temperature than (VPGVG)18, which is probably due to a higher flexibility of prolines and valines in poly(VGPVG). The occurrence of each residue in each type of secondary structural motifs (Turn, β-strands, and Helix) at 290 K and 350 K is presented in Table S3 and plotted in Figure S9.

In the forward sequence, VPGVG, Pro2-Gly3-Val4 segment mostly formed Turns and Gly5Val1 mostly formed β-strands. In reverse VGPVG sequence, residues adopt diverse secondary structures. For example, more than 8% of each residue in Gly2-Pro3-Val4 segment adopt Helix structures, while less than 1.1% of each residue in Pro2-Gly3-Val4 segment adopt Helix structures. This could be due to proline position within the sequence which has asymmetrical structure and restrains the conformational freedom of residues before it, which can break the symmetry of peptide chains. The secondary structures differ significantly due to the proximity of residues to proline, which demonstrate the importance of the sequence direction and the local amino acid order on the formation of the secondary structure of polypeptides. The reversing of amino acid order from poly(VPGVG) to poly(VGPVG) can be described at the molecular level as the mutation of Pro2-Gly3 to Gly2-Pro3. Statistical analysis was conducted on


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Table 1. Distance between Cα of Val1 and Cα of Val4 in pentamer (VPGVG) and (VGPVG) at 290 K and 350 K, respectively. The numbers listed in the third column are the probability of the distances below 7 Å. Average distance, Å

Probability (distances < 7 Å)

(VPGVG)18, 290 K

8.29±1.24

11.11%

(VPGVG)18, 350 K

8.41±1.49

33.33%

(VGPVG)18, 290 K

7.53±1.22

33.33%

(VGPVG)18, 350 K

8.60±1.33

11.11%

the distances (D1, D2) between the α-carbon atoms in Val1 and Val4 in forward and reverse sequences based on the last 40 ns trajectories. The segment VPGV or VGPV adopt β-turn structures when the distance between the α-carbon atoms in Val1 and Val4 is below 7 Å based on the distance criterion of β-turn structures.45 As listed in Table 1, 11.1% and 33.3% of VPGV segments form β-turns (D1< 7 Å) in forward sequence at 290 K and 350 K, respectively. Interestingly, these probabilities are reversed as 33.3% and 11.1% of VGPV segments form βturns (D2< 7 Å) in the backward sequence at 290 K and 350 K, respectively. The difference is due to the fact that the segment VPGV can intrinsically adopt type-II β-turn structures which increased as the temperature increased from 290 K and 350 K. Therefore, the “mutation” of Pro2-Gly3 to Gly2-Pro3 cause significant differences in the local residue interactions and consequently secondary structure formation, which is consistent with CD measurement (Figure 1).


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Interactions between each amino acid residue with their aqueous environment, determine the conformation of polypeptide and mediate further intermolecular interactions (Figure 4a). In general, the interactions between each amino acid and water molecules decreases as the temperature increased, except for Val4 in the reverse sequence VGPVG. The energy plots clearly confirm that the interactions between each residue type and water in the two sequences are not identical, although Pro2/3 and Val4-Gly5 in these two sequences can be argued to show very similar hydrophobicity. Val1 in the VPGVG interacts with water weaker than Val1 in VGPVG, possibly due to the steric restriction of Val1 in VPGVG by Pro2. The number of hydrogen bonds formed between each residue and water molecules reveals less structural heterogeneity in VGPVG than VPGVG (Figure 4b). The plot of SASA (Figure 4c) for each residue shows very little difference between Pro2, Val4 and Gly5 in VPGVG and Pro3, Val4 and Gly5 in VGPVG. However, the Val1 in VGPVG is more accessible by water molecules than Val1 in VPGVG and Gly2 in VGPVG is less accessible than Gly3 in VPGVG. The compensation between the SASA of these two residues explains the overall SASA trends of both ELP chains.


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Figure 4. Temperature dependence of properties of a single ELP chain: (a) Interaction energy of each residue in an ELP pentamer with water. (b) Number of hydrogen bonds formed between each residue in an ELP pentamer and water. (c) SASA of each residue in an ELP pentamer. The error bars represent the standard deviation.

Hydration water network We further characterized the structure of hydration water around the polypeptide chains by measuring the size of the largest H-bonded water network around each polypeptide (Figure 5a),


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which represents how polypeptide influence the water structure and dynamics46, the effect of orientational ordering of water molecules and water-water interactions near the polypeptide surface.21 In the hydration water network, all water molecules are considered to form hydrogen bonds when ROO ≤ 3.5 Å and φO1···O2-H2 ≤ 30° (Figure 5b). The probability distribution of the largest water network around the (VPGVG)18 and (VGPVG)18 at T = 290 K, 310 K, 330 K and 350 K are displayed in Figure 5a. For both polypeptides, the water-water H-bonds in the hydration water shell surrounding each polypeptide generally decreases with increasing temperature, indicating a more disordered water network, especially for (VGPVG)18. We observed a higher thermal stability of the H-bonded water network around (VPGVG)18 than that around (VGPVG)18, whereas the hydration water of (VGPVG)18 has a higher thermal-sensitivity than that of (VPGVG)18. This contrasts with a greater number of hydration water molecules in the water shell of (VGPVG)18 which also has a higher SASA; however, the water molecules form more hydrogen bonds with (VGPVG)18, thereby decreasing the water network in the hydration shell.


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Figure 5. (a) Probability distribution of the size Nmax of the largest water network around polypeptide. (b) A snapshot of (VPGVG)18 and the largest water network around it at 290 K Polypeptide interactions


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To examine the effect of temperature on the early stages of polypeptide aggregation, we performed simulations of two temperature-equilibrated polypeptides at 350 K. The simulation


25


set-up and MD simulation protocols were the same as described in our previous paper.

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21

The

distance between the center-of-mass of two chains (Dpp) (Figure 6a) shows that the both


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polypeptides aggregate during the simulations. The interaction energy (Epp) and number of interchain hydrogen bonds (Npp-double) profiles (Figure 6b and c) between the polypeptides for both


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cases are correlated with each other and show that the reverse sequence poly(VGPVG) form more compact aggregates with more inter-chain hydrogen bonds and a lower interaction energy


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between two chains than the forward sequence poly(VPGVG).

Figure 6. Interaction between two ELPs at 350 K. (a) Distance between the center-of-mass of two polypeptides, (b) peptide-peptide interaction energy and (c) the number of inter-chain hydrogen bonds.


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Discussions The “Pro-Gly” sequence motif has been demonstrated to be important for the elastomeric mechanical behaviors observed within a number of native protein-based polymers.54 It is reasonable to assume that it is also a recurrent if not dominant structural feature associated with the reversibility in the LCST behaviors of ELPs. This is because the type II β-turn per pentamer VPGVG involves proline-glycine pair as the corner residues. The presence of a hydrogen bond between Val1 and Val4 in VPGV is important for stabilization of the type-II β-turn structure formation. The peptide with Pro-Gly to Gly-Pro mutation still exhibits LCST behavior, but loses the propensity for the β-turn formation, which results in a more extended conformation (larger Rg and smaller npp) and a larger solvent exposure (larger SASA) of a peptide. The CD measurements of both soluble ELPs reveal a significant difference in the secondary structure between VPGVG and VGPVG peptides. Poly(VPGVG) has a negative “shelf” at 218 nm, which has historically been attributed to Pro-Gly β-turns in the polymer

38, 39

, and this peak

is absent in the CD spectrum of the reverse sequence, poly(VGPVG). Simulation results showed that, at all temperatures, (VGPVG)18 tends to form more Helix-structures than (VPGVG)18 and at higher temperatures a smaller number of β-strands and β-turns than (VPGVG)18. Such a dependence between amino acid order and secondary structures is consistent with a previous study that examined 1288 PDB structural fragments, and reported that about 21% of the protein sequence segments exhibits different secondary structure propensities in the forward and reverse directions, as assessed by the PREDATOR algorithm. 52, 53 The distinct coacervation process, which is exhibited by ELPs upon heating, is considered to be a complex and multistep transition in thermodynamics point of view.19, 55, 56 It was proposed that the coacervation process can be simplified into intra-molecular interaction and inter-molecular


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aggregation stages, and intra-molecular interactions during the initial chain folding prevail.

57, 58

Thus, it is reasonable to assume that the dissociation in the cooling process would occur in the reverse order of the two interaction stages; i.e. the order of intrachain interaction and interchain interaction is reversed. Upon cooling, the dissociation of the inter-chain aggregation occurs first. Collectively, the consecutive inter-chain hydrogen bonding arising in the poly(VGPVG) appeared to perturb the dissociation by the additional interchain interactions. In other words, each poly(VPGVG) aggregate directly and then quickly dissociates into individual chain when the solution is cooled to below the LCST, which is different from the dissociation mechanism of poly(VGPVG) aggregates where some aggregates are not able to completely dissociate upon cooling due to formation of additional strong inter-chain hydrogen bonds. Previously, the consistent but very small degree of hysteresis in the heating and cooling cycle was also observed in the PNIPAM water solution. 47-49 Based on FTIR data, it has been proposed that some of inter-chain hydrogen bonds could persist upon cooling, especially when the temperature is not far away from the phase transition temperature.50 Furthermore, a comparison between PNIPAM and PDEAM indicates that the hysteresis originates from additional hydrogen bonds formed in their collapsed state at temperatures higher than the LCST.51 We propose that the hysteretic thermal behavior observed for poly(VGPVG) (Figure 1) is because at high temperatures, a single poly(VGPVG) chain is in a loose conformation (Figure 2) that permits strong inter-molecular interactions within the aggregate (Figure 6) with formation of more interchain hydrogen bonds as compared to poly(VPGVG). These inter-chain hydrogen bonds can stabilize the aggregated phase by providing stable inter-chain contacts that are not easily disrupted by cooling. Moreover, more extended conformation and larger surface area in (VGPVG)18 increase the chances for the formation of intermolecular hydrogen bonds.


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Conclusions This paper presents results from a combined study using atomistic MD simulations and experimental observations (turbidity and CD measurements) on the effect of amino acid order on the structural and dynamics changes of ELP chains as a function of temperature. Our experimental observations show different thermal hysteresis behavior of the forward sequence, poly(VPGVG), and its reverse sequence, poly(VGPVG); whereas poly(VPGVG)s showed no thermal hysteresis in its LCST phase behavior, while the reverse sequence poly(VGPVG)s exhibits significant thermal hysteresis as the polypeptides remain aggregated despite significant undercooling below their Tt. The irreversible aggregation observed for poly(VGPVG)s is independent of concentration. Their behavior is also independent of both heating and cooling rates with nearly identical Tt and complete irreversibility observed for heating and cooling rates ranging from 0.5 – 5

C/min.

Our MD simulations show that both (VPGVG)18 and (VGPVG)18 show an increase in hydrophobicity with increased temperature, which is the common characteristic feature of neutral thermo-responsive polymers that exhibit LCST phase behavior. However, (VGPVG)18 is more hydrophilic than (VGPVG)18 at high temperatures despite the fact that both sequences have the same amino acid composition and molecular weight. (VPGVG)18 also assumes a more compact structure as indicated by a smaller Rg at high temperature then (VGPVG)18. The examination of the association kinetics revealed additional details about interchain association. The aggregation of two poly(VGPVG) molecules above the LCST can be regarded as the consequence of the competition between hydrophilic peptide–water and hydrophobic peptide-peptide interactions. We found that a pair of (VGPVG)18 peptides show stronger aggregation with a larger number of inter-chain hydrogen bonds as compared to (VPGVG)18,


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which is probably due to a more extended conformation and larger solvent exposure of a (VGPVG)18 peptide. Thus, thermal hysteresis exhibited by poly(VGPVG) may originate from formation of additional inter-chain hydrogen bonds that stabilize the aggregated phase by providing stable inter-chain contacts that are not easily disrupted by cooling. In summary, the conformational preferences of amino acids, which are impacted by sequence reversal, control the thermal hysteresis of ELPs. The change in rotational degrees of freedom within the sequence in (VPGVG)18 and (VGPVG)18 leads to the differences in the overall compaction as a function of temperature, which in turn control intermolecular interactions and hydrogen bonds formation within the aggregate, which consequently results in a thermal hysteresis. Supporting Information Available: Additional information on the simulation systems, convergence of simulations and analysis. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *[email protected] Funding Sources This work was supported by the NSF's Research Triangle MRSEC (DMR-1121107) and by a grant from the NIH TO A.C. (GM-61232) ACKNOWLEDGMENT


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This work was supported by the NSF's Research Triangle MRSEC (DMR-1121107) and by the NIH (GM-61232). Computer support was provided by the High Performance Computing Center at North Carolina State University.

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Table of Contents Graphic


39


Figure 1: Experimental comparison of LCST phase behavior of poly(VPGVG) and poly(VGPVG). (a,b) Temperature dependent turbidimetry for (VPGVG)40/80 and (VGPVG)40/80 (50 µM, PBS). Polymers with identical composition but sequence-reversed orientation (reverse sequence) have different LCST phase behaviors. (VPGVG)40 and (VPGVG)80 —canonical ELP sequences—shows reversible phase behavior, but the reverse sequence, poly(VGPVG), shows irreversible aggregation as a function of solution temperature. (c) The reverse sequence VGPVG has a slightly higher transition temperature across all measured concentrations despite the identical amino acid composition. (d) CD measurements of the soluble polymers (10 µM at 15⁰C in H2O) reveal a significant difference in their secondary structure. Poly(VPGVG) has a negative “shelf” at 218 nm, indicating the presence of beta turns 35, 36, and this peak is absent in the CD spectrum of the reverse sequence, poly(VGPVG). Both polymers otherwise appear highly disordered. 180x144mm (300 x 300 DPI)


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Figure 2. Temperature dependence of the properties of a single (VPGVG)18 and (VGPVG)18 ELP chain: (a) radius of gyration (Rg), (b) npw, the number of peptide-water hydrogen bonds, (c) solvent accessible surface area (SASA, (d) peptide-water interaction energy. Error bars represent the standard deviation. 159x128mm (300 x 300 DPI)



Figure 3. (a-d) Ramachandran plot (ϕ, ψ distributions) for Val1 and Val4 in (VPGVG)18 and (VGPVG)18, respectively, at 350 K, colored by intensities. The most populated regions on the Ramachandran plots are labelled. (e-f) Representative structures for (e) G(VPGVG)V segment and (f) G(VGPVG)V segment at 350 K built with the dihedral angles obtained from the highest intensity points in Ramachandran plots (Figure 3 and Figure S8). 142x95mm (300 x 300 DPI)


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Biomacromolecules

Figure 4. Temperature dependence of properties of a single ELP chain: (a) Interaction energy of each residue in an ELP pentamer with water. (b) Number of hydrogen bonds formed between each residue in an ELP pentamer and water. (c) SASA of each residue in an ELP pentamer. The error bars represent the standard deviation. 94x241mm (300 x 300 DPI)



Figure 5. (a) Probability distribution of the size Nmax of the largest water network around polypeptide. (b) A snapshot of (VPGVG)18 and the largest water network around it at 290 K 91x133mm (300 x 300 DPI)


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Biomacromolecules

Figure 6. Interaction between two ELPs at 350 K. (a) Distance between the center-of-mass of two polypeptides, (b) peptide-peptide interaction energy and (c) the number of inter-chain hydrogen bonds. 98x232mm (300 x 300 DPI)



88x35mm (300 x 300 DPI)


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Sequence Directionality Dramatically Affects LCST Behavior of Elastin

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