DOI: 10.1002/asia.201500438
Full Paper
Molecular Dynamics
A Molecular Dynamics Study on Controlling the Self-Assembly of b-Sheet Peptides with Designer Nanorings SeongByeong Park,[a] Myungsoo Lee,[b] and Seokmin Shin*[a] Abstract: Recently, a rational approach for constructing bbarrel protein mimics by the self-assembly of peptide-based building blocks has been demonstrated. We performed molecular dynamics simulations of nanoring formation by means of the self-assembly of designed b-sheet-forming peptides. Several factors contributing to the stability of the nanoring structures with respect to size were investigated. Our simulations predicted that an optimal nanoring size may be achieved by minimizing repulsions due to steric hin-
Introduction Bionanostructures, including peptide-based self-assembled nanostructures, have attracted enormous interest in recent years.[1] This interest reflects the fact that artificial bionanostructures can not only mimic the functional properties of biological systems such as proteins or subcellular organelles, but may also have properties that are unprecedented in nature. Designing synthetic self-assembling building blocks provides one of the most versatile approaches for constructing diverse artificial bionanostructures. To design optimal building blocks, it is necessary to understand the major driving forces responsible for a noncovalent self-assembly process during the formation of a particular bionanostructure. Noncovalent interactions play important roles in self-assembly processes; thus, the careful design of building blocks for self-assembling molecules is one of the key factors involved in fabricating non-natural bionanostructures. b-Barrel peptides are normally observed in cytoplasmic and transmembrane proteins. Cytoplasmic proteins are soluble in water, but transmembrane proteins are insoluble and amphiphilic. The green fluorescent protein is a well-studied cytoplas[a] S. Park, Prof. S. Shin Department of Chemistry Seoul National University Seoul 151-747 (Korea) E-mail: [email protected] [b] Prof. M. Lee State Key Laboratory of Supramolecular Structure and Materials College of Chemistry Jilin University Changchun 130012 (China) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/asia.201500438. Chem. Asian J. 2015, 10, 1684 – 1689
drance between bulky groups while maintaining favorable hydrogen-bond interactions between neighboring b-sheet chains. It was shown that mutations in a test peptide, in which all or half of the tryptophan residues were replaced by phenylalanine, could enable the assembly of stable nanoring structures with smaller pore sizes. Insights into the fundamental factors driving the formation of peptide-based nanostructures are expected to facilitate the design of novel functional bionanostructures.
mic protein that exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range.[2, 3] Transmembrane b-barrel proteins are often found in the outer membranes of bacteria, mitochondria, and chloroplasts. Fewer than 20 three-dimensional b-barrel structures have been described previously, although genomic databases contain thousands of b barrels.[4] In structural terms, a b barrel is a closed structure in which the first and last b sheets in a polypeptide are linked by hydrogen bonds. Adjacent b sheets in the b barrel are generally arranged in the antiparallel mode. Inspired by this natural structure, many b-barrel-mimicking peptides have been synthesized.[5] These artificial biopeptides have highlighted the potential for constructing artificial membrane pores or channels with chemical compositions and structures different from those found in nature.[4] Artificial biopeptides may potentially mimic natural proteins or cellular organelles, or even exhibit an enhancement of their functional properties. In addition, artificial biopeptides are anticipated to show characteristics that are unparalleled in nature. If synthetic pathways for forming artificial biopeptides such as b barrels become precisely defined, this could enable further breakthroughs for manipulating novel b-barrel structures. The b strands within b-barrel folds are composed of natural amino acids and are normally aligned roughly parallel to lipid bilayers, spanning the entire membrane.[4] Recently, we developed dual-function b-barrel protein mimics through the selfassembly of b-sheet-forming peptides.[6] The unique feature of the system was the formation of highly uniform and discrete water-soluble b-barrel nanoring structures with a hydrophobic interior. This structure and composition was highly similar to that found in natural b-barrel proteins, which in turn can coalesce into transmembrane b-barrel pores by simple manipulations of their molecular structure.
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Full Paper b-Sheet ribbons are organized in a cross-b structure, wherein each b strand runs perpendicular to the one-dimensional (1D) ribbon axis.[7] Previously, we proposed a fundamental design principle for constructing nanoring structures from b-sheet peptides. Considering that nanorings form highly curved structures, we hypothesized that the induction of curvature between adjacent b strands would force the 1D axis to bend. Based on this hypothesis, we designed T-shape b-sheet peptide building blocks such that bulky hydrophilic dendrons placed at the central region of b-sheet-forming peptides might induce curvature at the interface between b strands. The organic/peptide hybrid T-shape building blocks consisted of a bsheet-forming peptide and a hydrophilic oligoether dendron. The dendron was symmetrically placed on the side face of the peptide backbone. The artificially designed b-sheet peptide had a repeating structure of hydrophobic (tryptophan), positively charged (lysine), hydrophobic (tryptophan), and negatively charged (glutamic acid) amino acids, which has been found to promote hydrogen-bond arrangements conducive for b-sheet formation.[1b, 8] In the present study, we performed systematic simulations of b-barrel nanoring structures to provide a basic understanding of the roles of various intermolecular interactions during bionanostructure formation of self-assembling peptides. We also examined simple ways of controlling the sizes of nanoring structures based on these insights.
Results and Discussion The constant-temperature molecular dynamics (MD) simulations used for studying various polypeptides were performed under the condition of room temperature for sufficiently long durations to enable the examination of the stabilities of the initial structures. To quantify the degree of convergence, time evolutions of the radii of gyration (Rg) or root-mean-square deviation (RMSD) were computed. For T1 (Figure 1a), the initial structures examined were the planar antiparallel b sheets. The monolayers of these antiparallel b sheets were found to be unstable. The bilayers of T1 structures, with antiparallel configurations both within the plane and between the two planes, maintained stable structures during our simulations, as illustrated for the 24-mers shown in Figure 2. We also found that
Figure 2. Representative configuration of the bilayer structure for the 24mer of the T1 peptide. The left and right figures show the top and side views, respectively. The arrows represent antiparallel b sheets and the indole rings of the tryptophan residues are shown explicitly.
Figure 1. Molecular structures of the designed peptides: a) T1 (WKWEWY*WKWEW), b) T3 (WKFEWY*FKWEF), c) T3 mutant (WKWEWY*FKFEF), and d) T3 mutant (FKFEFY*FKFEF), in which Y* represents the tyrosine derivatives that have had their hydroxyl groups replaced by oligoether dendrons. Chem. Asian J. 2015, 10, 1684 – 1689
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Full Paper the indole rings of tryptophan (W) residues were alternatively arranged with respect to the plane containing the monomer backbones. Such an alternative arrangement of W residues appeared to be important for stabilizing the bilayer structure of T1. For wild-type T3 (Figure 1b), initial structures were constructed to mimic b-barrel nanoring structures. As mentioned above, the design principle for constructing such nanoring structures is based on the fact that the placement of bulky hydrophilic dendrons in the central region of b-sheet peptides induces curvature at the interface between the b strands. To determine the optimal number of monomers required to increase the stability of the resulting nanoring structures, we constructed several initial structures of differing sizes. Our results showed that smaller (12-mer and 24-mer) and bigger (50-mer) nanoring structures became unstable at the early stages of the simulations (Figure 3). However, for the 40-mer version of T3, the
Figure 3. Representative configurations of the nanoring structures for the a) 24-mer, b) 40-mer, and c) 50-mer of the T3 peptide. The arrows represent antiparallel b sheets and the indole rings of the tryptophan residues (blue) and the oligoether dendrons (red) are shown explicitly. The time dependence of the RMSD from the simulations for the three cases are also shown.
Conclusion
nanoring structure was found to be stable and maintained its structure during the MD simulations, as demonstrated by measuring structural quantities such as the Rg, RMSD, and atomic fluctuations. The simulation showed that the diameter for the inner pore was 3.5–4.5 nm, whereas the whole structure had a diameter of approximately 11 nm and a thickness of Chem. Asian J. 2015, 10, 1684 – 1689
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about 4 nm, which was consistent with the experimental observations. In the stable nanoring structures of T3, the indole rings of the Trp residues were located inside the pore, whereas the oligoether dendrons occupied the space outside the ring. The optimal size of the nanoring structures was determined by minimizing the repulsions due to steric hindrance between these bulky groups, both on the inside and outside of the pore structures. For the smaller initial structures (12-mer and 24-mer), crowding of indole rings inside the pore led to instability of the ring structures. The stability of the b-barrel nanoring structures was mainly due to hydrogen-bond interactions between neighboring b-sheet chains. To sustain stable structures, it is necessary that the neighboring chains are close enough to maintain favorable hydrogen-bond interactions. For the larger initial structure (50-mer), the average distances between the neighboring b sheets in the nanoring were too large to support strong hydrogen-bond interactions. Indeed, simple geometric considerations regarding the shape and size of the T3 monomer, optimized for minimal repulsion and maximal hydrogen bonding, would predict the formation of stable nanoring structures for the 40-mer version of the T3 peptide. The results described above suggest a means for controlling the sizes of b-barrel nanoring structures. Instead of the six Trp residues in the original b peptide, one can introduce residues with smaller side chains such as phenylalanine (F). We made W-to-F mutants of the T3 peptide in which all or half of the tryptophan residues were replaced by phenylalanine. The smaller benzene ring of phenylalanine relative to the indole ring of tryptophan allowed the nanorings to adopt smaller pore sizes. We performed simulations on nanoring structures with differing numbers of monomers for the all-W-to-F (Figure 1d) and the half-W-to-F (Figure 1c) mutants of the T3 peptide. It was found that the 20-mer of the all-W-to-F mutant and the 32-mer of the half-W-to-F mutant could form stable nanoring structures (Figure 4). Another interesting finding was that different arrangements in the half-W-to-F mutant of the T3 peptide, in which the Trp and Phe residues are alternatively placed along the peptide (WKFEWY*FKWEF), could not support stable nanoring structures. W-to-F mutants of the T3 peptide were found to form nanoring structures with somewhat smaller inner pore sizes. Compared with a pore size of about 4 nm for the nanoring of the original T3 peptide, the pore sizes for the nanoring structures of the all-W-to-F and the half-W-to-F mutants were approximately 2–3 nm and approximately 3– 4 nm, respectively.
We performed systematic MD simulations on b-barrel nanoring structures formed by various b-sheet peptides. The goal of the present study was to shed light on important factors that enhance the stabilities of such structures and to provide design principles for constructing well-defined bionanostructures. It was observed that the T3 peptide with bulky hydrophilic dendrons tended to form a nanoring structure with the indole rings of the Trp residues located inside the pore and the oli-
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Figure 4. Representative configurations of the nanoring structures for a) 20mer of all-W-to-F mutant, b) 32-mer of half-W-to-F mutant, and c) 40-mer of wild type for the T3 peptide. The left and right figures show the top and side views, respectively. The arrows represent antiparallel b sheets, and the indole rings of the tryptophan residues or the benzene rings of the phenylalanine residues (blue) and the oligoether dendrons (red) are shown explicitly.
goether dendrons occupying the space outside the ring. The size of the nanoring structure for the T3 peptide, predicted by the present simulations, was consistent with the experimental observations. The optimal size of a nanoring was determined by minimizing repulsions due to steric hindrance between bulky groups, both on the inside and outside of the pore structures, while maintaining favorable hydrogen-bond interactions between neighboring b-sheet chains. Based on our molecular modeling study with the T3 peptide, we proposed a simple way of controlling the sizes of the bbarrel nanoring structures. By introducing smaller side chains inside the pore structures, it was possible to obtain smaller nanoring structures due to the reduction of steric hindrance. We simulated W-to-F mutants of the T3 peptide in which all or half of the tryptophans were replaced by phenylalanine residues. It was found that different numbers of monomers for the all-W-to-F and the half-W-to-F mutants of T3 peptide could form stable nanoring structures. The pore sizes of the W-to-F mutant nanoring structures were smaller than those of the original T3 peptide. It was also noted that varying the position of the residues in the half-W-to-F mutant of the T3 peptide disrupted the stability of the nanoring structures. When smaller and larger side chains from the Trp and Phe residues were alternatively placed along the peptide (WKFEWY*FKWEF), it was less favorable to maintain optimal packing for both of the resiChem. Asian J. 2015, 10, 1684 – 1689
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dues. This result suggested that optimal packing of side chains inside the pore, which can provide favorable interactions, is also necessary to maintain nanoring structural stability. In conclusion, the formation of bionanostructures through peptide self-assembly is governed by a subtle interplay between unfavorable interactions, such as electrostatic repulsions and steric hindrance, and favorable interactions owing to hydrogen bonding and optimal packing. A basic understanding of these principles will facilitate the design and molecular characteristics of building blocks for such bionanostructures in such a way as to control the relative strengths of various interactions. It is interesting to note that the interior of the nanorings studied in the present work is hydrophobic. Experimental observation of stable nanorings for the T3 peptide suggested that instability due to the presence of a hydrophobic interior surface can be compensated by strong interactions such as hydrogen bonding. The fact that the hydrophobic part is buried inside the nanorings, instead of being exposed on the outside, may also contribute to the reduction of instability. Based on our simulations, we predicted that all-W-to-F and the half-Wto-F mutants of the T3 peptide could also form stable nanoring structures. It is expected that the predictions can be confirmed by future experiments. Our simulations showed that the T3 peptide and its mutants need a specific number of monomers to form stable nanorings. With a smaller and larger number of monomers, they might form other types of nanostructures other than nanoring structures. It is beyond the scope of the present simulations to investigate the formation of different structures from arbitrary (disordered) initial structures. Developing a coarse-grained model of the peptides and performing simulations on the spontaneous formation of various bionanostructures will be the subject of future studies.
Experimental Section Simulations of the b-Sheet Peptide T1 The b-sheet peptide T1 (WKWEWYWKWEW) monomers were designed in the xleap module of the AMBER molecular dynamics (MD) software package,[9] and were then duplicated several times to make polypeptides. The orientations and distances between backbones were controlled to construct antiparallel b sheets using the Sirius program.[10] The initial structures were minimized in the AMBER ff96 force field[11] using a combination of the steepest descent and the conjugate gradient algorithms,[12] and then heated in 10 steps of 50 K up to 500 K over 150 ps in the Berendsen thermostat[13] with all Ca atoms fixed to maintain the bilayer structure. MD simulations were equilibrated for 50 ps at 300 K in the Langevin thermostat[14] (collision frequency of 0.5 ps¢1) without any restraints, and MD production simulations were performed under the same conditions. The bond-stretching freedoms of the hydrogen atoms were removed by the SHAKE algorithm,[15] and time steps of 2 fs were applied over all simulations. The cut-off distance (20 æ) for non-bonded interactions were applied without periodic boundary conditions. The AMBER ff96 force field was applied using a modified generalized Born/surface area solvation model,[16] in which the effective Born radii were re-scaled to approximate the
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Full Paper interstitial spaces between atomic spheres to better assess the behaviors of various peptides.
Simulations of the Oligoether Dendron-Functionalized bSheet Peptide T3 b-Sheet peptides were designed to have tyrosine derivatives with attached oligoether dendrons. The force field parameters for the oligoether dendrons were determined for MD simulations. An initial structure of the oligoether dendron was prepared manually and capped by N-terminal acetylation (CH3CHO¢) and C-terminal amidation (¢NHCH3) for charge neutrality. This structure was geometrically optimized and subsequently confirmed by frequency computation by using density functional theory (DFT) at the B3LYP/6-31G(d) level.[17] It is noteworthy that geometries in the library of the Amber force field were optimized at the MP2/6-31G(d) level, and the DFT is comparable to the second-order Møller–Plesset (MP2) perturbation theory.[18] By using this optimized structure, electrostatic potentials were computed at the HF/6-31G(d) level, and charges in the oligoether dendron moiety were obtained using the restrained electrostatic potential (RESP) procedure[19] and the resp module of AMBER.[9] These new parameters and topologies were integrated into the AMBER force field 96 parameter set (ff96). Charges of the acetyl and amide capping groups were supplied from the AMBER ff96 force-field library without modification. Any missing parameters were supplied from the general AMBER force-field parameter set.[20] After the b-sheet peptide was generated, the hydroxyl group of the tyrosine residue was replaced with the prepared oligoether dendron. The resulting structure, utilized as a building block, was replicated to construct polypeptides with a specific number of peptides as candidates for forming a b-barrel-like structure. Once T3 (WKWEWYWKWEW) monomers were generated in the xleap module, they were replicated 11, 23, 39, or 49 times to yield 12mers, 24-mers, 40-mers, or 50-mers. The orientations and distances between the monomer backbones were adjusted to form closed anti-parallel b sheets, that is, nanorings, with the Sirius program. The initial structures were minimized by using a limited-memory Broyden–Fletcher–Goldfarb–Shanno quasi-Newton algorithm,[21] and heated in 10 steps of 50 K up to 500 K over 150 ps in the Berendsen thermostat, with the Ca atoms fixed to maintain the bbarrel nanoring-like structure. The equilibration and productionMD simulations were performed as done for T1. T3 mutants were generated from the wild-type T3 structure by introducing point mutations. Minimization, heating, equilibration, and production steps during MD simulations were performed similarly to the conditions used for T3. The Amber10 and the Gaussian 03[22] programs were used for MD simulations and quantum computations, respectively. Computations of the radii of gyration (Rg), atomic fluctuations, and RMSD values, as well as cluster analyses were performed by using analysis tools supplied in the AMBER software package.[9] The representative configurations of the bilayer structures (T1) and the nanoring structures (T3) were obtained by performing a cluster analysis of trajectories of the corresponding simulations. Figures 2–4 show the most populated structure of the representative configurations for different cases.
Acknowledgements This work was supported by the National Research Foundation of Korea (NRF) grants funded by the Korea government (MSIP) (no. 2007-0056095 (CMD), no. 2012M3C1A6035358 (EDISON), Chem. Asian J. 2015, 10, 1684 – 1689
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Manuscript received: April 28, 2015 Accepted article published: June 6, 2015 Final article published: July 7, 2015
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Molecular Dynamics
A Molecular Dynamics Study on Controlling the Self-Assembly of b-Sheet Peptides with Designer Nanorings SeongByeong Park,[a] Myungsoo Lee,[b] and Seokmin Shin*[a] Abstract: Recently, a rational approach for constructing bbarrel protein mimics by the self-assembly of peptide-based building blocks has been demonstrated. We performed molecular dynamics simulations of nanoring formation by means of the self-assembly of designed b-sheet-forming peptides. Several factors contributing to the stability of the nanoring structures with respect to size were investigated. Our simulations predicted that an optimal nanoring size may be achieved by minimizing repulsions due to steric hin-
Introduction Bionanostructures, including peptide-based self-assembled nanostructures, have attracted enormous interest in recent years.[1] This interest reflects the fact that artificial bionanostructures can not only mimic the functional properties of biological systems such as proteins or subcellular organelles, but may also have properties that are unprecedented in nature. Designing synthetic self-assembling building blocks provides one of the most versatile approaches for constructing diverse artificial bionanostructures. To design optimal building blocks, it is necessary to understand the major driving forces responsible for a noncovalent self-assembly process during the formation of a particular bionanostructure. Noncovalent interactions play important roles in self-assembly processes; thus, the careful design of building blocks for self-assembling molecules is one of the key factors involved in fabricating non-natural bionanostructures. b-Barrel peptides are normally observed in cytoplasmic and transmembrane proteins. Cytoplasmic proteins are soluble in water, but transmembrane proteins are insoluble and amphiphilic. The green fluorescent protein is a well-studied cytoplas[a] S. Park, Prof. S. Shin Department of Chemistry Seoul National University Seoul 151-747 (Korea) E-mail: [email protected] [b] Prof. M. Lee State Key Laboratory of Supramolecular Structure and Materials College of Chemistry Jilin University Changchun 130012 (China) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/asia.201500438. Chem. Asian J. 2015, 10, 1684 – 1689
drance between bulky groups while maintaining favorable hydrogen-bond interactions between neighboring b-sheet chains. It was shown that mutations in a test peptide, in which all or half of the tryptophan residues were replaced by phenylalanine, could enable the assembly of stable nanoring structures with smaller pore sizes. Insights into the fundamental factors driving the formation of peptide-based nanostructures are expected to facilitate the design of novel functional bionanostructures.
mic protein that exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range.[2, 3] Transmembrane b-barrel proteins are often found in the outer membranes of bacteria, mitochondria, and chloroplasts. Fewer than 20 three-dimensional b-barrel structures have been described previously, although genomic databases contain thousands of b barrels.[4] In structural terms, a b barrel is a closed structure in which the first and last b sheets in a polypeptide are linked by hydrogen bonds. Adjacent b sheets in the b barrel are generally arranged in the antiparallel mode. Inspired by this natural structure, many b-barrel-mimicking peptides have been synthesized.[5] These artificial biopeptides have highlighted the potential for constructing artificial membrane pores or channels with chemical compositions and structures different from those found in nature.[4] Artificial biopeptides may potentially mimic natural proteins or cellular organelles, or even exhibit an enhancement of their functional properties. In addition, artificial biopeptides are anticipated to show characteristics that are unparalleled in nature. If synthetic pathways for forming artificial biopeptides such as b barrels become precisely defined, this could enable further breakthroughs for manipulating novel b-barrel structures. The b strands within b-barrel folds are composed of natural amino acids and are normally aligned roughly parallel to lipid bilayers, spanning the entire membrane.[4] Recently, we developed dual-function b-barrel protein mimics through the selfassembly of b-sheet-forming peptides.[6] The unique feature of the system was the formation of highly uniform and discrete water-soluble b-barrel nanoring structures with a hydrophobic interior. This structure and composition was highly similar to that found in natural b-barrel proteins, which in turn can coalesce into transmembrane b-barrel pores by simple manipulations of their molecular structure.
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Full Paper b-Sheet ribbons are organized in a cross-b structure, wherein each b strand runs perpendicular to the one-dimensional (1D) ribbon axis.[7] Previously, we proposed a fundamental design principle for constructing nanoring structures from b-sheet peptides. Considering that nanorings form highly curved structures, we hypothesized that the induction of curvature between adjacent b strands would force the 1D axis to bend. Based on this hypothesis, we designed T-shape b-sheet peptide building blocks such that bulky hydrophilic dendrons placed at the central region of b-sheet-forming peptides might induce curvature at the interface between b strands. The organic/peptide hybrid T-shape building blocks consisted of a bsheet-forming peptide and a hydrophilic oligoether dendron. The dendron was symmetrically placed on the side face of the peptide backbone. The artificially designed b-sheet peptide had a repeating structure of hydrophobic (tryptophan), positively charged (lysine), hydrophobic (tryptophan), and negatively charged (glutamic acid) amino acids, which has been found to promote hydrogen-bond arrangements conducive for b-sheet formation.[1b, 8] In the present study, we performed systematic simulations of b-barrel nanoring structures to provide a basic understanding of the roles of various intermolecular interactions during bionanostructure formation of self-assembling peptides. We also examined simple ways of controlling the sizes of nanoring structures based on these insights.
Results and Discussion The constant-temperature molecular dynamics (MD) simulations used for studying various polypeptides were performed under the condition of room temperature for sufficiently long durations to enable the examination of the stabilities of the initial structures. To quantify the degree of convergence, time evolutions of the radii of gyration (Rg) or root-mean-square deviation (RMSD) were computed. For T1 (Figure 1a), the initial structures examined were the planar antiparallel b sheets. The monolayers of these antiparallel b sheets were found to be unstable. The bilayers of T1 structures, with antiparallel configurations both within the plane and between the two planes, maintained stable structures during our simulations, as illustrated for the 24-mers shown in Figure 2. We also found that
Figure 2. Representative configuration of the bilayer structure for the 24mer of the T1 peptide. The left and right figures show the top and side views, respectively. The arrows represent antiparallel b sheets and the indole rings of the tryptophan residues are shown explicitly.
Figure 1. Molecular structures of the designed peptides: a) T1 (WKWEWY*WKWEW), b) T3 (WKFEWY*FKWEF), c) T3 mutant (WKWEWY*FKFEF), and d) T3 mutant (FKFEFY*FKFEF), in which Y* represents the tyrosine derivatives that have had their hydroxyl groups replaced by oligoether dendrons. Chem. Asian J. 2015, 10, 1684 – 1689
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Full Paper the indole rings of tryptophan (W) residues were alternatively arranged with respect to the plane containing the monomer backbones. Such an alternative arrangement of W residues appeared to be important for stabilizing the bilayer structure of T1. For wild-type T3 (Figure 1b), initial structures were constructed to mimic b-barrel nanoring structures. As mentioned above, the design principle for constructing such nanoring structures is based on the fact that the placement of bulky hydrophilic dendrons in the central region of b-sheet peptides induces curvature at the interface between the b strands. To determine the optimal number of monomers required to increase the stability of the resulting nanoring structures, we constructed several initial structures of differing sizes. Our results showed that smaller (12-mer and 24-mer) and bigger (50-mer) nanoring structures became unstable at the early stages of the simulations (Figure 3). However, for the 40-mer version of T3, the
Figure 3. Representative configurations of the nanoring structures for the a) 24-mer, b) 40-mer, and c) 50-mer of the T3 peptide. The arrows represent antiparallel b sheets and the indole rings of the tryptophan residues (blue) and the oligoether dendrons (red) are shown explicitly. The time dependence of the RMSD from the simulations for the three cases are also shown.
Conclusion
nanoring structure was found to be stable and maintained its structure during the MD simulations, as demonstrated by measuring structural quantities such as the Rg, RMSD, and atomic fluctuations. The simulation showed that the diameter for the inner pore was 3.5–4.5 nm, whereas the whole structure had a diameter of approximately 11 nm and a thickness of Chem. Asian J. 2015, 10, 1684 – 1689
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about 4 nm, which was consistent with the experimental observations. In the stable nanoring structures of T3, the indole rings of the Trp residues were located inside the pore, whereas the oligoether dendrons occupied the space outside the ring. The optimal size of the nanoring structures was determined by minimizing the repulsions due to steric hindrance between these bulky groups, both on the inside and outside of the pore structures. For the smaller initial structures (12-mer and 24-mer), crowding of indole rings inside the pore led to instability of the ring structures. The stability of the b-barrel nanoring structures was mainly due to hydrogen-bond interactions between neighboring b-sheet chains. To sustain stable structures, it is necessary that the neighboring chains are close enough to maintain favorable hydrogen-bond interactions. For the larger initial structure (50-mer), the average distances between the neighboring b sheets in the nanoring were too large to support strong hydrogen-bond interactions. Indeed, simple geometric considerations regarding the shape and size of the T3 monomer, optimized for minimal repulsion and maximal hydrogen bonding, would predict the formation of stable nanoring structures for the 40-mer version of the T3 peptide. The results described above suggest a means for controlling the sizes of b-barrel nanoring structures. Instead of the six Trp residues in the original b peptide, one can introduce residues with smaller side chains such as phenylalanine (F). We made W-to-F mutants of the T3 peptide in which all or half of the tryptophan residues were replaced by phenylalanine. The smaller benzene ring of phenylalanine relative to the indole ring of tryptophan allowed the nanorings to adopt smaller pore sizes. We performed simulations on nanoring structures with differing numbers of monomers for the all-W-to-F (Figure 1d) and the half-W-to-F (Figure 1c) mutants of the T3 peptide. It was found that the 20-mer of the all-W-to-F mutant and the 32-mer of the half-W-to-F mutant could form stable nanoring structures (Figure 4). Another interesting finding was that different arrangements in the half-W-to-F mutant of the T3 peptide, in which the Trp and Phe residues are alternatively placed along the peptide (WKFEWY*FKWEF), could not support stable nanoring structures. W-to-F mutants of the T3 peptide were found to form nanoring structures with somewhat smaller inner pore sizes. Compared with a pore size of about 4 nm for the nanoring of the original T3 peptide, the pore sizes for the nanoring structures of the all-W-to-F and the half-W-to-F mutants were approximately 2–3 nm and approximately 3– 4 nm, respectively.
We performed systematic MD simulations on b-barrel nanoring structures formed by various b-sheet peptides. The goal of the present study was to shed light on important factors that enhance the stabilities of such structures and to provide design principles for constructing well-defined bionanostructures. It was observed that the T3 peptide with bulky hydrophilic dendrons tended to form a nanoring structure with the indole rings of the Trp residues located inside the pore and the oli-
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Figure 4. Representative configurations of the nanoring structures for a) 20mer of all-W-to-F mutant, b) 32-mer of half-W-to-F mutant, and c) 40-mer of wild type for the T3 peptide. The left and right figures show the top and side views, respectively. The arrows represent antiparallel b sheets, and the indole rings of the tryptophan residues or the benzene rings of the phenylalanine residues (blue) and the oligoether dendrons (red) are shown explicitly.
goether dendrons occupying the space outside the ring. The size of the nanoring structure for the T3 peptide, predicted by the present simulations, was consistent with the experimental observations. The optimal size of a nanoring was determined by minimizing repulsions due to steric hindrance between bulky groups, both on the inside and outside of the pore structures, while maintaining favorable hydrogen-bond interactions between neighboring b-sheet chains. Based on our molecular modeling study with the T3 peptide, we proposed a simple way of controlling the sizes of the bbarrel nanoring structures. By introducing smaller side chains inside the pore structures, it was possible to obtain smaller nanoring structures due to the reduction of steric hindrance. We simulated W-to-F mutants of the T3 peptide in which all or half of the tryptophans were replaced by phenylalanine residues. It was found that different numbers of monomers for the all-W-to-F and the half-W-to-F mutants of T3 peptide could form stable nanoring structures. The pore sizes of the W-to-F mutant nanoring structures were smaller than those of the original T3 peptide. It was also noted that varying the position of the residues in the half-W-to-F mutant of the T3 peptide disrupted the stability of the nanoring structures. When smaller and larger side chains from the Trp and Phe residues were alternatively placed along the peptide (WKFEWY*FKWEF), it was less favorable to maintain optimal packing for both of the resiChem. Asian J. 2015, 10, 1684 – 1689
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dues. This result suggested that optimal packing of side chains inside the pore, which can provide favorable interactions, is also necessary to maintain nanoring structural stability. In conclusion, the formation of bionanostructures through peptide self-assembly is governed by a subtle interplay between unfavorable interactions, such as electrostatic repulsions and steric hindrance, and favorable interactions owing to hydrogen bonding and optimal packing. A basic understanding of these principles will facilitate the design and molecular characteristics of building blocks for such bionanostructures in such a way as to control the relative strengths of various interactions. It is interesting to note that the interior of the nanorings studied in the present work is hydrophobic. Experimental observation of stable nanorings for the T3 peptide suggested that instability due to the presence of a hydrophobic interior surface can be compensated by strong interactions such as hydrogen bonding. The fact that the hydrophobic part is buried inside the nanorings, instead of being exposed on the outside, may also contribute to the reduction of instability. Based on our simulations, we predicted that all-W-to-F and the half-Wto-F mutants of the T3 peptide could also form stable nanoring structures. It is expected that the predictions can be confirmed by future experiments. Our simulations showed that the T3 peptide and its mutants need a specific number of monomers to form stable nanorings. With a smaller and larger number of monomers, they might form other types of nanostructures other than nanoring structures. It is beyond the scope of the present simulations to investigate the formation of different structures from arbitrary (disordered) initial structures. Developing a coarse-grained model of the peptides and performing simulations on the spontaneous formation of various bionanostructures will be the subject of future studies.
Experimental Section Simulations of the b-Sheet Peptide T1 The b-sheet peptide T1 (WKWEWYWKWEW) monomers were designed in the xleap module of the AMBER molecular dynamics (MD) software package,[9] and were then duplicated several times to make polypeptides. The orientations and distances between backbones were controlled to construct antiparallel b sheets using the Sirius program.[10] The initial structures were minimized in the AMBER ff96 force field[11] using a combination of the steepest descent and the conjugate gradient algorithms,[12] and then heated in 10 steps of 50 K up to 500 K over 150 ps in the Berendsen thermostat[13] with all Ca atoms fixed to maintain the bilayer structure. MD simulations were equilibrated for 50 ps at 300 K in the Langevin thermostat[14] (collision frequency of 0.5 ps¢1) without any restraints, and MD production simulations were performed under the same conditions. The bond-stretching freedoms of the hydrogen atoms were removed by the SHAKE algorithm,[15] and time steps of 2 fs were applied over all simulations. The cut-off distance (20 æ) for non-bonded interactions were applied without periodic boundary conditions. The AMBER ff96 force field was applied using a modified generalized Born/surface area solvation model,[16] in which the effective Born radii were re-scaled to approximate the
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Full Paper interstitial spaces between atomic spheres to better assess the behaviors of various peptides.
Simulations of the Oligoether Dendron-Functionalized bSheet Peptide T3 b-Sheet peptides were designed to have tyrosine derivatives with attached oligoether dendrons. The force field parameters for the oligoether dendrons were determined for MD simulations. An initial structure of the oligoether dendron was prepared manually and capped by N-terminal acetylation (CH3CHO¢) and C-terminal amidation (¢NHCH3) for charge neutrality. This structure was geometrically optimized and subsequently confirmed by frequency computation by using density functional theory (DFT) at the B3LYP/6-31G(d) level.[17] It is noteworthy that geometries in the library of the Amber force field were optimized at the MP2/6-31G(d) level, and the DFT is comparable to the second-order Møller–Plesset (MP2) perturbation theory.[18] By using this optimized structure, electrostatic potentials were computed at the HF/6-31G(d) level, and charges in the oligoether dendron moiety were obtained using the restrained electrostatic potential (RESP) procedure[19] and the resp module of AMBER.[9] These new parameters and topologies were integrated into the AMBER force field 96 parameter set (ff96). Charges of the acetyl and amide capping groups were supplied from the AMBER ff96 force-field library without modification. Any missing parameters were supplied from the general AMBER force-field parameter set.[20] After the b-sheet peptide was generated, the hydroxyl group of the tyrosine residue was replaced with the prepared oligoether dendron. The resulting structure, utilized as a building block, was replicated to construct polypeptides with a specific number of peptides as candidates for forming a b-barrel-like structure. Once T3 (WKWEWYWKWEW) monomers were generated in the xleap module, they were replicated 11, 23, 39, or 49 times to yield 12mers, 24-mers, 40-mers, or 50-mers. The orientations and distances between the monomer backbones were adjusted to form closed anti-parallel b sheets, that is, nanorings, with the Sirius program. The initial structures were minimized by using a limited-memory Broyden–Fletcher–Goldfarb–Shanno quasi-Newton algorithm,[21] and heated in 10 steps of 50 K up to 500 K over 150 ps in the Berendsen thermostat, with the Ca atoms fixed to maintain the bbarrel nanoring-like structure. The equilibration and productionMD simulations were performed as done for T1. T3 mutants were generated from the wild-type T3 structure by introducing point mutations. Minimization, heating, equilibration, and production steps during MD simulations were performed similarly to the conditions used for T3. The Amber10 and the Gaussian 03[22] programs were used for MD simulations and quantum computations, respectively. Computations of the radii of gyration (Rg), atomic fluctuations, and RMSD values, as well as cluster analyses were performed by using analysis tools supplied in the AMBER software package.[9] The representative configurations of the bilayer structures (T1) and the nanoring structures (T3) were obtained by performing a cluster analysis of trajectories of the corresponding simulations. Figures 2–4 show the most populated structure of the representative configurations for different cases.
Acknowledgements This work was supported by the National Research Foundation of Korea (NRF) grants funded by the Korea government (MSIP) (no. 2007-0056095 (CMD), no. 2012M3C1A6035358 (EDISON), Chem. Asian J. 2015, 10, 1684 – 1689
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Manuscript received: April 28, 2015 Accepted article published: June 6, 2015 Final article published: July 7, 2015
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