The Two‐Step Assemblies of Basic‐Amino‐Acid‐Rich Peptide with a Highly Charged Polyoxometalate

Two‐step assembly of a peptide from HPV16 L1 with a highly charged europium‐substituted polyoxometalate (POM) cluster, accompanying a great luminescence enhancement of the inorganic polyanions, is reported. The mechanism is discussed in detail by analyzing the thermodynamic parameters from isothermal titration calorimetry (ITC), time‐resolved fluorescent and NMR spectra. By comparing the actions of the peptide analogues, a binding process and model are proposed accordingly. The driving forces in each binding step are clarified, and the initial POM aggregation, basic‐sequence and hydrophobic C termini of peptide are revealed to contribute essentially to the two‐step assembly. The present study demonstrates both a meaningful preparation for bioinorganic materials and a strategy using POMs to modulate the assembly of peptides and even proteins, which could be extended to other proteins and/or viruses by using peptides and POMs with similar properties.     

Folding Dynamics of 10‐Residue β‐Hairpin Peptide Chignolin

Short peptides that fold into β‐hairpins are ideal model systems for investigating the mechanism of protein folding because their folding process shows dynamics typical of proteins. We performed folding, unfolding, and refolding molecular dynamics simulations (total of 2.7 μs) of the 10‐residue β‐hairpin peptide chignolin, which is the smallest β‐hairpin structure known to be stable in solution. Our results revealed the folding mechanism of chignolin, which comprises three steps. First, the folding begins with hydrophobic assembly. It brings the main chain together; subsequently, a nascent turn structure is formed. The second step is the conversion of the nascent turn into a tight turn structure along with interconversion of the hydrophobic packing and interstrand hydrogen bonds. Finally, the formation of the hydrogen‐bond network and the complete hydrophobic core as well as the arrangement of side‐chain–side‐chain interactions occur at approximately the same time. This three‐step mechanism appropriately interprets the folding process as involving a combination of previous inconsistent explanations of the folding mechanism of the β‐hairpin, that the first event of the folding is formation of hydrogen bonds and the second is that of the hydrophobic core, or vice versa.     

Molecular Design of β‐Sheet Peptide for the Multi‐Modal Analysis of Disease

Intermolecular forces constrain peptide conformation. However, the role of each intermolecular force in constraining peptide conformation remains poorly understood. In this work, we show that aromatic–aromatic interactions drive peptides into β‐sheets, and the hydrophobic effect determines the assembly speed of peptides. By using intermolecular forces to artificially control the assembly of β‐sheets, a multi‐modal analytical system was developed that allows five readouts and dual qualitative–quantitative analysis, and satisfies both point‐of‐care testing (POCT) and laboratory‐based testing. For Mycoplasma Pneumoniae diagnosis, this system eradicates misdiagnosis (from 30 % to 0 %) and broadens the linear range by three‐fold, both of which are critical for guiding therapy. This work not only illustrates exact roles of intermolecular forces in driving the formation of β‐sheets, but also provides a guideline for the construction of a multi‐modal analytical system for disease diagnosis.     

Folding induced assembly of polypeptide decorated gold nanoparticles

Reversible assembly of gold nanoparticles controlled by the homodimerization and folding of an immobilized de novo designed synthetic polypeptide is described. In solution at neutral pH, the polypeptide folds into a helix-loop-helix four-helix bundle in the presence of zinc ions. When immobilized on gold nanoparticles, the addition of zinc ions induces dimerization and folding between peptide monomers located on separate particles, resulting in rapid particle aggregation. The particles can be completely redispersed by removal of the zinc ions from the peptide upon addition of EDTA. Calcium ions, which do not induce folding in solution, have no effect on the stability of the peptide decorated particles. The contribution from folding on particle assembly was further determined utilizing a reference peptide with the same primary sequence but containing both D and L amino acids. Particles functionalized with the reference peptide do not aggregate, as the peptides are unable to fold. The two peptides, linked to the nanoparticle surface via a cysteine residue located in the loop region, form submonolayers on planar gold with comparable properties regarding surface density, orientation, and ability to interact with zinc ions. These results demonstrate that nanoparticle assembly can be induced, controlled, and to some extent tuned, by exploiting specific molecular interactions involved in polypeptide folding.     

How does a hydrocarbon staple affect peptide hydrophobicity?

Water is essential for the proper folding of proteins and the assembly of protein–protein/ligand complexes. How water regulates complex formation depends on the chemical and topological details of the interface. The dynamics of water in the interdomain region between an E3 ubiquitin ligase (MDM2) and three different peptides derived from the tumor suppressor protein p53 are studied using molecular dynamics. The peptides show bimodal distributions of interdomain water densities across a range of distances. The addition of a hydrocarbon chain to rigidify the peptides (in a process known as stapling) results in an increase in average hydrophobicity of the peptide–protein interface. Additionally, the hydrophobic staple shields a network of water molecules, kinetically stabilizing a water chain hydrogen‐bonded between the peptide and MDM2. These properties could result in a decrease in the energy barrier associated with dehydrating the peptide–protein interface, thereby regulating the kinetics of peptide binding. © 2015 Wiley Periodicals, Inc.     

Macrocyclic beta-sheet peptides that mimic protein quaternary structure through intermolecular beta-sheet interactions

This paper reports the design, synthesis, and characterization of a family of cyclic peptides that mimic protein quaternary structure through beta-sheet interactions. These peptides are 54-membered-ring macrocycles comprising an extended heptapeptide beta-strand, two Hao beta-strand mimics [JACS 2000, 122, 7654] joined by one additional alpha-amino acid, and two delta-linked ornithine beta-turn mimics [JACS 2003, 125, 876]. Peptide 3a, as the representative of these cyclic peptides, contains a heptapeptide sequence (TSFTYTS) adapted from the dimerization interface of protein NuG2 [PDB ID: 1mio]. 1H NMR studies of aqueous solutions of peptide 3a show a partially folded monomer in slow exchange with a strongly folded oligomer. NOE studies clearly show that the peptide self-associates through edge-to-edge beta-sheet dimerization. Pulsed-field gradient (PFG) NMR diffusion coefficient measurements and analytical ultracentrifugation (AUC) studies establish that the oligomer is a tetramer. Collectively, these experiments suggest a model in which cyclic peptide 3a oligomerizes to form a dimer of beta-sheet dimers. In this tetrameric beta-sheet sandwich, the macrocyclic peptide 3a is folded to form a beta-sheet, the beta-sheet is dimerized through edge-to-edge interactions, and this dimer is further dimerized through hydrophobic face-to-face interactions involving the Phe and Tyr groups. Further studies of peptides 3b-3n, which are homologues of peptide 3a with 1-6 variations in the heptapeptide sequence, elucidate the importance of the heptapeptide sequence in the folding and oligomerization of this family of cyclic peptides. Studies of peptides 3b-3g show that aromatic residues across from Hao improve folding of the peptide, while studies of peptides 3h-3n indicate that hydrophobic residues at positions R3 and R5 of the heptapeptide sequence are important in oligomerization.     

Effects of turn stability on the kinetics of refolding of a hairpin in a beta-sheet

As part of our continuing study of the effects of the turn sequence on the conformational stability as well as the mechanism of folding of a beta-sheet structure, we have undertaken a parallel investigation of the solution structure, conformational stability, and kinetics of refolding of the beta-sheet VFIVDGOTYTEV(D)PGOKILQ. The latter peptide is an analogue of the original Gellman beta-sheet VFITS(D)PGKTYTEV(D)PGOKILQ, wherein the TS(D)PGK turn sequence in the first hairpin has been replaced by VDGO. Thermodynamics studies revealed comparable conformational stability of the two peptides. However, unlike the Gellman peptide, which showed extremely rapid refolding of the first hairpin, early kinetic events associated with the refolding of the corresponding hairpin in the VDGO mutant were found to be significantly slower. A detailed study of the conformation of the modified peptide suggested that hydrophobic interactions might be contributing to its stability. Accordingly, we surmise that the early kinetic events are sensitive to whether the formation of the hairpin is nucleated at the turn or by sequestering of the hydrophobic residues across the strand, before structural rearrangements to produce the nativelike topology. Nucleation of the hairpin at the turn is expected to be intrinsically rapid for a strong turn. However, if the process must involve collapse of hydrophobic side chains, the nucleation should be slower as solvent molecules must be displaced to sequester the hydrophobic residues. These findings reflect the contribution of different forces toward nucleation of hairpins in the mechanism of folding of beta-sheets.     

Engineering the Ionic Self‐Assembly of Polyoxometalates and Facial‐Like Peptides

The self‐assembly behavior of polyoxometalates (PMs) and facial‐like cationic peptides carrying lysine residues were systematically investigated. Circular dichroism and UV/Vis spectra demonstrated that the multivalent electrostatic attractions between polyanionic PMs and short peptides with protonated lysine residues initiated the conformational transition of peptide molecules from random‐coil to β‐sheet state, and subsequently the co‐assembly. TEM and atomic force microscopy (AFM) measurements showed that uniform nanofibers formed with decreasing size of the PMs or increasing the intermolecular forces of the peptides, such as through hydrogen‐bonding, hydrophobic, and/or π–π interactions. Additionally, the stability of the nanostructures can be improved by rational suppression of the electrostatic repulsion of the shell peptides covering the surface of the nanostructures. These results provide new insight into understanding the ionic self‐assembly of peptides and PMs and controlling their final morphology.     

Thermally reversible hydrogels via intramolecular folding and consequent self-assembly of a de novo designed peptide

A small de novo designed peptide (MAX3) is described that exhibits complete thermoreversible self-assembly into a hydrogel network. Importantly, a prerequisite to hydrogelation is that the peptide must first fold into a conformation conducive to self-assembly. At ambient temperature, MAX3 is unfolded, resulting in a low viscosity aqueous solution. On increasing the temperature, the peptide undergoes a unimolecular folding event, affording an amphiphilic beta-hairpin that consequently self-assembles into a hydrogel network. Increasing the temperature serves to dehydrate the nonpolar residues of the unfolded peptide and trigger folding via hydrophobic collapse. Cooling the resultant hydrogel results in beta-hairpin unfolding and consequent complete dissolution of the hydrogel. The temperature at which folding and consequent self-assembly into a rigid hydrogel occur can be tuned by altering the hydrophobicity of the peptides.     

Metal-coupled folding of Cys2His2 zinc-finger

Zinc-fingers, which widely exist in eukaryotic cell and play crucial roles in life processes, depend on the binding of zinc ion for their proper folding. To computationally study the zinc-coupled folding of the zinc-fingers, charge transfer and metal induced protonation/deprotonation effects have to be considered. Here, by attempting to implicitly account for such effects in classical molecular dynamics and performing intensive simulations with explicit solvent for the peptides with and without zinc binding, we investigate the folding of the Cys2His2-type zinc-finger motif and the coupling between the peptide folding and zinc binding. We find that zinc ion not only stabilizes the native structure but also participates in the whole folding process. It binds to the peptide at an early stage of folding and directs or modulates the folding and stabilizations of the component beta-hairpin and alpha-helix. Such a crucial role of zinc binding is mediated by the packing of the conserved hydrophobic residues. We also find that the packing of the hydrophobic residues and the coordination of the native ligands are coupled. Meanwhile, the processes of zinc binding, mis-ligation, ligand exchange, and zinc induced secondary structure conversion as well as the water behavior due to the involvement of zinc ion are characterized. Our results are in good agreement with related experimental observations and provide significant insight into the general mechanisms of the metal cofactor dependent protein folding and other metal-induced conformational changes of biological importance.     

Self-assembly of multidomain peptides: balancing molecular frustration controls conformation and nanostructure

A series of nine, frustrated, multidomain peptides is described in which forces favoring self-assembly into a nanofiber versus those favoring disassembly could be easily modified. The peptides are organized into an ABA block motif in which the central B block is composed of alternating hydrophilic and hydrophobic amino acids (glutamine and leucine, respectively). This alternation allows the amino acid side chains to segregate on opposite sides of the peptide backbone when it is in a fully extended beta-sheet conformation. In water, packing between two such peptides stabilizes the extended conformation by satisfying the desire of the leucine side chains to exclude themselves from the aqueous environment. Once in this conformation intermolecular backbone hydrogen bonding can readily take place between additional peptides eventually growing into high aspect ratio fibers. B block assembly may continue infinitely or until monomeric peptides are depleted from solution which results in an insoluble precipitate. Block A consists of a variable number of positively charged lysine residues whose electrostatic repulsion at pH 7 works against the desire of the B block to assemble. Here we show that balancing the forces of block A against B allows the formation of controlled length, individually dispersed, and fully soluble nanofibers with a width of 6 +/- 1 nm and length of 120 +/- 30 nm. Analysis by infrared, circular dichroism, and vitreous ice cryo-transmission electron microscopy reveals that the relative sizes of blocks A and B dictate the peptide secondary structure which in turn controls the resulting nanostructure. The system described epitomizes the use of molecular frustration in the design of finite self-assembled structures. These materials, and ones based on their architecture, may find applications where nanostructured control over fiber architecture and chemical functionality is required.     

Fabrication of Chiral Materials via Self‐Assembly and Biomineralization of Peptides

With different scales of chirality, chiral materials have various particular properties and potential applications in many fields. Peptides are the fundamental building units of biological systems, and a variety of ordered functional nanostructures are produced through self‐assembly and biomineralization of peptides in nature. This Personal Account describes chiral silica materials fabricated by using amphiphilic peptides as building blocks. Three particular biomineralization approaches are described based on different kinds of geometry of amphiphilic peptides: the influence of the specific amino acid proline in the peptide sequence, the hydrophilicity of amphiphilic peptides, and different kinds of hydrophobic tails in amphiphilic peptides. These strategies are useful for designing peptides toward the bottom‐up synthesis of nanomaterials as well as improving the understanding of the mechanism of peptide self‐assembly.     

Examination of the folding of a short alanine-based helical peptide with salt bridges using molecular dynamics simulation

A molecular dynamics simulation of the folding of a short alanine-based helical peptide of 17 residues with three Glu...Lys (i, i + 4) salt bridge pairs, referred to as the AEK17 peptide, was carried out. The simulation gave an estimated simulation folding time of 2.5 ns, shorter than 12 ns for an alanine-based peptide of 16 residues with three Lys residues only, referred to as the AK16 peptide, simulated previously. After folded, the AEK17 peptide had a helical content of 77%, in excellent agreement with the experimentally determined value of 80%. An examination of the folding pathways of AEK17 indicated that the peptide proceeded via three-turn helix conformations more than the helix-turn-helix conformation in the folding pathways. An analysis of interactions indicated that the formation of hydrogen bonds between Lys residue side chains and backbone carbonyls is a major factor in the abundant conformation of the three-turn helix intermediate. The substitution of three Ala with Glu residues reduces the extent of hydrophobic interaction in alanine-based AK peptides with the result that the breaking of the interactions of Lys epsilon-NH3+(side chain)...C=O(backbone) is a major activation action for the AEK17 to achieve a complete fold, in contrast to the AK16 peptide, in which breaking non-native hydrophobic interaction is the rate-determining step.     

Copper(II)‐Mediated Self‐Assembly of Hairpin Peptides and Templated Synthesis of CuS Nanowires

The self‐assembly of peptides and proteins under well‐controlled conditions underlies important nanostructuring processes that could be harnessed in practical applications. Herein, the synthesis of a new hairpin peptide containing four histidine residues is reported and the self‐assembly process mediated by metal ions is explored. The work involves the combined use of circular dichroism, NMR spectroscopy, UV/Vis spectroscopy, AFM, and TEM to follow the structural and morphological details of the metal‐coordination‐mediated folding and self‐assembly of the peptide. The results indicate that by forming a tetragonal coordination geometry with four histidine residues, copper(II) ions selectively trigger the peptide to fold and then self‐assemble into nanofibrils. Furthermore, the copper(II)‐bound nanofibrils template the synthesis of CuS nanowires, which display a near‐infrared laser‐induced thermal effect.     

Folding and translocation of the undecamer of poly-L-leucine across the water-hexane interface. A molecular dynamics study.

The undecamer of poly-L-leucine at the water-hexane interface is studied by molecular dynamics simulations. This represents a simple model relevant to folding and insertion of hydrophobic peptides into membranes. The peptide, initially placed in a random coil conformation on the aqueous side of the system, rapidly translocates toward the hexane phase and undergoes interfacial folding into an alpha-helix in the subsequent 36 ns. Folding is nonsequential and highly dynamic. The initially formed helical segment at the N-terminus of the undecamer becomes transiently broken and, subsequently, reforms before the remainder of the peptide folds from the C-terminus. The formation of intramolecular hydrogen bonds during the folding of the peptide is preceded by a dehydration of the participating polar groups, as they become immersed in hexane. Folding proceeds through a short-lived intermediate, a 3(10)-helix, which rapidly interconverts to an alpha-helix. Both helices contribute to the equilibrium ensemble of folded structures. The helical peptide is largely buried in hexane, yet remains adsorbed at the interface. Its preferred orientation is parallel to the interface, although the perpendicular arrangement with the N-terminus immersed in hexane is only slightly less favorable. In contrast, the reversed orientation is highly unfavorable, because it would require dehydration of C-terminus carbonyl groups that do not participate in intramolecular hydrogen bonding. For the same reason, the transfer of the undecamer from the interface to the bulk hexane is also unfavorable. The results suggest that hydrophobic peptides fold in the interfacial region and, simultaneously, translocate into the nonpolar side of the interface. It is further implied that peptide insertion into the membrane is accomplished by rotating from the parallel to the perpendicular orientation, most likely in such a way that the N-terminus penetrates the bilayer.     

Expected and unexpected results from combined beta-hairpin design elements

A model beta-hairpin dodecapeptide [EFGWVpGKWTIK] was designed by including a favorable D-ProGly Type II' beta-turn sequence and a Trp-zip interaction, while also incorporating a beta-strand unfavorable glycine residue in the N-terminal strand. This peptide is highly folded and monomeric in aqueous solution as determined by combined analysis with circular dichroism and 1H NMR spectroscopy. A peptide representing the folded conformation of the model beta-hairpin [cyclic(EFGWVpGKWTIKpG)] and a linear peptide representing the unfolded conformation [EFGWVPGKWTIK] yield unexpected relative deviations between the CD and 1H NMR spectroscopic results that are attributed to variations in the packing interactions of the aromatic side chains. Mutational analysis of the model beta-hairpin indicates that the Trp-zip interaction favors folding and stability relative to an alternate hydrophobic cluster between Trp and Tyr residues [EFGYVpGKWTIK]. The significance of select diagonal interactions in the model beta-hairpin was tested by rearranging the cross-strand hydrophobic interactions to provide a folded peptide [EWFGIpGKTYWK] displaying evidence of an unusual backbone conformation at the hydrophobic cluster. This unusual conformation does not appear to be a result of the glycine residue in the beta-strand, as replacement with a serine results in a peptide [EWFSIpGKTYWK] with a similar and seemingly characteristic CD spectrum. However, an alternate arrangement of hydrophobic residues with a Trp-zip interaction in a similar position to the parent beta-hairpin [EGFWVpGKWITK] results in a folded beta-hairpin conformation. The differences between side chain packing of these peptides precludes meaningful thermodynamic analysis and illustrates the caution necessary when interpreting beta-hairpin folding thermodynamics that are driven, at least in part, by aromatic cross strand interactions.     

Controlled aggregation of peptide-substituted perylene-bisimides

Controlled aggregation of perylene bisimides in multiple modes has been achieved via symmetric substitution with peptides. Using optical probes of aggregation, the balance of hydrophobic and electrostatic forces are found to play a key role in directing self assembly and are exploited via solvent, pH, and specific extrinsic ion effects.     

Molecular dynamics simulation of folding of a short helical peptide with many charged residues

A molecular dynamics simulation of the folding of conantokin-T (con-T), a short helical peptide with 5 helical turns of 21 amino acids with 10 charged residues, was carried out to examine folding pathways for this peptide and to predict the folding rate. In the 18 trajectories run at 300 K, 16 trajectories folded, with an averaged folding time of approximately 50 ns. Two trajectories did not fold in up to 200 ns simulation. The folded structure in folded trajectories is in good agreement with experimental structure. An analysis of the trajectories showed that, at the beginning of a few nanoseconds, helix formation started from residues 5-9 with assistance of a hydrophobic clustering involving Tyr5, Met8, and Leu9. The peptide formed a U-shape mainly due to charge-charge interactions between charged residues at the N- and C-terminus segments. In the next approximately 10 ns, several nonnative charge-charge interactions were broken and nonnative Gla10-Lys18 (this denotes a salt bridge between Gal10 and Lys18) and/or Gla10-Lys19 interactions appeared more frequently in this folding step and the peptide became a fishhook J-shape. From this structure, the peptide folded to the folded state in 7 of all 16 folded trajectories in approximately 15 ns. Alternatively, in approximately 30 ns, the con-T went to a conformation in an L-shape with 4 helical turns and a kink at the Arg13 and Gla14 segment in the other 9 trajectories. Con-T in the L-shape then required another approximately 15 ns to fold into the folded state. In addition, in overall folding times, the former 7 trajectories folded faster with the total folding times all shorter than 45 ns, while the latter 9 trajectories folded at a time longer than 45 ns, resulting in an average folding time of approximately 50 ns. Two major folding intermediates found in 2 nonfolded trajectories are stabilized by charge clusters of 5 and 6 charged residues, respectively. With inclusion of friction and solvent-solvent interactions, which were ignored in the present GB/SA solvation model, the folding time obtained above should be multiplied by a factor of 1.25-1.7 according to a previous, similar simulation study. This results in a folding time of 65-105 ns, slightly shorter than the folding time of 127 ns for an alanine-based peptide of the same length. This suggests that the energy barrier of folding for this type of peptides with many charged residues is slightly lower than alanine-based helical peptides by less than 1 kcal/mol.     

Contribution of individual zinc ligands to metal binding and peptide folding of zinc finger peptides

Little is known about the contribution of individual zinc-ligating amino acid residues for coupling between zinc binding and protein folding in zinc finger domains. To understand such roles of each zinc ligand, four zinc finger mutant peptides corresponding to the second zinc finger domain of Sp1 were synthesized. In the mutant peptides, glycine was substituted for one of four zinc ligands. Their metal binding and folding properties were spectroscopically characterized and compared to those of the native zinc finger peptide. In particular, the electronic charge-transfer and d-d bands of the Co(II)-substituted peptide complexes were used to examine the metal coordination number and geometry. Fluorescence emission studies revealed that the mutant peptides are capable of binding zinc despite removing one ligand. Circular dichroism results clearly showed the induction of an alpha-helix by zinc binding. In addition, the structures of certain mutant zinc finger peptides were simulated by molecular dynamics calculation. The information indicates that His23 and the hydrophobic core formed between the alpha-helix and the beta-sheet play an essential role in alpha-helix induction. This report demonstrates that each ligand does not contribute equally to alpha-helix formation and coordination geometry in the zinc finger peptide.     

Substrate-facilitated assembly of elastin-like peptides: studies by variable-temperature in situ atomic force microscopy

Extended duration in situ variable-temperature atomic force microscopy performed on a series of coacervating hydrophobic peptides based on human elastin revealed a novel multistage surface assembly process. Formation of epitaxially arranged peptide rods was preceded by rapid expansion of a two-dimensional film on the nucleating substrate, a process facilitated by specific hydrophobic peptide-substrate and intrapeptide interactions.