Impact of Peptide Sequence on Functional siRNA Delivery and Gene Knockdown with Cyclic Amphipathic Peptide Delivery Agents.

Short-interfering RNA (siRNA) oligonucleotide therapeutics that modify gene expression by accessing RNA-interference (RNAi) pathways have great promise for the treatment of a range of disorders; however, their application in clinical settings has been limited by significant challenges in cellular delivery. Herein, we report a structure-function study using a series of modified cyclic amphipathic cell-penetrating peptides (CAPs) to determine the impact of peptide sequence on (1) siRNA-binding efficiency, (2) cellular delivery and knockdown efficiency, and (3) the endocytic uptake mechanism. Nine cyclic peptides of the general sequence Ac-C[XZ]4CG-NH2 in which X residues are hydrophobic/aromatic (Phe, Tyr, Trp, or Leu) and Z residues are charged/hydrophilic (Arg, Lys, Ser, or Glu) are assessed along with one acyclic peptide, Ac-(WR)4G-NH2. Cyclization is enforced by intramolecular disulfide bond formation between the flanking Cys residues. Binding analyses indicate that strong cationic character and the presence of aromatic residues that are competent to participate in CH-π interactions lead to CAP sequences that most effectively interact with siRNA. CAP-siRNA binding increases in the following order as a function of CAP hydrophobic/aromatic content: His < Phe < Tyr < Trp. Both cationic charge and disulfide-constrained cyclization of CAPs improve uptake of siRNA in vitro. Net neutral CAPs and an acyclic peptide demonstrate less-efficient siRNA translocation compared to the cyclic, cationic CAPs tested. All CAPs tested facilitated efficient siRNA target gene knockdown of at least 50% (as effective as a lipofectamine control), with the best CAPs enabling >80% knockdown. Significantly, gene knockdown efficiency does not strongly correlate with CAP-siRNA internalization efficiency but moderately correlates with CAP-siRNA-binding affinity. Finally, utilization of small-molecule inhibitors and targeted knockdown of essential endocytic pathway proteins indicate that most CAP-siRNA nanoparticles facilitate siRNA delivery through clathrin- and caveolin-mediated endocytosis. These results provide insight into the design principles for CAPs to facilitate siRNA delivery and the mechanisms by which these peptides translocate siRNA into cells. These studies also demonstrate the nature of the relationships between peptide-siRNA binding, cellular delivery of siRNA cargo, and functional gene knockdown. Strong correlations between these properties are not always observed, which illustrates the complexity in the design of optimal next-generation materials for oligonucleotide delivery.

Defining the Landscape of the Pauling-Corey Rippled Sheet: An Orphaned Motif Finding New Homes.

When peptides are mixed with their mirror images in an equimolar ratio, two-dimensional periodic structural folds can form, in which extended peptide strands are arrayed with alternating chirality. The resultant topography class, termed the rippled ß-sheet, was introduced as a theoretical concept by Pauling and Corey in 1953. Unlike other fundamental protein structural motifs identified around that time, including the α-helix and the pleated ß-sheet, it took several decades before conclusive experimental data supporting the proposed rippled ß-sheet motif were gained. Much of the key experimental evidence was provided over the course of the past decade through the concurrent efforts of our three laboratories. Studies that focused on developing new self-assembling hydrogel materials have shown that certain amphiphilic peptides form fibrils and hydrogel networks that are more rigid and have a higher thermodynamic stability when made from racemic peptide mixtures as opposed to pure enantiomers. Related interrogation of assemblies composed of mixtures of l- and d-amphiphilic peptides confirmed that the resulting fibrils were composed of alternating l/d peptides consistent with rippled ß-sheets. It was also demonstrated that mirror-image amyloid beta (Aß) could act as a molecular chaperone to promote oligomer-to-fibril conversion of the natural Aß enantiomer, which was found to reduce Aß neurotoxicity against different neuronal cell models. With a cross-disciplinary approach that combines experiment and theory, our three laboratories have demonstrated the unique biophysical, biochemical, and biological properties that arise upon mixing of peptide enantiomers, in consequence of rippled ß-sheet formation. In this Account, we give an overview of the early history of the rippled ß-sheet and provide a detailed structural description/definition of this motif relative to the pleated ß-sheet. We then summarize the key findings, obtained on three unique sets of aggregating mirror-image peptide pairs through independent efforts of our three laboratories, and use these results to delineate the landscape of the rippled ß-sheet structural motif to inspire future studies. Peptide sequence parameters that favor rippled ß-sheet assembly are described, along with the accompanying kinetic and thermodynamic properties, as well as the resulting emergent physical properties of the assemblies. The Account then concludes with a brief overview of some key unresolved challenges in this nascent field. There is much potential for future applications of this unique supramolecular motif in the realm of materials design and biomedical research. We hope this Account will stimulate much-needed discussion of this fascinating structural class to eventually produce a fully quantitative, rational framework for the molecular engineering of rippled ß-sheets in the future.

Anion Effects on the Supramolecular Self-Assembly of Cationic Phenylalanine Derivatives.

Supramolecular hydrogels have emerged as a class of promising biomaterials for applications such as drug delivery and tissue engineering. Self-assembling peptides have been well studied for such applications, but low molecular weight (LMW) amino acid-derived gelators have attracted interest as low-cost alternatives with similar emergent properties. Fluorenylmethyloxycarbonyl-phenylalanine (Fmoc-Phe) is one such privileged motif often chosen due to its inherent self-assembly potential. Previously, we developed cationic Fmoc-Phe-DAP gelators that assemble into hydrogel networks in aqueous NaCl solutions of sufficient ionic strength. The chloride anions in these solutions screen the cationic charge of the gelators to enable self-assembly to occur. Herein, we report the effects of varying the anions of sodium salts on the gelation potential, nanoscale morphology, and hydrogel viscoelastic properties of Fmoc-Phe-DAP and two of its fluorinated derivatives, Fmoc-3F-Phe-DAP and Fmoc-F5-Phe-DAP. It was observed that both the anion identity and gelator structure had a significant impact on the self-assembly and gelation properties of these derivatives. Changing the anion identity resulted in significant polymorphism of the nanoscale morphology of the assembled states that was dependent on the chemical structure of the gelator. The emergent viscoelastic character of the hydrogel networks was also found to be reliant on the anion identity and gelator structure. These results demonstrate the complex interplay between the gelator and environment that have a profound and often unpredictable impact on both self-assembly properties and emergent viscoelasticity in supramolecular hydrogels formed by LMW compounds. This work also illustrates the current lack of understanding that limits the rational design of potential biomaterials that will be in contact with complex biological fluids and provides motivation for additional research to correlate the chemical structure of LMW gelators with the structure and emergent properties of the resulting supramolecular assemblies as a function of environment.

Side-chain halogen effects on self-assembly and hydrogelation of cationic phenylalanine derivatives.

Low molecular weight (LMW) supramolecular hydrogels have great potential as next-generation biomaterials for drug delivery, tissue engineering, and regenerative medicine. The design of LMW gelators is complicated by the lack of understanding regarding how the chemical structure of the gelator correlates to self-assembly potential and emergent hydrogel material properties. The fluorenylmethyloxycarbonyl-phenylalanine (Fmoc-Phe) motif is a privileged scaffold that is prone to undergo self-assembly into self-supporting hydrogel networks. Cationic Fmoc-Phe-DAP derivatives modified with diaminopropane (DAP) at the C-terminus have been developed that self-assemble into hydrogel networks in aqueous solutions of sufficient ionic strength. We report herein the impact of side-chain halogenation on the self-assembly and hydrogelation properties of Fmoc-Phe-DAP derivatives. A systematic study of the self-assembly and hydrogelation of monohalogenated Fmoc-Phe-DAP derivatives with F, Cl, or Br atoms in the ortho, meta, or para positions of the phenyl side chain reveal significant differences in self-assembly and gelation potential, nanoscale assembly morphology, and hydrogel viscoelastic properties as a function of halogen identity and substitution position. These results demonstrate the profound impact that subtle changes to the chemical scaffold can have on the behavior of LMW supramolecular gelators and illustrate the ongoing difficulty of predicting the emergent self-assembly and hydrogelation behavior of LMW gelators that differ even modestly in chemical structure.

Capacity for increased surface area in the hydrophobic core of ß-sheet peptide bilayer nanoribbons.

Amphipathic peptides with amino acids arranged in alternating patterns of hydrophobic and hydrophilic residues efficiently self-assemble into ß-sheet bilayer nanoribbons. Hydrophobic side chain functionality is effectively buried in the interior of the putative bilayer of these nanoribbons. This study investigates consequences on self-assembly of increasing the surface area of aromatic side chain groups that reside in the hydrophobic core of nanoribbons derived from Ac-(XKXE)2 -NH2 peptides (X = hydrophobic residue). A series of Ac-(XKXE)2 -NH2 peptides incorporating aromatic amino acids of increasing molecular volume and steric profile (X = phenylalanine [Phe], homophenylalanine [Hph], tryptophan [Trp], 1-naphthylalanine [1-Nal], 2-naphthylalanine [2-Nal], or biphenylalanine [Bip]) were assessed to determine substitution effects on self-assembly propensity and on morphology of the resulting nanoribbon structures. Additional studies were conducted to determine the effects of incorporating amino acids of differing steric profile in the hydrophobic core (Ac-X1 KFEFKFE-NH2 and Ac-(X1,5 KFE)-NH2 peptides, X = Trp or Bip). Spectroscopic analysis by circular dichroism (CD) and Fourier transform infrared (FT-IR) spectroscopy indicated ß-sheet formation for all variants. Self-assembly rate increased with peptide hydrophobicity; increased molecular volume of the hydrophobic side chain groups did not appear to induce kinetic penalties on self-assembly rates. Transmission electron microscopy (TEM) imaging indicated variation in fibril morphology as a function of amino acid in the X positions. This study confirms that hydrophobicity of amphipathic Ac-(XKXE)2 -NH2 peptides correlates to self-assembly propensity and that the hydrophobic core of the resulting nanoribbon bilayers has a significant capacity to accommodate sterically demanding functional groups. These findings provide insight that may be used to guide the exploitation of self-assembled amphipathic peptides as functional biomaterials.

Impact of gelation method on thixotropic properties of phenylalanine-derived supramolecular hydrogels.

Supramolecular hydrogels formed by noncovalent self-assembly of low molecular weight (LMW) agents are promising next-generation biomaterials. Thixotropic shear response and mechanical stability are two emergent properties of hydrogels that are critical for biomedical applications including drug delivery and tissue engineering in which injection of the hydrogel will be necessary. Herein, we demonstrate that the emergent thixotropic properties of supramolecular phenylalanine-derived hydrogels are dependent on the conditions in which they are formulated. Specifically, hydrogels formed from fluorenylmethoxycarbonyl (Fmoc) modified phenylalanine derivatives, 3-fluorophenylalanine (Fmoc-3F-Phe) and pentafluorophenylalanine (Fmoc-F5-Phe), were characterized as a function of gelation conditions to examine how shear response and mechanical stability properties correlate to mode of gelation. Two distinct methods of gelation were compared. First, spontaneous self-assembly and gelation was triggered by a solvent exchange method in which a concentrated solution of the gelator in dimethylsulfoxide was diluted in water. Second, gelation was promoted by dissolution of the gelator in water at basic pH followed by gradual pH adjustment from basic to mildly acidic by the hydrolysis of glucono-delta-lactone. Hydrogels formed under solvent exchange conditions were mechanically unstable and poorly shear-responsive whereas hydrogels formed by gradual acidification were temporally stable and had highly shear-responsive viscoelastic character. These studies confirm that gelation environment and mechanism have a significant influence on the emergent properties of supramolecular hydrogels and offer insight into how gelation conditions can be used to tune hydrogel properties for specific applications.

Strategy to Identify Improved N-Terminal Modifications for Supramolecular Phenylalanine-Derived Hydrogelators.

Supramolecular hydrogels formed by self-assembly of low molecular weight (LMW) compounds have been identified as promising materials for applications in tissue engineering and regenerative medicine. In many cases, the relationship between the chemical structure of the gelator and the emergent hydrogel properties is poorly understood. As a result, empirical screening strategies instead of rational design approaches are often relied upon to tune the emergent properties of the gels. Herein, we describe a novel strategy to identify improved phenylalanine (Phe) derived gelators using a focused empirical approach. Fluorenylmethoxycarbonyl (Fmoc) protected Phe derivatives are a privileged class of gelators that spontaneously self-assemble into fibrils that entangle to form a hydrogel network upon dissolution into water. However, the Fmoc group has been shown to have toxicity drawbacks for potential biological applications, requiring the identification of new N-terminal modifications that promote efficient self-assembly but lack the shortcomings of the Fmoc group. We previously discovered that fibrils in Fmoc-p-nitrophenylalanine (Fmoc-4-NO2-Phe) hydrogels transition to crystalline microtubes after several hours by a mechanism that involves the hierarchical assembly and fusion of the hydrogel fibrils. We hypothesized that this hierarchical crystallization behavior could form the basis of a screening approach to identify alternative N-terminal functional groups to replace Fmoc in Phe-derived LMW gelators. Specifically, screening N-terminal modifying groups for 4-NO2-Phe that stabilize the hydrogel state by preventing subsequent hierarchical crystallization would facilitate empirical identification of functional Fmoc replacements. To test this approach, we screened a small series of 4-NO2-Phe derivatives with various N-terminal modifying groups to determine if any provided stable LMW supramolecular hydrogels. All but one of the 4-NO2-Phe derivatives assembled into crystalline forms. Only the 1-naphthaleneacetic acid (1-Nap) 4-NO2-Phe derivative self-assembled into a stable hydrogel network. Additional Phe derivatives were modified by N-terminal 1-Nap groups to confirm the general potential of 1-Nap as a suitable replacement for Fmoc, and all derivatives formed stable hydrogels under similar conditions to their Fmoc-Phe counterparts. These results illustrate the potential of this approach to identify next-generation Phe-derived LMW gelators with improved emergent properties.

Multicomponent peptide assemblies.

Self-assembled peptide nanostructures have been increasingly exploited as functional materials for applications in biomedicine and energy. The emergent properties of these nanomaterials determine the applications for which they can be exploited. It has recently been appreciated that nanomaterials composed of multicomponent coassembled peptides often display unique emergent properties that have the potential to dramatically expand the functional utility of peptide-based materials. This review presents recent efforts in the development of multicomponent peptide assemblies. The discussion includes multicomponent assemblies derived from short low molecular weight peptides, peptide amphiphiles, coiled coil peptides, collagen, and ß-sheet peptides. The design, structure, emergent properties, and applications for these multicomponent assemblies are presented in order to illustrate the potential of these formulations as sophisticated next-generation bio-inspired materials.

Rippled ß-Sheet Formation by an Amyloid-ß Fragment Indicates Expanded Scope of Sequence Space for Enantiomeric ß-Sheet Peptide Coassembly.

In 1953, Pauling and Corey predicted that enantiomeric ß-sheet peptides would coassemble into so-called "rippled" ß-sheets, in which the ß-sheets would consist of alternating l- and d-peptides. To date, this phenomenon has been investigated primarily with amphipathic peptide sequences composed of alternating hydrophilic and hydrophobic amino acid residues. Here, we show that enantiomers of a fragment of the amyloid-ß (Aß) peptide that does not follow this sequence pattern, amyloid-ß (16-22), readily coassembles into rippled ß-sheets. Equimolar mixtures of enantiomeric amyloid-ß (16-22) peptides assemble into supramolecular structures that exhibit distinct morphologies from those observed by self-assembly of the single enantiomer pleated ß-sheet fibrils. Formation of rippled ß-sheets composed of alternating l- and d-amyloid-ß (16-22) is confirmed by isotope-edited infrared spectroscopy and solid-state NMR spectroscopy. Sedimentation analysis reveals that rippled ß-sheet formation by l- and d-amyloid-ß (16-22) is energetically favorable relative to self-assembly into corresponding pleated ß-sheets. This work illustrates that coassembly of enantiomeric ß-sheet peptides into rippled ß-sheets is not limited to peptides with alternating hydrophobic/hydrophilic sequence patterns, but that a broader range of sequence space is available for the design and preparation of rippled ß-sheet materials.

Balancing hydrophobicity and sequence pattern to influence self-assembly of amphipathic peptides.

Amphipathic peptides with alternating polar and nonpolar amino acid sequences efficiently self-assemble into functional ß-sheet fibrils as long as the nonpolar residues have sufficient hydrophobicity. For example, the Ac-(FKFE)2 -NH2 peptide rapidly self-assembles into ß-sheet bilayer nanoribbons, while Ac-(AKAE)2 -NH2 fails to self-assemble under similar conditions due to the significantly reduced hydrophobicity and ß-sheet propensity of Ala relative to Phe. Herein, we systematically explore the effect of substituting only two of the four Ala residues at various positions in the Ac-(AKAE)2 -NH2 peptide with amino acids of increasing hydrophobicity, ß-sheet potential, and surface area (including Phe, 1-naphthylalanine (1-Nal), 2-naphthylalanine (2-Nal), cyclohexylalanine (Cha), and pentafluorophenylalanine (F5 -Phe)) on the self-assembly propensity of the resulting sequences. It was found that double Phe variants, regardless of the position of substitution, failed to self-assemble under the conditions used in this study. In contrast, all double 1-Nal and 2-Nal variants readily self-assembled, albeit at differing rates depending on the substitution patterns. To determine whether this was due to hydrophobicity or side chain surface area, we also prepared double Cha and F5 -Phe variant peptides (both side chain groups are more hydrophobic than Phe). Each of these variants also underwent effective self-assembly, with the aromatic F5 -Phe peptides doing so with greater efficiency. These findings provide insight into the role of amino acid hydrophobicity and sequence pattern on self-assembly proclivity of amphipathic peptides and on how targeted substitutions of nonpolar residues in these sequences can be exploited to tune the characteristics of the resulting self-assembled materials.

Investigating the effects of peptoid substitutions in self-assembly of Fmoc-diphenylalanine derivatives.

Low molecular weight agents that undergo self-assembly into fibril networks with hydrogel properties are promising biomaterials. Most low molecular weight hydrogelators are discovered empirically or serendipitously due to imperfect understanding of the mechanisms of self-assembly, the packing structure of self-assembled materials, and how the self-assembly process corresponds to emergent hydrogelation. Herein, the mechanisms of self-assembly and hydrogelation of N-fluorenylmethoxycarbonyl diphenylalanine (Fmoc-PhePhe), a well-studied low molecular weight hydrogelator, is probed by systematic comparison with derivatives in which Phe residues are replaced by corresponding N-benzyl glycine peptoid (Nphe) analogs. Peptoids are peptidomimetics that shift display of side chain functionality from the α-carbon to the terminal nitrogen. This alters the hydrogen bonding capacity, the side chain presentation geometry, amide cis/trans isomerization equilibrium, and ß-sheet potential of the peptoid relative to the corresponding amino acid in the context of peptidic polymers. It was found that amino acid/peptoid hybrids Fmoc-Phe-Nphe and Fmoc-Nphe-Phe have altered fibril self-assembly propensity and reduced hydrogelation capacity relative to the parent dipeptide, and that fibril self-assembly of the dipeptoid, Fmoc-Nphe-Nphe, is completely curtailed. These findings provide insight into the potential of low molecular weight peptoids and peptide/peptoid hybrids as hydrogelation agents and illuminate the importance of hydrogen bonding and π-π interaction geometry in facilitating self-assembly of Fmoc-Phe-Phe.

Self-Assembly, Hydrogelation, and Nanotube Formation by Cation-Modified Phenylalanine Derivatives.

Fluorenylmethoxycarbonyl-protected phenylalanine (Fmoc-Phe) derivatives are a privileged class of molecule that spontaneously self-assemble into hydrogel fibril networks. Fmoc-Phe-derived hydrogels are typically formed by dilution of the hydrogelator from an organic cosolvent into water, by dissolution of the hydrogelator under basic aqueous conditions followed by adjustment of the pH with acid, or by other external triggering forces, including sonication and heating. These conditions complicate biological applications of these hydrogels. Herein, we report C-terminal cation-modified Fmoc-Phe derivatives that are positively charged across a broad range of pH values and that can self-assemble and form hydrogel networks spontaneously without the need to adjust pH or to use an organic cosolvent. In addition, these cationic Fmoc-Phe derivatives are found to self-assemble into novel sheet-based nanotube structures at higher concentrations. These nanotube structures are unique to C-terminal cationic Fmoc-Phe derivatives; the parent Fmoc-Phe carboxylic acids form only fibril or worm-like micelle structures. Nanotube formation by the cationic Fmoc-Phe molecules is dependent on positive charge at the C-terminus, since at basic pH where the positive charge is reduced only fibrils/worm-like micelles are formed and nanotube formation is suppressed. These studies provide an important example of Fmoc-Phe derivatives that can elicit hydrogelation without organic cosolvent or pH modification and also provide insight into how subtle modification of structure can perturb the self-assembly pathways of Fmoc-Phe derivatives.

Modulating Supramolecular Peptide Hydrogel Viscoelasticity Using Biomolecular Recognition.

Self-assembled peptide-based hydrogels are emerging materials that have been exploited for wound healing, drug delivery, tissue engineering, and other applications. In comparison to synthetic polymer hydrogels, supramolecular peptide-based gels have advantages in biocompatibility, biodegradability, and ease of synthesis and modification. Modification of the emergent viscoelasticity of peptide hydrogels in a stimulus responsive fashion is a longstanding goal in the development of next-generation materials. In an effort to selectively modulate hydrogel viscoelasticity, we report herein a method to enhance the elasticity of ß-sheet peptide hydrogels using specific molecular recognition events between functionalized hydrogel fibrils and biomolecules. Two distinct biomolecular recognition strategies are demonstrated: oligonucleotide Watson-Crick duplex formation between peptide nucleic acid (PNA) modified fibrils with a bridging oligonucleotide and protein-ligand recognition between mannose modified fibrils with concanavalin A. These methods to modulate hydrogel elasticity should be broadly adaptable in the context of these materials to a wide variety of molecular recognition partners.

Display of functional proteins on supramolecular peptide nanofibrils using a split-protein strategy.

The display of functional proteins on self-assembled peptide nanofibrils is challenging since the steric bulk of proteins attached to simple self-assembling peptides often impedes incorporation into nanofibrils. Herein is described a split-protein strategy to tether functional proteins to preassembled peptide nanofibrils. In this strategy, a short affinity motif peptide derived from a split protein system is appended to a self-assembly motif (the amphipathic Ac-(FKFE)2-NH2 peptide) to form an affinity-assembly fusion peptide. The small size of the affinity motif allows the affinity-assembly fusion peptide to be readily incorporated into peptide nanofibrils that display the affinity motif when the affinity-assembly peptide is coassembled with Ac-(FKFE)2-NH2. Introduction of the split-protein that is complementary to the affinity motif to the assembled nanofibrils results in efficient, multivalent attachment of functional proteins to the peptide nanofibrils. This strategy is demonstrated with two split-protein systems, ribonuclease S' (RNase S') and split green fluorescent protein (GFP).

Substituent Effects on the Self-Assembly/Coassembly and Hydrogelation of Phenylalanine Derivatives.

Supramolecular hydrogels derived from the self-assembly of organic molecules have been exploited for applications ranging from drug delivery to tissue engineering. The relationship between the structure of the assembly motif and the emergent properties of the resulting materials is often poorly understood, impeding rational approaches for the creation of next-generation materials. Aromatic π-π interactions play a significant role in the self-assembly of many supramolecular hydrogelators, but the exact nature of these interactions lacks definition. Conventional models that describe π-π interactions rely on quadrupolar electrostatic interactions between neighboring aryl groups in the π-system. However, recent experimental and computational studies reveal the potential importance of local dipolar interactions between elements of neighboring aromatic rings in stabilizing π-π interactions. Herein, we examine the nature of π-π interactions in the self- and coassembly of Fmoc-Phe-derived hydrogelators by systematically varying the electron-donating or electron-withdrawing nature of the side chain benzyl substituents and correlating these effects to the emergent assembly and gelation properties of the systems. These studies indicate a significant role for stabilizing dipolar interactions between neighboring benzyl groups in the assembled materials. Additional evidence for specific dipolar interactions is provided by high-resolution crystal structures obtained from dynamic transition of gel fibrils to crystals for several of the self-assembled/coassembled Fmoc-Phe derivatives. In addition to electronic effects, steric properties also have a significant effect on the interaction between neighboring benzyl groups in these assembled systems. These findings provide significant insight into the structure-function relationship for Fmoc-Phe-derived hydrogelators and give cues for the design of next-generation materials with desired emergent properties.

Spontaneous Transition of Self-assembled Hydrogel Fibrils into Crystalline Microtubes Enables a Rational Strategy To Stabilize the Hydrogel State.

Hydrogel fibril and crystal formation are related self-assembly processes that provide materials with distinct emergent properties. The relationship between fibril and crystal growth is poorly understood, and efforts to engineer controlled hydrogelation vs crystallization via small molecule self-assembly currently depend on empirical approaches. Herein, we report the dynamic transition of self-assembled hydrogel fibrils of a phenylalanine (Phe) derivative, Fmoc-p-nitrophenylalanine (Fmoc-4-NO2-Phe), to crystalline microtubes. As has been shown with other Fmoc-Phe derivatives, Fmoc-4-NO2-Phe spontaneously self-assembles into amyloid-like fibrils that form an entangled hydrogel network when suspended in water. However, Fmoc-4-NO2-Phe fibrils uniquely transform over time into crystalline microtubes. Hydrogel fibrils appear to be a kinetic state with microtube crystals more thermodynamically favored. This dynamic transition from fibril to crystal has enabled a high-resolution structural analysis of the packing orientation of these self-assembled materials. Taking cues from this structural analysis, we demonstrate a rational strategy for stabilization of the kinetic Fmoc-4-NO2-Phe hydrogel fibrils. These results represent significant advances in our understanding of the dynamic nature of self-assembly processes and in our ability to rationally engineer these processes to provide materials with desired emergent properties.

Peptide Self-Assembly into Amyloid Fibrils: Unbiased All-Atom Simulations.

Protein self-assembly plays an important role in biological systems, accounting for the formation of mesoscopic structures that can be highly symmetric as in the capsid of viruses or disordered as in molecular condensates or exhibit a one-dimensional fibrillar morphology as in amyloid fibrils. Deposits of the latter in tissues of individuals with degenerative diseases like Alzheimer's and Parkinson's has motivated extensive efforts to understand the sequence of molecular events accounting for their formation. These studies aim to identify on-pathway intermediates that may be the targets for therapeutic intervention. This detailed knowledge of fibril formation remains obscure, in part due to challenges with experimental analyses of these processes. However, important progress is being achieved for short amyloid peptides due to advances in our ability to perform completely unbiased all-atom simulations of the self-assembly process. This perspective discusses recent developments, their implications, and the hurdles that still need to be overcome to further advance the field.

Seminal plasma accelerates semen-derived enhancer of viral infection (SEVI) fibril formation by the prostatic acid phosphatase (PAP248-286) peptide.

Amyloid fibrils contained in semen, known as SEVI, or semen-derived enhancer of viral infection, have been shown to increase the infectivity of HIV dramatically. However, previous work with these fibrils has suggested that extensive time and nonphysiologic levels of agitation are necessary to induce amyloid formation from the precursor peptide (a proteolytic cleavage product of prostatic acid phosphatase, PAP(248-286)). Here, we show that fibril formation by PAP(248-286) is accelerated dramatically in the presence of seminal plasma (SP) and that agitation is not required for fibrillization in this setting. Analysis of the effects of specific SP components on fibril formation by PAP(248-286) revealed that this effect is primarily due to the anionic buffer components of SP (notably inorganic phosphate and sodium bicarbonate). Divalent cations present in SP had little effect on the kinetics of fibril formation, but physiologic levels of Zn(2+) strongly protected SEVI fibrils from degradation by seminal proteases. Taken together, these data suggest that in the in vivo environment, PAP(248-286) is likely to form fibrils efficiently, thus providing an explanation for the presence of SEVI in human semen.

Sequence length determinants for self-assembly of amphipathic ß-sheet peptides.

Amphipathic peptides composed of alternating hydrophobic and hydrophilic amino acids are a privileged class of peptide, which have a high propensity to self-assemble into ß-sheet fibrils. The Ac-(FKFE)2-NH2 peptide has been extensively studied and forms putative ß-sheet bilayer fibrils in which the hydrophobic Phe side chains are organized to a single face of each constituent sheet; upon bilayer formation, these hydrophobic benzyl groups are sequestered in the hydrophobic core of the resulting fibril. In order for the Phe side chains to be uniformly displayed on one face of Ac-(FKFE)2-NH2 ß-sheets, an antiparallel packing orientation in which one amino acid residue is unpaired must be adopted. Based on molecular models, we hypothesized that truncated seven amino acid derivatives of Ac-(FKFE)2-NH2 in which either the N-terminal Phe residue (Ac-KFEFKFE-NH2) or the C-terminal Glu residue (Ac-FKFEFKF-NH2) is eliminated should readily self-assemble into ß-sheet bilayers in which all hydrogen bond and hydrophobic/charge interactions are satisfied. We found, however, that these minute changes in peptide sequence have unanticipated and dramatic effects on the self-assembly of each peptide. Ac-FKFEFKF-NH2 self-assembled into fibrils with unique morphology relative to the parent peptide, whereas the Ac-KFEFKFE-NH2 peptide had a strongly reduced propensity to self-assemble, even failing to self-assemble altogether under some conditions. These findings provide significant insight into the effect of sequence length and strand registry as well as hydrophobicity and charge on the self-assembly of simple amphipathic peptides to illuminate the possibility of tuning self-assembly processes and the resulting structures with minute changes to peptide sequence.

Effects of varied sequence pattern on the self-assembly of amphipathic peptides.

Amphipathic peptides have an increased propensity to self-assemble into amyloid-like ß-sheet fibrils when their primary sequence pattern consists of alternating hydrophobic and hydrophilic amino acids. These fibrils adopt a bilayer architecture composed of two ß-sheets laminated to bury the hydrophobic side chains of the ß-sheet in the bilayer interior, leaving the hydrophilic side chains exposed at the bilayer surface. In this study, the effects of altering the sequence pattern of amphipathic peptides from strictly alternating hydrophobic/hydrophilic repeats to more complex patterning of hydrophobic and hydrophilic residues on self-assembly of the resulting sequences is reported. Self-assembly of the Ac-(FKFE)2-NH2 peptide was compared to that of four related sequences with varied amino acid sequence patterning: Ac-(FK)2(FE)2-NH2, Ac-KEFFFFKE-NH2, Ac-(KFFE)2-NH2, and Ac-FFKEKEFF-NH2. The Ac-(FKFE)2-NH2 and Ac-(FK)2(FE)2-NH2 peptides effectively self-assembled at high (1.0 mM) and low (0.2 mM) concentrations (pH 3-4) into ß-sheet nanoribbons that were 8 and 4 nm wide, respectively. The Ac-KEFFFFKE-NH2 peptide failed to self-assemble at low concentration (pH 3-4), but self-assembled into distinct nanotapes that were ~20 nm in width at high concentration. Ac-(KFFE)2-NH2 and Ac-FFKEKEFF-NH2 failed to self-assemble into fibril/tape-like materials at either high or low concentration at pH 3-4, although Ac-FFKEKEFF-NH2 formed micelle-like aggregates at higher concentrations. At neutral pH, similar self-assembly behavior was observed for each peptide as was observed at acidic pH. An exception was the Ac-FFKEKEFF-NH2 peptide, which formed ~20 nm nanotapes at neutral pH. These results indicate that amino acid sequence patterns exert a profound influence on self-assembly propensity and morphology of the resulting materials even when the overall hydrophobicity or charge of the related peptides are identical. Sequence pattern variation can thus be exploited as a variable in the creation of novel materials composed of self-assembled peptides.