MagicWand: a single, designed peptide that assembles to stable, ordered alpha-helical fibers

We describe a straightforward single-peptide design that self-assembles into extended and thickened nano-to-mesoscale fibers of remarkable stability and order. The basic chassis of the design is the well-understood dimeric alpha-helical coiled-coil motif. As such, the peptide has a heptad sequence repeat, abcdefg , with isoleucine and leucine residues at the a and d sites to ensure dimerization. In addition, to direct staggered assembly of peptides and to foster fibrillogenesisthat is, as opposed to blunt-ended discrete speciesthe terminal quarters of the peptide are cationic and the central half anionic with lysine and glutamate, respectively, at core-flanking e and g positions. This +,-,-,+ arrangement gives the peptide its name, MagicWand (MW). As judged by circular dichroism (CD) spectra, MW assembles to alpha-helical structures in the sub-micromolar range and above. The thermal unfolding of MW is reversible with a melting temperature >70 degrees C at 100 muM peptide concentration. Negative-stain transmission electron microscopy (TEM) of MW assemblies reveals stiff, straight, fibrous rods that extended for tens of microns. Moreover, different stains highlight considerable order both perpendicular and parallel to the fiber long axis. The dimensions of these features are consistent with bundles of long, straight coiled alpha-helical coiled coils with their axes aligned parallel to the long axis of the fibers. The fiber thickening indicates inter-coiled-coil interactions. Mutagenesis of the outer surface of the peptide i.e., at the b and f positionscombined with stability and microscopy measurements, highlights the role of electrostatic and cation-pi interactions in driving fiber formation, stability and thickening. These findings are discussed in the context of the growing number of self-assembling peptide-based fibrous systems.     

Dynamic light scattering study of self-assembly of HPMA hybrid graft copolymers

The time course of self-assembly of a hybrid hydrogel system was investigated using dynamic light scattering (DLS) techniques. The self-assembling system consisted of a hydrophilic synthetic N-(2-hydroxypropyl)methacrylamide (HPMA) polymer backbone and a pair of oppositely charged peptide grafts (CCE and CCK). These two distinct pentaheptad peptides were anticipated to act as physical cross-linkers by the formation of antiparallel coiled-coil heterodimers. Equimolar mixture of HPMA graft copolymers CCE-P and CCK-P solutions (where P is the HPMA copolymer backbone) with total concentration from 1.25 to 10 mg/mL were measured at a scattering angle 90 degrees and room temperature. A critical extension of average relaxation time was observed with increasing concentration and incubation time. To reveal the role of coiled-coil grafts in the self-assembly process, a pair of modified random coil peptides, CCEw and CCKy, was designed. The DLS evaluation of HPMA copolymer conjugates (CCEw-P and CCKy-P) at total concentration of 10 mg/mL demonstrated that no association occurred after 28 h of incubation. Moreover, addition of a competing peptide (CCK) or a denaturant (guanidium chloride, GndHCl) to the self-assembled CCE-P/CCK-P hydrogels resulted in partial disassembly or collapse of the hydrogel clusters. These results correlated to changes in the secondary structure of peptides (grafts) as measured by circular dichroism spectroscopy (CD). These investigations supported the hypothesis that the self-assembly of CCE-P/CCK-P into hybrid hydrogels is mediated by the formation of coiled-coil heterodimers.     

Removing an interhelical salt bridge abolishes coiled-coil formation in a de novo designed peptide

Alpha-helical coiled coils represent a common protein oligomerization motif that are mainly stabilized by hydrophobic interactions occurring along their coiled-coil interface, the so-called hydrophobic seam. We have recently de novo designed and optimized a series of two-heptad repeat long coiled-coil peptides which are further stabilized by a complex network of inter- and intrahelical salt bridges. Here we have extended the de novo design of such two heptad-repeat long peptides by removing the central and most important g-e' Arg to Glu (g-e'RE) ionic interhelical interaction and replacing these residues by alanine residues. The effect of the missing interhelical ionic interaction on coiled-coil formation and stability has been analyzed by CD spectroscopy, analytical ultracentrifugation, and X-ray crystallography. We show that the peptide, while being highly alpha-helical, is no longer able to form a parallel coiled-coil structure but rather assumes an octameric globular helical assembly devoid of any coiled-coil interactions.     

Metal-induced folding of a designed metalloprotein

The metal-induced assembly of a designed peptide-based rubredoxin model is described. The C16C19-GGY peptide has the sequence Ac-K(IEALEGK)(2)(CEACEGK)(IEALEGK)GGY-amide in which the presence of the Cys-X-X-Cys metal binding domain of rubredoxin was used to place cysteine residues at the hydrophobic "a" and "d" positions upon formation of a homodimeric alpha-helical coiled-coil. Circular dichroism spectroscopy shows that the apopeptide exists as a random coil and assembles into a coiled-coil in the presence of Cd(2+). Metal binding is monitored by the appearance of a new LMCT band at 238 nm. UV-Vis titrations and SDS-PAGE experiments are used to show that this designed metalloprotein exists as a metal-bridged coiled-coil dimer.     

Identification of a region of fast skeletal troponin T required for stabilization of the coiled-coil formation with troponin I

We have previously identified evolutionarily conserved heptad hydrophobic repeat (HR) domains in all isoprotein members of troponin T (TnT) and troponin I (TnI), two subunits of the Ca(2+)-regulatory troponin complex. Our suggestion that the HR domains are involved in the formation of a coiled-coil heterodimer of TnT and TnI has been recently confirmed by the crystal structure of the core domain of the human cardiac troponin complex. Here we studied a series of recombinant deletion mutants of the fast skeletal TnT to determine the minimal sequence required for stable coiled-coil formation with the HR domain of the fast skeletal TnI. Using circular dichroism spectroscopy, we measured the alpha helical content of the coiled-coil formed by the various TnT peptides with TnI HR domain. Sedimentation equilibrium experiments confirmed that the individual peptides of TnT were monomeric but formed heterodimers when mixed with HR domain of TnI. Isothermal titration calorimetry was then used to directly measure the affinity of the TnT peptides for the TnI HR domain. Surprisingly we found that the HR regions alone of the fast skeletal TnT and TnI, as defined earlier, were insufficient to form a coiled-coil. Furthermore we showed that an additional 14 amino acid residues N-terminal to the conserved HR region (TnT residues 165-178) are essential for the stable coiled-coil formation. We discuss the implication of our finding in the fast skeletal troponin isoform in the light of the crystal structure of the cardiac isoform.     

Detection of alpha-helical coiled-coil dimer formation by spin-labeled synthetic peptides: a model parallel coiled-coil peptide and the antiparallel coiled coil formed by a replica of the ProP C-terminus

Electron paramagnetic resonance spectroscopy was used to determine relative peptide orientation within homodimeric, alpha-helical coiled-coil structures. Introduction of cysteine (Cys) residues into peptides/proteins for spin labeling allows detection of their oligomerization from exchange broadening or dipolar interactions between residues within 25 A of each other. Two synthetic peptides containing Cys substitutions were used: a 35-residue model peptide and the 30-residue ProP peptide. The model peptide is known to form a stable, parallel homodimeric coiled coil, which is partially destabilized by Cys substitutions at heptad a and d positions (peptides C30a and C33d). The ProP peptide, a 30-residue synthetic peptide, corresponds to residues 468-497 of osmoregulatory transporter ProP from Escherichia coli. It forms a relatively unstable, homodimeric coiled coil that is predicted to be antiparallel in orientation. Cys was introduced in heptad g positions of the ProP peptide, near the N-terminus (K473C, creating peptide C473g) or closer to the center of the sequence (E480C, creating peptide C480g). In contrast to the destabilizing effect of Cys substitution at the core heptad a or d positions of model peptides C30a and C33d, circular dichroism spectroscopy showed that Cys substitutions at the heptad g positions of the ProP peptide had little or no effect on coiled-coil stability. Thermal denaturation analysis showed that spin labeling increased the stability of the coiled coil for all peptides. Strong exchange broadening was detected for both C30a and C33d, in agreement with a parallel structure. EPR spectra of C480g had a large hyperfine splitting of about 90 G, indicative of strong dipole-dipole interactions and a distance between spin-labeled residues of less than 9 A. Spin-spin interactions were much weaker for C473g. These results supported the hypothesis that the ProP peptide primarily formed an antiparallel coiled coil, since formation of a parallel dimer should result in similar spin-spin interactions for the spin-labeled Cys at both sites.     

The role of the N-terminal heptad repeat of HIV-1 in the actual lipid mixing step as revealed by its substitution with distant coiled coils

The gp41 glycoprotein of HIV-1 is considered to be responsible for the actual fusion process between the virus and the host membranes. According to a prevailing model, gp41 trimer organization, directed by the N-terminal coiled-coil region (NHR), is essential for steps involved in the actual merging of viral and cellular membranes. This study addresses a major question: Is the specific sequence of the NHR obligatory for the fusion process, or can it be replaced by distant coiled coils that form different oligomeric states in solution? For this purpose we synthesized three known GCN4 coiled-coil mutants that oligomerize in solution into either dimers, trimers, or tetramers. These peptides were chemically ligated to the fusion peptide thereby creating three chimera peptides with different oligomeric tendencies in solution. These peptides were investigated, together with the 70-mer wild-type peptide (N70), regarding their structure in solution and membrane by using circular dichroism (CD) and FTIR spectroscopies, their ability to induce vesicle fusion, and their ability to bind phospholipid membranes by using surface plasmon resonance (SPR). Our results suggest that local assembly of fusion peptides, facilitated by coiled-coil oligomers, increases lipid mixing ability, probably by facilitating stronger binding of the fusion peptides to the opposing membrane as revealed by SPR. However, N70 is significantly more active than the other chimeras. Overall, the data indicate a correlation between the distinct conformation of N70 in solution and in membranes and its enhanced lipid mixing relative to the GCN4 chimeras.     

The effect of a side chain-backbone swap on protein stability.

To assess the relative importance of backbone hydrogen bonding (H-bonding) vs. side chain hydrophobicity in protein structural formation, a method called side chain-backbone swap is proposed. Such a method swaps the side chain and backbone portions of certain amino acid residues, such as Asp, Glu, Asn, Gln, Lys, and Arg. Such a swap retains the sequence of a polypeptide and preserves the identity of the backbone linkage. On the other hand, the swap disrupts backbone H-bonding geometry because of the introduction of extra methylene groups into the peptide backbone. In this project, we chose the two-stranded alpha-helical coiled-coil to implement side chain-backbone swap. A pair of 36-residue peptides was designed. The two peptides have identical sequence with four residues in each heptad repeat occupied by glutamyl residues. Each glutamic acid was incorporated either as alpha-glutamyl residue (the peptide is denoted as alpha-Glu-36) or as gamma-glutamyl residue (the peptide is denoted as gamma-Glu-36). The inter-conversion between the two peptides constitutes a side chain-backbone swap. Residues constituting the hydrophobic core of the coiled-coil, however, are left unchanged. The peptide pair was characterized by circular dichroism spectroscopy, reversed-phase liquid chromatography (RPLC), and two-dimensional nuclear magnetic resonance (NMR). The results indicate that alpha-Glu-36 is a two-stranded alpha-helical coiled-coil while gamma-Glu-36 lacks stable structural elements. It is concluded that, at least for coiled-coils where hydrophobic interactions are predominantly long-range, local backbone H-bonding is a required for structural formation, consistent with a hierarchic folding mechanism. The methodological implication of side chain-backbone swap is also discussed.     

Design, synthesis and structural characterization of model heterodimeric coiled-coil proteins.

We report the design and synthesis of model heterodimeric coiled-coil proteins and the packing contribution of interchain hetero-hydrophobic side-chains to coiled-coil stability. The heterodimeric coiled-coils are obtained by oxidizing two 35-residue polypeptide chains, each containing a cysteine residue at position 2 and differing in amino acid sequences in the hydrophobic positions ("a" and "d") responsible for the formation and stabilization of the coiled-coil. In each peptide, a single Ala residue was substituted for Leu at position "a" or "d". The formation and stability of heterodimeric coiled-coils were investigated by circular dichroism studies in the presence and absence of guanidine hydrochloride and compared to the corresponding homodimeric coiled-coils. The coiled-coil proteins with an Ala substitution at position "a" were less stable than those with an Ala substitution at position "d" in both the homodimeric (Ala-Ala interchain interactions) and heterodimeric (Leu-Ala interchain interactions ) coiled-coils. The 70-residue disulfide bridged peptides (homo- and heterodimeric coiled-coils) can be readily separated by reversed-phase chromatography (RPC) even though they have identical amino acid compositions as well as in the hydrophobic "a" and "d" positions. The elution of the 70-residue peptides prior to their corresponding 35-residue monomers suggests that these proteins are retaining a large portion of their coiled-coil structure during RPC at pH2 and their retention behavior correlates with protein stability.     

A distinct 14 residue site triggers coiled-coil formation in cortexillin I.

We have investigated the process of the assembly of the Dictyostelium discoideum cortexillin I oligomerization domain (Ir) into a tightly packed, two-stranded, parallel coiled-coil structure using a variety of recombinant polypeptide chain fragments. The structures of these Ir fragments were analyzed by circular dichroism spectroscopy, analytical ultracentrifugation and electron microscopy. Deletion mapping identified a distinct 14 residue site within the Ir coiled coil, Arg311-Asp324, which was absolutely necessary for dimer formation, indicating that heptad repeats alone are not sufficient for stable coiled-coil formation. Moreover, deletion of the six N-terminal heptad repeats of Ir led to the formation of a four- rather than a two-helix structure, suggesting that the full-length cortexillin I coiled-coil domain behaves as a cooperative folding unit. Most interestingly, a 16 residue peptide containing the distinct coiled-coil 'trigger' site Arg311-Asp324 yielded approximately 30% helix formation as monomer, in aqueous solution. pH titration and NaCl screening experiments revealed that the peptide's helicity depends strongly on pH and ionic strength, indicating that electrostatic interactions by charged side chains within the peptide are critical in stabilizing its monomer helix. Taken together, these findings demonstrate that Arg311-Asp324 behaves as an autonomous helical folding unit and that this distinct Ir segment controls the process of coiled-coil formation of cortexillin I.     

Role of hydrophobic clusters in the stability of alpha-helical coiled coils and their conversion to amyloid-like beta-sheets

We designed a library of short peptides using standard rules for coiled-coil assembly. Depending on the composition of amino acids in the non-interacting region of the coiled coil (positions b, c, and f) these peptides are able to convert from alpha-helical to beta-sheet secondary structure. This type of transition is observed in amyloid-like proteins and is a key feature associated with many types of neurodegenerative diseases. Studies on peptides that are 14 amino acids in length indicated that positioning hydrophobic amino acids at an f position within a heptad repeat accelerated the rate of conformational conversion as compared to that at a c position. We believe that this occurs because of the formation of a hydrophobic pocket that preferentially stabilizes beta-sheets over alpha-helices. This effect was also observed in longer 21 amino acid peptides. Our study shows that the relative rates of structural conversion correlate with the formation of a continuous three-amino-acid hydrophobic patch consisting of amino acids in the d, f, and a positions and not on the secondary structure propensities of the individual amino acids. The sequence-structure relationship observed in this study will be used to help understand the mechanism of amyloid fiber formation and design future coiled-coil and beta-sheet-forming peptide systems.     

Design and crystal structure of bacteriophage T4 mini-fibritin NCCF

Fibritin is a fibrous protein that forms "whiskers" attached to the neck of bacteriophage T4. Whiskers interact with the long tail fibers regulating the assembly and infectivity of the virus. The fibritin trimer includes the N-terminal domain responsible for attachment to the phage particle and for the collar formation, the central domain forming a 500 A long segmented coiled-coil structure, and the C-terminal "foldon" domain. We have designed a "mini" fibritin with most of the coiled-coil domain deleted, and solved its crystal structure. The non-helical N-terminal part represents a new protein fold that tightly interacts with the coiled-coil segment forming a single domain, as revealed by calorimetry. The analysis of the crystal structure and earlier electron microscopy data on the collar-whisker complex suggests the necessity of other proteins to participate in the collar formation. Crystal structure determination of the N-terminal domain of fibritin is the first step towards elucidating the detailed structure and assembly mechanism of the collar-whisker complex.     

Reversible assembly of helical filaments by de novo designed minimalist peptides

We have designed a series of 15 short, helical de novo peptides consisting of lysine, isoleucine, and alanine. We have termed this the KIA series. These peptides differ only in their hydrophobic interface, and thus their self-association is largely a consequence of hydrophobic interactions. One of these peptides, KIA13, forms insoluble helical fibers at specific NaCl concentrations. We have used CD spectroscopy, turbidity assays, and in situ tapping mode atomic force microscopy to characterize the reversible assembly pathway for this peptide. It is unfolded at low NaCl concentration, and forms helical, soluble fibers resembling a coiled-coil conformation at intermediate NaCl concentrations, and rope-like insoluble fibers at high NaCl concentrations. Reducing the NaCl concentration completely reverses this process. Another peptide from the KIA series specifically inhibits the formation of the insoluble KIA13 fibers, and reverses the process to some extent. This work sheds light onto protein fibrillogenesis and offers intriguing possibilities for the use of these types of peptides in drug delivery and biomaterials applications.     

Effects of the sequence and size of non-polar residues on self-assembly of amphiphilic peptides

Peptides with alternating hydrophobic and polar amino acids have been shown to form stable beta-sheet secondary structures and self-assemble into hydrogel-like matrices in the presence of physiological salt concentrations. We hypothesized that the sequence and steric size differences of non-polar residues can affect the balance of peptide intermolecular forces in solution that drive self-assembly. To test this hypothesis, we designed a library of artificial amphiphilic peptides based on the sequence (FEFEFKFK)2 by substituting combinations of the non-polar residues glycine, alanine, valine, leucine and isoleucine for phenylalanine. Peptide structure and self-assembly were characterized using scanning electron microscopy, the Thioflavin T assay, transmission electron microscopy, X-ray fiber diffraction and circular dichroism spectroscopy. The sequence and steric size of non-polar residues are shown to cause variations in peptide secondary structures and create significant differences in the matrix morphology of self-assembled peptides.     

Design of a heterotetrameric coiled coil

We have successfully designed a simple peptide sequence that forms highly stable coiled-coil heterotetramers. Our model system is based on the GCN4-pLI parallel coiled-coil tetramer, first described by Kim and coworkers (Harbury et al., Science 1993;262:1401–1407). We introduced glutamates at all of the e and c heptad positions of one sequence (ecE) and lysines at the same positions in a second sequence (ecK). Based on a modeling study, these sidechains are close enough in space to form structure-stabilizing salt bridges. We show that ecE and ecK are highly unstable by themselves but form very stable parallel helical tetramers when mixed, as judged by circular dichroism, analytical ultracentrifugation, and disulfide crosslinking studies. The origin of the difference in stabilities between the homomeric structures and the heteromeric structures comes from a combination of the relief of electrostatic repulsions with concomitant formation of electrostatic attractive interactions based on pH and NaCl screening experiments. We quantify the stability of the heterotetrameric coiled coil from a thermodynamic analysis and compare the finding to other similar coiled-coil systems.     

The Positively Charged Region of the Myosin IIC Non-helical Tailpiece Promotes Filament Assembly

The motor protein, non-muscle myosin II (NMII), must undergo dynamic oligomerization into filaments to participate in cellular processes such as cell migration and cytokinesis. A small non-helical region at the tail of the long coiled-coil region (tailpiece) is a common feature of all dynamically assembling myosin II proteins. In this study, we investigated the role of the tailpiece in NMII-C self-assembly. We show that the tailpiece is natively unfolded, as seen by circular dichroism and NMR experiments, and is divided into two regions of opposite charge. The positively charged region (Tailpiece_1946–1967) starts at residue 1946 and is extended by seven amino acids at its N terminus from the traditional coiled-coil ending proline (Tailpiece_1953–1967). Pull-down and sedimentation assays showed that the positive Tailpiece_1946–1967 binds to assembly incompetent NMII-C fragments inducing filament assembly. The negative region, residues 1968–2000, is responsible for NMII paracrystal morphology as determined by chimeras in which the negative region was swapped between the NMII isoforms. Mixing the positive and negative peptides had no effect on the ability of the positive peptide to bind and induce filament assembly. This study provides molecular insight into the role of the structurally disordered tailpiece of NMII-C in shifting the oligomeric equilibrium of NMII-C toward filament assembly and determining its morphology.     

A designed system for assessing how sequence affects alpha to beta conformational transitions in proteins.

The role of amino acid sequence in conformational switching observed in prions and proteins associated with amyloid diseases is not well understood. To study alpha to beta conformational transitions, we designed a series of peptides with structural duality; namely, peptides with sequence features of both an alpha-helical leucine zipper and a beta-hairpin. The parent peptide, Template-alpha, was designed to be a canonical leucine-zipper motif and was confirmed as such using circular dichroism spectroscopy and analytical ultracentrifugation. To introduce beta-structure character into the peptide, glutamine residues at sites away from the leucine-zipper dimer interface were replaced by threonine to give Template-alphaT. Unlike the parent peptide, Template-alphaT underwent a heat-inducible switch to beta-structure, which reversibly formed gels containing amyloid-like fibrils. In contrast to certain other natural proteins where destabilization of the native states facilitate transitions to amyloid, destabilization of the leucine-zipper form of Template-alphaT did not promote a transformation. Cross-linking the termini of the peptides compatible with the alternative beta-hairpin design, however, did promote the change. Furthermore, despite screening various conditions, only the internally cross-linked form of the parent, Template-alpha, peptide formed amyloid-like fibrils. These findings demonstrate that, in addition to general properties of the polypeptide backbone, specific residue placements that favor beta-structure promote amyloid formation.     

Characterization of mesoscale coiled-coil peptide-porphyrin complexes

Photoelectronically conductive self-assembling peptide-porphyrin assemblies have great potential in their use as biomaterials, owing largely to their environmentally responsive properties. We have successfully designed a coiled-coil peptide that can self-assemble to form mesoscale filaments and serve as a scaffold for porphyrin interaction. In our earlier work, peptide-porphyrin-based biomaterials were formed at neutral pH, but the structures were irregular at the nano- to microscale size range, as judged by atomic force microscopy. We identified a pH in which mesoscale fibrils were formed, taking advantage of the types of porphyrin interactions that are present in well-characterized J-aggregates. We used UV-visible spectroscopy, circular dichroism spectropolarimetry, fluorescence spectroscopy, and atomic force microscopy to characterize these self-assembling biomaterials. We propose a new assembly paradigm that arises from a set of unique porphyrin-porphyrin and porphyrin-peptide interactions whose structure may be readily modulated by changes in pH or peptide concentration.