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Membrane-peptide interactions play critical roles in many cellular and organismic functions, including protection from infection, remodeling of membranes, signaling, and ion transport. Peptides interact with membranes in a variety of ways: some associate with membrane surfaces in either intrinsically disordered conformations or well-defined secondary structures. Peptides with sufficient hydrophobicity can also insert vertically as transmembrane monomers, and many associate further into membrane-spanning helical bundles. Indeed, some peptides progress through each of these stages in the process of forming oligomeric bundles. In each case, the structure of the peptide and the membrane represent a delicate balance between peptide-membrane and peptide-peptide interactions. We will review this literature from the perspective of several biologically important systems, including antimicrobial peptides and their mimics, α-synuclein, receptor tyrosine kinases, and ion channels. We also discuss the use of de novo design to construct models to test our understanding of the underlying principles and to provide useful leads for pharmaceutical intervention of diseases.

The design of peptides that assemble in membranes to form functional ion channels is challenging. Specifically, hydrophobic interactions must be designed between the peptides and at the peptide-lipid interfaces simultaneously. Here, we take a multi-step approach towards this problem. First, we use rational de novo design to generate water-soluble α-helical barrels with polar interiors, and confirm their structures using high-resolution X-ray crystallography. These α-helical barrels have water-filled lumens like those of transmembrane channels. Next, we modify the sequences to facilitate their insertion into lipid bilayers. Single-channel electrical recordings and fluorescent imaging of the peptides in membranes show monodisperse, cation-selective channels of unitary conductance. Surprisingly, however, an X-ray structure solved from the lipidic cubic phase for one peptide reveals an alternative state with tightly packed helices and a constricted channel. To reconcile these observations, we perform computational analyses to compare the properties of possible different states of the peptide.

One of the fundamental events in protein folding is α-helix formation, which involves sequential development of a series of helical hydrogen bonds between the backbone C═O group of residues i and the -NH group of residues i + 4. While we now know a great deal about α-helix folding dynamics, a key question that remains to be answered is where the productive helical nucleation event occurs. Statistically, a helical nucleus (or the first helical hydrogen-bond) can form anywhere within the peptide sequence in question; however, the one that leads to productive folding may only form at a preferred location. This consideration is based on the fact that the α-helical structure is inherently asymmetric, due to the specific alignment of the helical hydrogen bonds. While this hypothesis is plausible, validating it is challenging because there is not an experimental observable that can be used to directly pinpoint the location of the productive nucleation process. Therefore, in this study we combine several techniques, including peptide cross-linking, laser-induced temperature-jump infrared spectroscopy, and molecular dynamics simulations, to tackle this challenge. Taken together, our experimental and simulation results support an α-helix folding mechanism wherein the productive nucleus is formed at the N-terminus, which propagates toward the C-terminal end of the peptide to yield the folded structure. In addition, our results show that incorporation of a cross-linker can lead to formation of differently folded conformations, underscoring the need for all-atom simulations to quantitatively assess the proposed cross-linking design.

Amyloids adopt 'cross-β' structures composed of long, twisted fibrils with β-strands running perpendicular to the fibril axis. Recently, a toxic peptide was proposed to form amyloid-like cross-α structures in solution, with a planar bilayer-like assembly observed in the crystal structure. Here we crystallographically characterize designed peptides that assemble into spiraling cross-α amyloid-like structures, which resemble twisted β-amyloid fibrils. The peptides form helical dimers, stabilized by packing of small and apolar residues, and the dimers further assemble into cross-α amyloid-like fibrils with superhelical pitches ranging from 170 Å to 200 Å. When a small residue that appeared critical for packing was converted to leucine, it resulted in structural rearrangement to a helical polymer. Fluorescently tagged versions of the designed peptides form puncta in mammalian cells, which recover from photobleaching with markedly different kinetics. These structural folds could be potentially useful for directing in vivo protein assemblies with predetermined spacing and stabilities.

Biomaterials derived via programmable supramolecular protein assembly provide a viable means of constructing precisely defined structures. Here, we present programmed superstructures of AuPt nanoparticles (NPs) on carbon nanotubes (CNTs) that exhibit distinct electrocatalytic activities with respect to the nanoparticle positions via rationally modulated peptide-mediated assembly. De novo designed peptides assemble into six-helix bundles along the CNT axis to form a suprahelical structure. Surface cysteine residues of the peptides create AuPt-specific nucleation site, which allow for precise positioning of NPs onto helical geometries, as confirmed by 3-D reconstruction using electron tomography. The electrocatalytic model system, i.e., AuPt for oxygen reduction, yields electrochemical response signals that reflect the controlled arrangement of NPs in the intended assemblies. Our design approach can be expanded to versatile fields to build sophisticated functional assemblies.

Although helices play key roles in peptide-protein and protein-protein interactions, the helical conformation is generally unstable for short peptides (10-15 residues) in aqueous solution in the absence of their binding partners. Thus, stabilizing the helical conformation of peptides can lead to increases in binding potency, specificity, and stability towards proteolytic degradation. Helices have been successfully stabilized by introducing side chain-to-side chain crosslinks within the central portion of the helix. However, this approach leaves the ends of the helix free, thus leading to fraying and exposure of the non-hydrogen-bonded amide groups to solvent. Here, we develop a "capped-strapped" peptide strategy to stabilize helices by embedding the entire length of the helix within a macrocycle, which also includes a semirigid organic template as well as end-capping interactions. We have designed a ten-residue capped-strapped helical peptide that behaves like a miniprotein, with a cooperative thermal unfolding transition and T m ≈70 °C, unprecedented for helical peptides of this length. The NMR structure determination confirmed the design, and X-ray crystallography revealed a novel quaternary structure with implications for foldamer design., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Computationally designed transmembrane α-helical peptides (CHAMP) have been used to compete for helix-helix interactions within the membrane, enabling the ability to probe the activation of the integrins αIIbβ3 and αvβ3. Here, this method is extended towards the design of CHAMP peptides that inhibit the association of the α5β1 transmembrane (TM) domains, targeting the Ala-X3-Gly motif within α5. Our previous design algorithm was performed alongside a new workflow implemented within the widely used Rosetta molecular modeling suite. Peptides from each computational approach activated integrin α5β1 but not αVβ3 in human endothelial cells. Two CHAMP peptides were shown to directly associate with an α5 TM domain peptide in detergent micelles to a similar degree as a β1 TM peptide does. By solution-state nuclear magnetic resonance, one of these CHAMP peptides was shown to bind primarily the integrin β1 TM domain, which itself has a Gly-X3-Gly motif. The second peptide associated modestly with both α5 and β1 constructs, with slight preference for α5. Although the design goal was not fully realized, this work characterizes novel CHAMP peptides activating α5β1 that can serve as useful reagents for probing integrin biology.

For many voltage-gated ion channels (VGICs), creation of a properly functioning ion channel requires the formation of specific protein-protein interactions between the transmembrane pore-forming subunits and cystoplasmic accessory subunits. Despite the importance of such protein-protein interactions in VGIC function and assembly, their potential as sites for VGIC modulator development has been largely overlooked. Here, we develop meta-xylyl (m-xylyl) stapled peptides that target a prototypic VGIC high affinity protein-protein interaction, the interaction between the voltage-gated calcium channel (Ca V ) pore-forming subunit α-interaction domain (AID) and cytoplasmic β-subunit (Ca V β). We show using circular dichroism spectroscopy, X-ray crystallography, and isothermal titration calorimetry that the m-xylyl staples enhance AID helix formation are structurally compatible with native-like AID:Ca V β interactions and reduce the entropic penalty associated with AID binding to Ca V β. Importantly, electrophysiological studies reveal that stapled AID peptides act as effective inhibitors of the Ca V α 1 :Ca V β interaction that modulate Ca V function in an Ca V β isoform-selective manner. Together, our studies provide a proof-of-concept demonstration of the use of protein-protein interaction inhibitors to control VGIC function and point to strategies for improved AID-based Ca V modulator design.

We present a strategy for designed self-assembly of peptides into two-dimensional monolayer crystals on the surface of graphene and graphite. As predicted by computation, designed peptides assemble on the surface of graphene to form very long, parallel, in-register β-sheets, which we call β-tapes. Peptides extend perpendicularly to the long axis of each β-tape, defining its width, with hydrogen bonds running along the axis. Tapes align on the surface to create highly regular microdomains containing 4-nm pitch striations. Moreover, in agreement with calculations, the atomic structure of the underlying graphene dictates the arrangement of the β-tapes, as they orient along one of six directions defined by graphene's sixfold symmetry. A cationic-assembled peptide surface is shown here to strongly adhere to DNA, preferentially orienting the double helix along β-tape axes. This orientational preference is well anticipated from calculations, given the underlying peptide layer structure. These studies illustrate how designed peptides can amplify the Ångstrom-level atomic symmetry of a surface onto the micrometer scale, further imparting long-range directional order onto the next level of assembly. The remarkably stable nature of these assemblies under various environmental conditions suggests applications in enzymelike catalysis, biological interfaces for cellular recognition, and two-dimensional platforms for studying DNA-peptide interactions., (Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Learning to engineer self-assembly would enable the precise organization of molecules by design to create matter with tailored properties. Here we demonstrate that proteins can direct the self-assembly of buckminsterfullerene (C60) into ordered superstructures. A previously engineered tetrameric helical bundle binds C60 in solution, rendering it water soluble. Two tetramers associate with one C60, promoting further organization revealed in a 1.67-Å crystal structure. Fullerene groups occupy periodic lattice sites, sandwiched between two Tyr residues from adjacent tetramers. Strikingly, the assembly exhibits high charge conductance, whereas both the protein-alone crystal and amorphous C60 are electrically insulating. The affinity of C60 for its crystal-binding site is estimated to be in the nanomolar range, with lattices of known protein crystals geometrically compatible with incorporating the motif. Taken together, these findings suggest a new means of organizing fullerene molecules into a rich variety of lattices to generate new properties by design.

Using the single-chain due ferri (DFsc) peptide scaffold, the differential oxidase and oxygenase reactivities of two 4A→4G variants, one with two histidines at the diiron center (G4DFsc) and the other with three histidines (3His-G4DFsc(Mut3)), are explored. By controlling the reaction conditions, the active form responsible for 4-aminophenol (4-AP) oxidase activity in both G4DFsc and 3His-G4DFsc(Mut3) is determined to be the substrate-bound biferrous site. Using circular dichroism (CD), magnetic CD (MCD), and variable-temperature, variable-field (VTVH) MCD spectroscopies, 4-AP is found to bind directly to the biferrous sites of the DF proteins. In G4DFsc, 4-AP increases the coordination of the biferrous site, while in 3His-G4DFsc(Mut3), the coordination number remains the same and the substrate likely replaces the additional bound histidine. This substrate binding enables a two-electron process where 4-AP is oxidized to benzoquinone imine and O2 is reduced to H2O2. In contrast, only the biferrous 3His variant is found to be active in the oxygenation of p-anisidine to 4-nitroso-methoxybenzene. From CD, MCD, and VTVH MCD, p-anisidine addition is found to minimally perturb the biferrous centers of both G4DFsc and 3His-G4DFsc(Mut3), indicating that this substrate binds near the biferrous site. In 3His-G4DFsc(Mut3), the coordinative saturation of one iron leads to the two-electron reduction of O2 at the second iron to generate an end-on hydroperoxo-Fe(III) active oxygenating species.

There are many opportunities to use macromolecules, such as peptides and oligonucleotides, for intracellular applications. Despite this, general methods for delivering these molecules to the cytosol in a safe and efficient manner are not available. Efforts to develop a variety of intracellular drug delivery systems such as viral vectors, lipoplexes, nanoparticles, and amphiphilic peptides have been made, but various challenges such as delivery efficiency, toxicity, and controllability remain. A central challenge is the ability to selectively perturb, not destroy, the membrane to facilitate cargo introduction. Herein, we describe our efforts to design and characterize peptides that form pores inside membranes at acidic pH, so-called pH-switchable pore formation (PSPF) peptides, as a potential means for facilitating cargo translocation through membranes. Consistent with pore formation, these peptides exhibit low-pH-triggered selective release of ATP and miRNA, but not hemoglobin, from red blood cells. Consistent with these observations, biophysical studies (tryptophan fluorescence, circular dichroism, size-exclusion chromatography, analytical ultracentrifugation, and attenuated total reflectance Fourier transformed infrared spectroscopy) show that decreased pH destabilizes the PSPF peptides in aqueous systems while promoting their membrane insertion. Together, these results suggest that reduced pH drives insertion of PSPF peptides into membranes, leading to target-specific escape through a proposed pore formation mechanism.

Brilacidin (PMX30063) has shown potent bactericidal activity against drug-resistant and -susceptible strains of multiple Gram-negative and Gram-positive pathogens. In this study, we demonstrate that brilacidin causes membrane depolarization in the Gram-positive bacterium Staphylococcus aureus, to an extent comparable to that caused by the lipopeptidic drug daptomycin. Transcriptional profiling of Staphylococcus aureus by deep sequencing shows that the global response to brilacidin treatment is well correlated to those of treatment with daptomycin and the cationic antimicrobial peptide LL37 and mostly indicates abrogation of cell wall and membrane functions. Furthermore, the upregulation of various chaperones and proteases by brilacidin and daptomycin indicates that cytoplasmic protein misfolding stress may be a contributor to the mechanism of action of these drugs. These stress responses were orchestrated mainly by three two-component systems, GraSR, VraSR, and NsaSR, which have been implicated in virulence and drug resistance against other clinically available antibiotics., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Enzymes fold into unique three-dimensional structures, which underlie their remarkable catalytic properties. The requirement to adopt a stable, folded conformation is likely to contribute to their relatively large size (>10,000 Da). However, much shorter peptides can achieve well-defined conformations through the formation of amyloid fibrils. To test whether short amyloid-forming peptides might in fact be capable of enzyme-like catalysis, we designed a series of seven-residue peptides that act as Zn(2+)-dependent esterases. Zn(2+) helps stabilize the fibril formation, while also acting as a cofactor to catalyse acyl ester hydrolysis. These results indicate that prion-like fibrils are able to not only catalyse their own formation, but they can also catalyse chemical reactions. Thus, they might have served as intermediates in the evolution of modern-day enzymes. These results also have implications for the design of self-assembling nanostructured catalysts including ones containing a variety of biological and non-biological metal ions.

The design of β-peptide foldamers targeting the transmembrane (TM) domains of complex natural membrane proteins has been a formidable challenge. A series of β-peptides was designed to stably insert in TM orientations in phospholipid bilayers. Their secondary structures and orientation in the phospholipid bilayer was characterized using biophysical methods. Computational methods were then devised to design a β-peptide that targeted a TM helix of the integrin α(IIb)β(3). The designed peptide (β-CHAMP) interacts with the isolated target TM domain of the protein and activates the intact integrin in vitro.

Transmembrane (TM) β-peptides comprised of acyclic β(3)-amino acids demonstrate equilibrium between 12- and 14-helical structures in an environment- and sequence-dependent manner. Circular dichroism (CD) spectra of TM β(3)-peptides may be described as linear combinations of the 12- and 14-helical CD spectra. The apparent malleability of β(3)-substituted acyclic β-peptides has practical implications for foldamer design, as it suggests that both the 14-helix and 12-helix might be reasonable platforms for molecular recognition.

Two-dimensional (2D) vibrational echo spectroscopy has previously been applied to structural determination of small peptides. Here we extend the technique to a more complex, biologically important system: the homodimeric transmembrane dimer from the α chain of the integrin α(IIb)β(3). We prepared micelle suspensions of the pair of 30-residue chains that span the membrane in the native structure, with varying levels of heavy ((13)C=(18)O) isotopes substituted in the backbone of the central 10th through 20th positions. The constraints derived from vibrational coupling of the precisely spaced heavy residues led to determination of an optimized structure from a range of model candidates: Glycine residues at the 12th, 15th, and 16th positions form a tertiary contact in parallel right-handed helix dimers with crossing angles of -58° ± 9° and interhelical distances of 7.7 ± 0.5 angstroms. The frequency correlation established the dynamical model used in the analysis, and it indicated the absence of mobile water associated with labeled residues. Delocalization of vibrational excitations between the helices was also quantitatively established.

There is a general need for the engineering of protein-like molecules that organize into geometrically specific superstructures on molecular surfaces, directing further functionalization to create richly textured, multilayered assemblies. Here we describe a computational approach whereby the surface properties and symmetry of a targeted surface define the sequence and superstructure of surface-organizing peptides. Computational design proceeds in a series of steps that encode both surface recognition and favorable intersubunit packing interactions. This procedure is exemplified in the design of peptides that assemble into a tubular structure surrounding single-walled carbon nanotubes (SWNTs). The geometrically defined, virus-like coating created by these peptides converts the smooth surfaces of SWNTs into highly textured assemblies with long-scale order, capable of directing the assembly of gold nanoparticles into helical arrays along the SWNT axis.

The first computationally designed self-assembling oligomer consisting of exclusively β-amino acids (βAAs) is presented. The packing of a β-3(14) helix into coiled-coils of varying stoichiometries as a function of amino acid sequence is examined. β-Peptides with hVal repeating every third residue in the sequence appeared to have a strong propensity to pack into hexameric bundles. The designed sequence was synthesized and characterized with CD spectroscopy, NMR, and analytical ultracentrifugation, suggesting that the peptide adopts a well-folded hexameric structure.

Synthetic foldamers consisting of beta-amino acids offer excellent model systems for examining the effect of backbone flexibility on the dynamics of protein folding. Herein, we study the folding-unfolding kinetics of a beta-peptide that folds into a 14-helical structure in water. We find that the T-jump induced relaxation kinetics of this peptide occur on the nanosecond time scale and are noticeably slower than those of alanine-based alpha-helical peptides, and additionally, the relaxation rates show a weaker dependence on temperature. These differences appear to indicate that the folding energy landscapes of these peptides are different. In addition, we find that the amide I' band of this beta-peptide exhibits a sharp feature at approximately 1612 cm(-1), which we believe is a distinct infrared reporter of 14-helix.

The forces that define the interactions of transmembrane helices have been evaluated using a model membrane-soluble peptide (MS1), whose packing is modeled on the two-stranded coiled-coil from GCN4. The thermodynamic stability of water-soluble coiled-coils depends on the side chain at the buried "a" position of the repeat, favoring large hydrophobic residues over small side chains. Here we show that just the opposite is true for the membrane-soluble peptide. Analytical ultracentrifugation and equilibrium disulfide interchange show that the stability of MS1 is greatest when Gly is at each "a" position of the heptad repeat (MS1-Gly), followed by Ala > Val > Ile. Moreover, MS1-Gly has a strong tendency to form antiparallel dimers, MS1-Ala forms a mixture of parallel and antiparallel dimers, while MS1-Val and MS1-Ile have a preference to form parallel dimers. Calculations based on exhaustive conformational searching and rotamer optimization were in excellent agreement with experiments, in terms of the overall stability of the structures and the preference for parallel vs antiparallel packing. The MS1-Gly helices are able to achieve more favorable and uniform packing in an antiparallel dimer, while MS1-Val and MS1-Ile have more favorable van der Waals interactions in a parallel dimer. Finally, the electrostatic component arising from the partial charges of the backbones become significant in the antiparallel MS1-Gly and MS1-Ala conformations, due to close packing of the helices. Thus, van der Waals interactions and electrostatic interactions contribute to the stability and orientational preferences of the dimers.

Helix-helix association within a membrane environment represents one of the fundamental processes in membrane protein folding. However, studying the kinetics of such processes has been difficult because most membrane proteins are insoluble in aqueous solution. Here we present a stopped-flow fluorescence study of the membrane-interaction kinetics of a designed, water-soluble transmembrane (TM) peptide, anti-alpha(IIb), which is known to dimerize in phospholipid bilayers. We show that by using two fluorescent amino acids, tryptophan and p-cyanophenylalanine, we are able to kinetically dissect distinct phases in the peptide-membrane interaction, representing membrane binding, membrane insertion, and TM helix-helix association. Our results further show that the last process occurs on a time scale of seconds, indicating that the association of two TM helices is an intrinsically slow event.

The de novo design of molecular switching peptides is of increasing interest because it tests and extends our fundamental understanding of this process while laying the groundwork for the creation of new chemical and biological sensors. Here, an alpha-helical amphiphilic cell-lytic peptide, mastoparan X, was engineered to bind divalent cations. Binding of Zn(II) or Ni(II) to the designed peptide Mst-HH stabilizes the lytic amphiphilic structure and increases the activity of the peptide. Although both Zn(II) and Ni(II) activate Mst-HH for membrane lysis, they appear to do so via different mechanisms. Additionally, a series of metal binding-site mutants were synthesized to assess the relationship of charge and helical propensity to the toxicity and switchability. Additionally, by changing the characteristics of the metal-binding ligands, we can vary the selectivity of the site.

Compared with human platelets, rodent platelets are less responsive to peptides and peptidomimetics containing an arginine-glycine-aspartic acid (RGD) motif. Using chimeric human-rat alphaIIbbeta3 molecules, we found that this difference in Arg-Gly-Asp-Ser (RGDS) sensitivity was the result of amino acid substitutions at residues 157, 159, and 162 in the W3:4-1 loop and an Asp-His replacement at residue 232 in the W4:4-1 loop of the alphaIIb beta propeller. Introducing the entire rat W3:4-1 and W4:4-1 loops into human alphaIIbbeta3 also decreased the inhibitory effect of the disintegrins, echistatin and eristostatin, and the alphaIIbbeta3 antagonists, tirofiban and eptifibatide, on fibrinogen binding, whereas the specific point mutations did not. This suggests that RGDS interacts with alphaIIb in a different manner than with these small molecules. None of these species-based substitutions affected the ability of alphaIIbbeta3 to interact with RGD-containing macromolecules. Thus, human von Willebrand factor contains an RGD motif and binds equally well to adenosine diphosphate-stimulated human and rodent platelets, implying that other motifs are responsible for maintaining ligand binding affinity. Many venoms contain RGD-based toxins. Our data suggest that these species amino acids differences in the alphaIIb beta-propeller represent an evolutionary response by rodents to maintain hemostasis while concurrently protecting against RGD-containing toxins.

DFsc is a single chain de novo designed four-helix bundle peptide that mimics the core protein fold and primary ligand set of various binuclear non-heme iron enzymes. DFsc and the E11D, Y51L, and Y18F single amino acid variants have been studied using a combination of near-IR circular dichroism (CD), magnetic circular dichroism (MCD), variable temperature variable field MCD (VTVH MCD), and X-ray absorption (XAS) spectroscopies. The biferrous sites are all weakly antiferromagnetically coupled with mu-1,3 carboxylate bridges and one 4-coordinate and one 5-coordinate Fe, very similar to the active site of class I ribonucleotide reductase (R2) providing open coordination positions on both irons for dioxygen to bridge. From perturbations of the MCD and VTVH MCD the iron proximal to Y51 can be assigned as the 4-coordinate center, and XAS results show that Y51 is not bound to this iron in the reduced state. The two open coordination positions on one iron in the biferrous state would become occupied by dioxygen and Y51 along the O(2) reaction coordinate. Subsequent binding of Y51 functions as an internal spectral probe of the O(2) reaction and as a proton source that would promote loss of H(2)O(2). Coordination by a ligand that functions as a proton source could be a structural mechanism used by natural binuclear iron enzymes to drive their reactions past peroxo biferric level intermediates.

Antimicrobial peptides are small cationic amphiphiles that play an important role in the innate immune system. Given their broad specificity, they appear to be ideal therapeutic agents. As a result, over the last decade, there has been considerable interest in developing them as intravenously administered antibiotics. However, it has proven difficult to accomplish this goal with peptide-based structures. Although it has been possible to solve some relatively simple problems such as susceptibility to proteolysis, more severe problems have included the expense of the materials, toxicity, limited efficacy, and limited tissue distribution. In an effort to overcome these problems, we developed small synthetic oligomers designed to adopt amphiphilic conformations and exhibit potent antimicrobial activity while being nontoxic to host cells. One class of these synthetic mimics of antimicrobial peptides (SMAMPs) is being developed as intravenous antibiotics.

We have developed a computational design strategy based on the alpha-helical coiled-coil to generate modular peptide motifs capable of assembling into metalloporphyrin arrays of varying lengths. The current study highlights the extension of a two-metalloporphyrin array to a four-metalloporphyrin array through the incorporation of a coiled-coil repeat unit. Molecular dynamics simulations demonstrate that the initial design evolves rapidly to a stable structure with a small rmsd compared to the original model. Biophysical characterization reveals elongated proteins of the desired length, correct cofactor stoichiometry, and cofactor specificity. The successful extension of the two-porphyrin array demonstrates how this methodology serves as a foundation to create linear assemblies of organized electrically and optically responsive cofactors.

We recently reported a computational method (CHAMP) for designing sequence-specific peptides that bind to the membrane-embedded portions of transmembrane proteins. We successfully applied this method to design membrane-spanning peptides targeting the transmembrane domains of the alpha IIb subunit of integrin alpha IIbbeta 3. Previously, we demonstrated that these CHAMP peptides bind specifically with reasonable affinity to isolated transmembrane helices of the targeted transmembrane region. These peptides also induced integrin alpha IIbbeta 3 activation due to disruption of the helix-helix interactions between the transmembrane domains of the alpha IIb and beta 3 subunits. In this paper, we show the direct interaction of the designed anti-alpha IIb CHAMP peptide with isolated full-length integrin alpha IIbbeta 3 in detergent micelles. Further, the behavior of the designed peptides in phospholipid bilayers is essentially identical to their behavior in detergent micelles. In particular, the peptides assume a membrane-spanning alpha-helical conformation that does not disrupt bilayer integrity. The activity and selectivity of the CHAMP peptides were further explored in platelets, comfirming that anti-alpha IIb activates wild-type alpha IIbbeta 3 in whole cells as a result of its disruption of the protein-protein interactions between the alpha and beta subunits in the transmembrane regions. These results demonstrate that CHAMP is a successful chemical biology approach that can provide specific tools for probing the transmembrane domains of proteins.

We present a new design of peptide-polymer conjugates where a polymer chain is covalently linked to the side chain of a helix bundle-forming peptide. The effect of conjugated polymer chains on the peptide structure was examined using a de novo designed three-helix bundle and a photoactive four-helix bundle. Upon attachment of poly(ethylene glycol) to the exterior of the coiled-coil helix bundle, the peptide secondary structure was stabilized and the tertiary structure, that is, the coiled-coil helix bundle, was retained. When a heme-binding peptide as an example is used, the new peptide-polymer conjugate architecture also preserves the built-in functionalities within the interior of the helix bundle. It is expected that the conjugated polymer chains act to mediate the interactions between the helix bundle and its external environment. Thus, this new peptide-polymer conjugate design strategy may open new avenues to macroscopically assemble the helix bundles and may enable them to function in nonbiological environments.

Many antimicrobial peptides undergo a coil-to-helix transition upon binding to membranes. While this conformational transition is critical for function, little is known about the underlying mechanistic details. Here, we explore the membrane-mediated folding mechanism of an antimicrobial peptide, mastoparan X. Using stopped-flow fluorescence techniques in conjunction with a fluorescence resonance energy transfer (FRET) pair, p-cyanophenylalanine (donor) and tryptophan (acceptor), we were able to probe, albeit in an indirect manner, the membrane-mediated folding kinetics of this peptide. Our results show that the association of mastoparan X with model lipid vesicles proceeds with biphasic kinetics. The first step shows a large change in the FRET signal, indicating that the helix forms early in the time course of the interaction, while the second step where a further increase in tryptophan fluorescence is observed presumably reflects deeper insertion of the peptide into the bilayer. Additional kinetic studies on a double mutant of mastoparan X, designed to form a nucleation site for alpha-helix formation through coordination with a metal ion (e.g., Zn2+ or Ni2+), indicate that while the coil-to-helix transition occurs in the first step, it follows the rate-determining docking of the peptide onto the membrane surface. Taken together, these results indicate that the initial association of the peptide with the membrane occurs in a nonhelical conformation, which rapidly converts to a helical state within the anisotropic environment of the bilayer surface.

The alpha-helix is a ubiquitous structural element in proteins, and a number of studies have addressed the mechanism of helix formation and melting in simple peptides. However, fundamental issues remain to be resolved, particularly the temperature (T) dependence of the rate. In this work, we report application of a novel kHz repetition rate solid-state tunable NIR (pump) and deep UV Raman (probe) laser system to study the dynamics of helix unfolding in Ac-GSPEA3KA4KA4-CO-D-Arg-CONH2, a peptide designed for helix stabilization in aqueous solution. Its T-dependent UV resonance Raman (UVRR) spectra, excited at 197 nm for optimal enhancement of amide vibrations, were decomposed into variable contributions from helix and coil spectra. The helix fractions derived from the UVRR spectra and from far UV CD spectra were coincident at low T but deviated increasingly at high T, the UVRR curve giving higher helix content. This difference is consistent with the greater sensitivity of UVRR spectra to local conformation than CD. After a laser-induced T-jump, the UVRR-determined helix fractions defined monoexponential decays, with time-constants of approximately 120 ns, independent of the final T (Tf = 18-61 degrees C), provided the initial T (Ti) was held constant (6 degrees C). However, there was also a prompt loss of helicity, whose amplitude increased with increasing Tf, thereby defining an initial enthalpic phase, distinct from the subsequent entropic phase. These phases are attributed to disruption of H-bonds followed by reorientation of peptide links, as the chain is extended. When Ti was raised in parallel with Tf (10 degrees C T-jumps), the prompt phase merged into an accelerating slow phase, an effect attributable to the shifting distribution of initial helix lengths. Even greater acceleration with rising Ti has been reported in T-jump experiments monitored by IR and fluorescence spectroscopies. This difference is attributable to the longer range character of these probes, whose responses are therefore more strongly weighted toward the H-bond-breaking enthalpic process.

Reverse micelles formed by sodium bis(2-ethylhexyl) sulfosuccinate (AOT) in isooctane (IO) and water have long been used as a means to provide a confined aqueous environment for various applications. In particular, AOT reverse micelles have often been used as a template to mimic membrane-water interfaces. While earlier studies have shown that membrane-binding peptides can indeed be incorporated into the polar cavity of AOT reverse micelles where they mostly fold into an alpha-helical structure, the underlying interactions leading to the ordered conformation are however not well understood. Herein, we have used circular dichroism (CD) and infrared (IR) spectroscopies in conjunction with a local IR marker (i.e., the CN group of a non-natural amino acid, p-cyano-phenylalanine) and a global IR reporter (i.e., the amide I' band of the peptide backbone) to probe the conformation as well as the hydration status of an antimicrobial peptide, mastoparan x (MPx), in AOT reverse micelles of different water contents. Our results show that at, w0=6, MPx adopts an alpha-helical conformation with both the backbone and hydrophobic side chains mostly dehydrated, whereas its backbone becomes partially hydrated at w0=20. In addition, our results suggest that the amphipathic alpha-helix so formed orients itself in such a manner that its positively charged, lysine-rich, hydrophilic face points toward the negatively charged AOT head groups, while its hydrophobic face is directed toward the polar interior of the water pool. This picture is in marked contrast to that observed for the binding of MPx to phospholipid bilayers wherein the hydrophobic surface of the bound alpha-helix is buried deeper into the membrane interior.

A variety of methods exist for the design or selection of antibodies and other proteins that recognize the water-soluble regions of proteins; however, companion methods for targeting transmembrane (TM) regions are not available. Here, we describe a method for the computational design of peptides that target TM helices in a sequence-specific manner. To illustrate the method, peptides were designed that specifically recognize the TM helices of two closely related integrins (alphaIIbbeta3 and alphavbeta3) in micelles, bacterial membranes, and mammalian cells. These data show that sequence-specific recognition of helices in TM proteins can be achieved through optimization of the geometric complementarity of the target-host complex.

Polar residues play important roles in the association of transmembrane helices and the stabilities of membrane proteins. Although a single Ser residue in a transmembrane helix is unable to mediate a strong association of the helices, the cooperative interactions of two or more appropriately placed serine hydroxyl groups per helix has been hypothesized to allow formation of a "serine zipper" that can stabilize transmembrane helix association. In particular, a heptad repeat Sera Xxx Xxx Leud Xxx Xxx Xxx (Xxx is a hydrophobic amino acid) appears in both antiparallel helical pairs of polytopic membrane proteins as well as the parallel helical dimerization motif found in the murine erythropoietin receptor. To examine the intrinsic conformational preferences of this motif independent of its context within a larger protein, we synthesized a peptide containing three copies of a SeraLeud heptad motif. Computational results are consistent with the designed peptide adopting either a parallel or antiparallel structure, and conformational search calculations yield the parallel dimer as the lowest energy configuration, which is also significantly more stable than the parallel trimer. Analytical ultracentrifugation indicated that the peptide exists in a monomer-dimer equilibrium in dodecylphosphocholine micelles. Thiol disulfide interchange studies showed a preference for forming parallel dimers in micelles. In phospholipid vesicles, only the parallel dimer was formed. The stability of the SerZip peptide was studied in vesicles prepared from phosphatidylcholine (PC) lipids of different chain length: POPC (C16:0C18:1 PC) and DLPC (C12:0PC). The stability was greater in POPC, which has a good match between the length of the hydrophobic region of the peptide and the bilayer length. Finally, mutation to Ala of the Ser residues in the SerZip motif gave rise to a relatively small decrease in the stability of the dimer, indicating that packing interactions rather than hydrogen-bonding provided the primary driving force for association.

Polar interactions have a profound influence on membrane stability and structure. A membrane-solubilized GCN4 peptide, MS-1, is used to study the impact of polar networks. Amide functionalities from amino acid side chains have been shown to promote peptide oligomerization, but lacked specificity. Herein, the hydrogen bonding interactions of an Asn side chain are coupled with the hydroxyl of Ser or Thr to generate a polar network. Analytical ultracentrifugation and fluorescence resonance energy transfer studies indicate that a trimer assembly is established where each membrane-embedded hydrogen bond contributes 1 kcal mol-1.

Polypeptides incorporating D-amino acids occasionally occur in nature and are an important class of pharmaceutical molecules. With the use of heterochiral Monte Carlo (HCMC), a method inspired by the de novo design of proteins, we develop peptide scaffolds for interacting with a molecular target, a left-handed alpha-helix. The HCMC approach concurrently seeks to optimize a peptide sequence, its internal conformation, and its docked conformation with a target surface. Several major classes of interactions are observed: (1) homochiral interactions between two alphaL helices, (2) heterochiral interactions between an alphaL and an alphaR helix, and (3) heterochiral interactions between the alphaL target and novel nonhelical structures. We explore the application of HCMC to simulating the preferential enantioselectivity of heterochiral complexes. Implications for biomimetic design in molecular recognition are discussed.

[structure: see text] Many peptides bind to calmodulin (CaM) in a helical conformation. Here we describe a group of synthetic inhibitors of CaM based on an arylamide scaffold that is intended to mimic smMLCK, a CaM-binding helical peptide. Compound 1 showed a K(i) value of 7.10 +/- 1.48 nM in a fluorescence polarization assay that monitors the strong association of CaM and its peptide ligand mastoparan X. ((1)H,(15)N)-HSQC NMR spectroscopy experiments suggested that 1 binds to CaM in an analogous fashion to that of smMLCK.

To determine if occupancy of interfacial pockets in oligomeric proteins by volatile anesthetic molecules can allosterically regulate oligomerization equilibria, variants of a three-helix bundle peptide able to form higher oligomers were studied with analytical ultracentrifugation, hydrogen exchange and modeling. Halothane shifted the oligomerization equilibria towards the oligomer only in a mutation predicted to create sufficient volume in the hexameric pocket. Other mutations at this residue, predicted to create a too small or too polar pocket, were unaffected by halothane. Inhaled anesthetic modulation of oligomerization interactions is a novel and potentially generalizable biophysical basis for some anesthetic actions.

The relationship between monomer chirality and polymer structure has been studied using both theoretical and experimental methods. Atomistic models, such as the ones employed in computational protein folding and design, can be used to study the relationship between monomer chirality and the properties of polypeptides. Using a simulated evolution approach that combines side-chain epimerization with backbone flexibility, we recapitulate the relationship between basic forces that drive secondary structure formation and sequence homochirality. Additionally, we find heterochiral motifs including a C-terminal helix capping interaction and stable helix-reversals that result in bent helix structures. Our studies show that simulated evolution of chirality with backbone flexibility can be a powerful tool in the design of novel heteropolymers with tuned stereochemical properties.

A peptide containing glycine at a and d positions of a heptad motif was synthesized to investigate the possibility that membrane-soluble peptides with a Gly-based, left-handed helical packing motif would associate. Based on analytical ultracentrifugation in C14-betaine detergent micelles, the peptide did associate in a monomer-dimer equilibrium, although the association constant was significantly less than that reported for the right-handed dimer of the glycophorin A transmembrane peptide in similar detergents. Fluorescence resonance energy transfer (FRET) experiments conducted on peptides labeled at their N-termini with either tetramethylrhodamine (TMR) or 7-nitrobenz-2-oxa-1,3-diazole (NBD) also indicated association. However, analysis of the FRET data using the usual assumption of complete quenching for NBD-TMR pairs in the dimer could not be quantitatively reconciled with the analytical ultracentrifugation-measured dimerization constant. This led us to develop a general treatment for the association of helices to either parallel or antiparallel structures of any aggregation state. Applying this treatment to the FRET data, constraining the dimerization constant to be within experimental uncertainty of that measured by analytical ultracentrifugation, we found the data could be well described by a monomer-dimer equilibrium with only partial quenching of the dimer, suggesting that the helices are most probably antiparallel. These results also suggest that a left-handed Gly heptad repeat motif can drive membrane helix association, but the affinity is likely to be less strong than the previously reported right-handed motif described for glycophorin A.

An idealized, water-soluble D(2)-symmetric diheme protein is constructed based on a mathematical parametrization of the backbone coordinates of the transmembrane diheme four-helix bundle in cytochrome bc(1). Each heme is coordinated by two His residues from diagonally apposed helices. In the model, the imidazole rings of the His ligands are held in a somewhat unusual perpendicular orientation as found in cytochrome bc(1), which is maintained by a second-shell hydrogen bond to a Thr side chain on a neighboring helix. The resulting peptide is unfolded in the apo state but assembles cooperatively upon binding to heme into a well-folded tetramer. Each tetramer binds two hemes with high affinity at low micromolar concentrations. The equilibrium reduction midpoint potential varies between -76 mV and -124 mV vs SHE in the reducing and oxidizing direction, respectively. The EPR spectrum of the ferric complex indicates the presence of a low-spin species, with a g(max) value of 3.35 comparable to those obtained for hemes b of cytochrome bc(1) (3.79 and 3.44). This provides strong support for the designed perpendicular orientation of the imidazole ligands. Moreover, NMR spectra show that the protein exists in solution in a unique conformation and is amenable to structural studies. This protein may provide a useful scaffold for determining how second-shell ligands affect the redox potential of the heme cofactor.

We studied here the binding of the mastoparan X peptide to a zwitterionic lipid bilayer (POPC) and demonstrated that nitrile-derivatized amino acids can be used to determine the hydration state (or change in hydration state) of specific sites of membrane-interactive peptides (upon binding). We have also shown that polarized ATR-FTIR measurements can further be used to uncover information regarding the spatial orientation of individual side chains as well as their conformational preference within the lipid bilayer.

The de novo design of peptides that switch their oligomerization state in response to a chemical stimulus is of interest, both as a tool for understanding the basis of molecular switching as well as development of reagents for the study of signal transduction in cells. The target of the current study is the design of a series of peptides that undergo a transition from an unstructured monomer to a four-helical bundle upon phosphorylation by the enzyme cyclic AMP-dependent protein kinase (PKA). The designed peptides are based on the 20-residue Lac repressor tetramerization domain. Beginning with this structure, we introduced a phosphorylation site near the N terminus. Phosphorylation leads to a 2-4.6 kcal/mol increase in the stability of the tetramer, depending on the design. The most successful switches were designed such that phosphorylation would increase the stability of the individual helices and also relieve an unfavorable electrostatic interaction in the tetramer.

Thermodynamics studies aimed at quantitatively characterizing free energy effects of amino acid substitutions are not restricted to two state systems, but do require knowing the number of states involved in the equilibrium under consideration. Using analytical ultracentrifugation and NMR methods, we show here that a membrane-soluble peptide, MS1, designed by modifying the sequence of the water-soluble coiled-coil GCN4-P1, exhibits a reversible monomer-dimer-trimer association in detergent micelles with a greater degree of cooperativity in C14-betaine than in dodecyl phosphocholine detergents.

The design of polymers and oligomers that mimic the complex structures and remarkable biological properties of proteins is an important endeavor with both fundamental and practical implications. Recently, a number of nonnatural peptides with designed sequences have been elaborated to provide biologically active structures; in particular, facially amphiphilic peptides built from beta-amino acids have been shown to mimic both the structures as well as the biological function of natural antimicrobial peptides such as magainins and cecropins. However, these natural peptides as well as their beta-peptide analogues are expensive to prepare and difficult to produce on a large scale, limiting their potential use to certain pharmaceutical applications. We therefore have designed a series of facially amphiphilic arylamide polymers that capture the physical and biological properties of this class of antimicrobial peptides, but are easy to prepare from inexpensive monomers. The design process was aided by molecular calculations with density functional theory-computed torsional potentials. This new class of amphiphilic polymers may be applied in situations where inexpensive antimicrobial agents are required.

The helix-coil transition kinetics of an alpha-helical peptide were investigated by time-resolved infrared spectroscopy coupled with laser-induced temperature-jump initiation method. Specific isotope labeling of the amide carbonyl groups with 13C at selected residues was used to obtain site-specific information. The relaxation kinetics following a temperature jump, obtained by probing the amide I' band of the peptide backbone, exhibit nonexponential behavior and are sensitive to both initial and final temperatures. These data are consistent with a conformation diffusion process on the folding energy landscape, in accord with a recent molecular dynamics simulation study.

Site-specific protein cleavage is essential for many protein-production protocols and typically requires proteases. We report the development of a chemical protein-cleavage method that is achieved through the use of a sequence-specific nickel-assisted cleavage (SNAC)-tag. We demonstrate that the SNAC-tag can be inserted before both water-soluble and membrane proteins to achieve fusion protein cleavage under biocompatible conditions with efficiency comparable to that of enzymes, and that the method works even when enzymatic cleavages fail.

De novo design provides an attractive approach, which allows one to test and refine the principles guiding metalloproteins in defining the geometry and reactivity of their metal ion cofactors. Although impressive progress has been made in designing proteins that bind transition metal ions including iron-sulfur clusters, the design of tetranuclear clusters with oxygen-rich environments remains in its infancy. In previous work, we described the design of homotetrameric four-helix bundles that bind tetra-Zn2+ clusters. The crystal structures of the helical proteins were in good agreement with the overall design, and the metal-binding and conformational properties of the helical bundles in solution were consistent with the crystal structures. However, the corresponding apo-proteins were not fully folded in solution. In this work, we design three peptides, based on the crystal structure of the original bundles. One of the peptides forms tetramers in aqueous solution in the absence of metal ions as assessed by CD and NMR. It also binds Zn2+ in the intended stoichiometry. These studies strongly suggest that the desired structure has been achieved in the apo state, providing evidence that the peptide is able to actively impart the designed geometry to the metal cluster.