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Hydrophobic interactions govern how proteins fold and interact with other molecules, but the impact of nearby polar functionality on the effective hydrophobicity of nonpolar surfaces remains unclear. Here we use a common protein quaternary structure motif, the parallel coiled-coil dimer, to ask whether the identity of basic residues (arginine vs lysine; guanidinium vs ammonium) arrayed along one side of the constituent α-helices influences the favorability of dimerization driven by burial of hydrophobic surface on the other side of each helix. Significant sequence redesign was necessary to achieve the desired juxtaposition of nonpolar and cationic functionality, because we needed to eliminate charged side chains from positions flanking the nonpolar helix surface. Natural and designed sequences that form coiled coils are almost universally rich in acidic and basic residues at these flanking positions. Our arginine coiled-coil dimer was moderately more stable than the lysine analogue, which contrasts with behavior previously observed with helical β-amino acid oligomers bearing guanidinium versus ammonium groups. We attribute this backbone-dependent difference to variations in the extent to which the helical propensities of α- and β-residues can be modulated by design. These findings highlight the challenge of identifying noncovalent forces that direct structure formed by a flexible backbone.

β-Amino acids have a backbone that is expanded by one carbon atom relative to α-amino acids, and β residues have been investigated as subunits in protein-like molecules that adopt discrete and predictable conformations. Two classes of β residue have been widely explored in the context of generating α-helix-like conformations: β 3 -amino acids, which are homologous to α-amino acids and bear a side chain on the backbone carbon adjacent to nitrogen, and residues constrained by a five-membered ring, such the one derived from trans-2-aminocyclopentanecarboxylic acid (ACPC). Substitution of α residues with their β 3  homologues within an α-helix-forming sequence generally causes a decrease in conformational stability. Use of a ring-constrained β residue, however, can offset the destabilizing effect of α→β substitution. Here we extend the study of α→β substitutions, involving both β 3 and ACPC residues, to short loops within a small tertiary motif. We start from previously reported variants of the Pin1 WW domain that contain a two-, three-, or four-residue β-hairpin loop, and we evaluate α→β replacements at each loop position for each variant. By referral to the ϕ,ψ angles of the native structure, one can choose a stereochemically appropriate ACPC residue. Use of such logically chosen ACPC residues enhances conformational stability in several cases. Crystal structures of three β-containing Pin1 WW domain variants show that a native-like tertiary structure is maintained in each case., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Molecules intended to antagonize protein-protein interactions or augment polypeptide-based signaling must bind tightly to large and specific surfaces on target proteins. Some types of unnatural oligomers with discrete folding propensities ('foldamers') have recently been shown to display this capability. This review covers important recent advances among several classes of foldamers, including α-peptides with secondary structures stabilized by covalent bonds, d-α-peptides, α/β-peptides and oligo-oxopiperazines. Recent advances in this area have involved enhancing membrane permeability to provide access to intracellular protein targets, improving pharmacokinetics and duration of action in vivo, and developing strategies appropriate for targeting large and irregularly-shaped protein surfaces., Competing Interests: statement JWC and SHG are co-inventors on a patent application covering several of the α/β-peptides discussed in this review. SHG is a co-inventor on several other foldamer patent applications. SHG is a co-founder of Longevity Biotech, Inc., which is pursuing biomedical applications of α/β-peptides., (Copyright © 2016. Published by Elsevier Ltd.)

Synthetic peptides that contain backbone modifications but nevertheless adopt folded structures similar to those of natural polypeptides are of fundamental interest and may provide a basis for biomedical applications. Such molecules can, for example, mimic the ability of natural prototypes to bind to specific target macromolecules but resist degradation by proteases. We have previously shown that oligomers containing mixtures of α- and β-amino acid residues ("α/β-peptides") can mimic the α-helix secondary structure, and that properly designed α/β-peptides can bind to proteins that evolved to bind to α-helical partners. Here we report fundamental studies that support the long-range goal of extending the α/β approach to tertiary structures. We have evaluated the impact of single α → β modifications on the structure and stability of the small and well-studied villin headpiece subdomain (VHP). The native state of this 35-residue polypeptide contains several α-helical segments packed around a small hydrophobic core. We examined α → β substitution at four solvent-exposed positions, Asn19, Trp23, Gln26 and Lys30. In each case, both the β(3) homologue of the natural α residue and a cyclic β residue were evaluated. All α → β(3) substitutions caused significant destabilization of the tertiary structure as measured by variable-temperature circular dichroism, although at some of these positions, replacing the β(3) residue with a cyclic β residue led to improved stability. Atomic-resolution structures of four VHP analogues were obtained via quasiracemic crystallization. These findings contribute to a fundamental α/β-peptide knowledge-base by confirming that β(3)-amino acid residues can serve as effective structural mimics of homologous α-amino acid residues within a natural tertiary fold, which should support rational design of functional α/β analogues of natural poly-α-peptides.

Interactions between polypeptide chains containing amino acid residues with opposite absolute configurations have long been a source of interest and speculation, but there is very little structural information for such heterochiral associations. The need to address this lacuna has grown in recent years because of increasing interest in the use of peptides generated from d amino acids (d peptides) as specific ligands for natural proteins, e.g., to inhibit deleterious protein-protein interactions. Coiled-coil interactions, between or among α-helices, represent the most common tertiary and quaternary packing motif in proteins. Heterochiral coiled-coil interactions were predicted over 50 years ago by Crick, and limited experimental data obtained in solution suggest that such interactions can indeed occur. To address the dearth of atomic-level structural characterization of heterochiral helix pairings, we report two independent crystal structures that elucidate coiled-coil packing between l- and d-peptide helices. Both structures resulted from racemic crystallization of a peptide corresponding to the transmembrane segment of the influenza M2 protein. Networks of canonical knobs-into-holes side-chain packing interactions are observed at each helical interface. However, the underlying patterns for these heterochiral coiled coils seem to deviate from the heptad sequence repeat that is characteristic of most homochiral analogs, with an apparent preference for a hendecad repeat pattern.

Peptide-based agents derived from well-defined scaffolds offer an alternative to antibodies for selective and high-affinity recognition of large and topologically complex protein surfaces. Here, we describe a strategy for designing oligomers containing both α- and β-amino acid residues ("α/β-peptides") that mimic several peptides derived from the three-helix bundle "Z-domain" scaffold. We show that α/β-peptides derived from a Z-domain peptide targeting vascular endothelial growth factor (VEGF) can structurally and functionally mimic the binding surface of the parent peptide while exhibiting significantly decreased susceptibility to proteolysis. The tightest VEGF-binding α/β-peptide inhibits the VEGF165-induced proliferation of human umbilical vein endothelial cells. We demonstrate the versatility of this strategy by showing how principles underlying VEGF signaling inhibitors can be rapidly extended to produce Z-domain-mimetic α/β-peptides that bind to two other protein partners, IgG and tumor necrosis factor-α. Because well-established selection techniques can identify high-affinity Z-domain derivatives from large DNA-encoded libraries, our findings should enable the design of biostable α/β-peptides that bind tightly and specifically to diverse targets of biomedical interest. Such reagents would be useful for diagnostic and therapeutic applications.

Infrared spectroscopy is playing an important role in the elucidation of amyloid fiber formation, but the coupling models that link spectra to structure are not well tested for parallel β-sheets. Using a synthetic macrocycle that enforces a two stranded parallel β-sheet conformation, we measured the lifetimes and frequency for six combinations of doubly (13)C═(18)O labeled amide I modes using 2D IR spectroscopy. The average vibrational lifetime of the isotope labeled residues was 550 fs. The frequencies of the labels ranged from 1585 to 1595 cm(-1), with the largest frequency shift occurring for in-register amino acids. The 2D IR spectra of the coupled isotope labels were calculated from molecular dynamics simulations of a series of macrocycle structures generated from replica exchange dynamics to fully sample the conformational distribution. The models used to simulate the spectra include through-space coupling, through-bond coupling, and local frequency shifts caused by environment electrostatics and hydrogen bonding. The calculated spectra predict the line widths and frequencies nearly quantitatively. Historically, the characteristic features of β-sheet infrared spectra have been attributed to through-space couplings such as transition dipole coupling. We find that frequency shifts of the local carbonyl groups due to nearest neighbor couplings and environmental factors are more important, while the through-space couplings dictate the spectral intensities. As a result, the characteristic absorption spectra empirically used for decades to assign parallel β-sheet secondary structure arises because of a redistribution of oscillator strength, but the through-space couplings do not themselves dramatically alter the frequency distribution of eigenstates much more than already exists in random coil structures. Moreover, solvent exposed residues have amide I bands with >20 cm(-1) line width. Narrower line widths indicate that the amide I backbone is solvent protected inside the macrocycle. This work provides calculated and experimentally verified couplings for parallel β-sheets that can be used in structure-based models to simulate and interpret the infrared spectra of β-sheet containing proteins and protein assemblies, such as amyloid fibers.

Elucidating relationships between the amino-acid sequences of proteins and their three-dimensional structures, and uncovering non-covalent interactions that underlie polypeptide folding, are major goals in protein science. One approach toward these goals is to study interactions between selected residues, or among constellations of residues, in small folding motifs. The α-helical coiled coil has served as a platform for such studies because this folding unit is relatively simple in terms of both sequence and structure. Amino acid side chains at the helix-helix interface of a coiled coil participate in so-called "knobs-into-holes" (KIH) packing whereby a side chain (the knob) on one helix inserts into a space (the hole) generated by four side chains on a partner helix. The vast majority of sequence-stability studies on coiled-coil dimers have focused on lateral interactions within these KIH arrangements, for example, between an a position on one helix and an a' position of the partner in a parallel coiled-coil dimer, or between a--d' pairs in an antiparallel dimer. More recently, it has been shown that vertical triads (specifically, a'--a--a' triads) in antiparallel dimers exert a significant impact on pairing preferences. This observation provides impetus for analysis of other complex networks of side-chain interactions at the helix-helix interface. Here, we describe a combination of experimental and bioinformatics studies that show that d'--d--d' triads have much less impact on pairing preference than do a'--a--a' triads in a small, designed antiparallel coiled-coil dimer. However, the influence of the d'--d--d' triad depends on the lateral a'--d interaction. Taken together, these results strengthen the emerging understanding that simple pairwise interactions are not sufficient to describe side-chain interactions and overall stability in antiparallel coiled-coil dimers; higher-order interactions must be considered as well.

A new strategy for rapid evaluation of sequence-stability relationships in the parallel coiled-coil motif is described. The experimental design relies upon thiol-thioester exchange equilibria, an approach that is particularly well suited to examination of heterodimeric systems. Our model system has been benchmarked by demonstrating that it can quantitatively reproduce previously reported trends in interhelical a-a' side-chain pairing preferences at the coiled-coil interface. This new tool has been used to explore the role of Coulombic interactions between a core position on one helix and a flanking position on the other helix (a-g'). This type of interhelical contact has received relatively little attention to date. Our results indicate that such interactions can influence coiled-coil partner preferences.

We describe the use of parallel and split-and-mix library synthesis strategies for exploration of structure-activity relationships among peptidic foldamer ligands for the BH3-recognition cleft of the anti-apoptotic protein Bcl-xL. This effort began with a chimeric (alpha/beta+alpha)-peptide oligomer (composed of an alpha/beta-peptide segment and an alpha-peptide segment) that we previously identified to bind tightly to the target cleft on Bcl-xL. The side chains that interact with Bcl-xL were varied in a 1000-member one-bead-one-compound library. Fluorescence polarization (FP) screening identified four new analogues with binding affinities similar to that of the lead compound but no analogues with enhanced affinity. These results suggested that significant improvements in affinity were unlikely in this series. We then used library synthesis to examine backbone variations in the C-terminal alpha-peptide segment of the lead compound. These studies provided an opportunity for direct comparison of parallel and split-and-mix synthesis formats for foldamer libraries with respect to synthetic variability and assay sensitivity. We found that compounds from both the parallel and one-bead-one-compound libraries could be reliably screened in a competition FP assay without purification of library members. Our findings should facilitate the use of combinatorial library synthesis for exploration of foldamers as inhibitors of protein-protein interactions.

Reliable predictive rules that relate protein sequence to structure would facilitate postgenome predictive biology and the engineering and de novo design of peptides and proteins. Through a combination of experiment and analysis of the protein data bank (PDB), we have deciphered and rationalized new rules for helix-helix interfaces of a common protein-folding and association motif, the antiparallel dimeric coiled coil. These interfaces are defined by a specific pattern of interactions among largely hydrophobic side chains often referred to as knobs-into-holes (KIH) packing: a knob from one helix inserts into a hole formed by four residues on the partner. Previous work has focused on lateral interactions within the KIH motif, for example, between an a position on one helix and a d' position on the other in an antiparallel coiled coil. We show that vertical interactions within the KIH motif, such as a'-a-a', are energetically important as well. The experimental and database analyses concur regarding preferred vertical combinations, which can be rationalized as leading to favorable side-chain interactions that we call constellations. The findings presented here highlight an unanticipated level of complexity in coiled-coil interactions, and our analysis of a few specific constellations illustrates a general, multipronged approach to addressing this complexity.

The equilibrium unfolding properties of four model protein systems were characterized using SUPREX (stability of unpurified proteins from rates of H/D exchange). SUPREX is an H/D exchange- and mass spectrometry-based technique for measuring the free energy (DeltaGf) and m-value (deltaDeltaGf/delta[denaturant]) associated with the folding/unfolding reaction of a protein. The model proteins in this study (calmodulin, carbonic anhydrase II, RmlB, Bcl-xL) were chosen to test the applicability of SUPREX to the thermodynamic analysis of larger (> approximately 15 kDa) or multidomain proteins. In the absence of ligand, DeltaGf and m-values for these proteins could not be evaluated using the conventional data acquisition and analysis methods previously established for SUPREX. However, ligand-bound forms of the proteins were amenable to conventional SUPREX analyses, and it was possible to evaluate reasonably accurate and precise binding free energies of selected ligands. In some cases, protein-ligand dissociation constants (Kd values) could also be ascertained. The SUPREX-derived binding free energies and Kd values evaluated here were in good agreement with those reported on the same complexes using other techniques.

We report the first high-resolution structure for a protein containing a fluorinated side chain. Recently we carried out a systematic evaluation of phenylalanine to pentafluorophenylalanine (Phe --> F(5)-Phe) mutants for the 35-residue chicken villin headpiece subdomain (c-VHP), the hydrophobic core of which features a cluster of three Phe side chains (residues 6, 10, and 17). Phe --> F(5)-Phe mutations are interesting because aryl-perfluoroaryl interactions of optimal geometry are intrinsically more favorable than either aryl-aryl or perfluoroaryl-perfluoroaryl interactions, and because perfluoroaryl units are more hydrophobic than are analogous aryl units. Only one mutation, Phe10 --> F(5)-Phe, was found to provide enhanced tertiary structural stability relative to the native core (by approximately 1 kcal/mol, according to guanidinium chloride denaturation studies). The NMR structure of this mutant, described here, reveals very little variation in backbone conformation or side chain packing relative to the wild type. Thus, although Phe --> F(5)-Phe mutations offer the possibility of greater tertiary structural stability from side chain-side chain attraction and/or side chain desolvation, the constraints associated with the native c-VHP fold apparently prevent the modified polypeptide from taking advantage of this possibility. Our findings are important because they complement several studies that have shown that fluorination of saturated side chain carbon atoms can provide enhanced conformational stability.

Both parallel and antiparallel alpha-helical coiled-coil dimers are common among proteins; however, biophysical scrutiny has focused almost entirely on parallel dimers. We describe the development of a model system that enables efficient and systematic analysis of hydrophobic packing between antiparallel alpha-helices. Our findings reveal significant differences in packing preferences between parallel and antiparallel coiled-coils.

Protegrins are short (16-18 residues) cationic peptides from porcine leukocytes that display potent, broad-spectrum antimicrobial activity. Protegrin-1 (PG-1), one of five natural homologues, adopts a rigid beta-hairpin structure that is stabilized by two disulfide bonds. We have previously employed the principles of beta-hairpin design to develop PG-1 variants that lack disulfide bonds but nevertheless display potent antimicrobial activity [Lai, J. R., Huck, B. R., Weisblum, B., and Gellman, S. H. (2002) Biochemistry 41, 12835-12842.]. The activity of these disulfide-free variants, however, is attenuated in the presence of salt, and the activity of PG-1 itself is not. Salt-induced inactivation of host-defense peptides, such as human defensins, is thought to be important in some pathological situations (e.g., cystic fibrosis), and the variation in salt-sensitivity among our PG-1 analogues offers a model system with which to explore the origins of these salt effects. We find that the variations in antimicrobial activity among our peptides are correlated with the folding propensities of these molecules and with the extent to which the peptides induce leakage of contents from synthetic liposomes. Comparable correlations were observed between folding and hemolytic activity. The extent to which added salt reduces antimicrobial activity parallels salt effects on vesicle perturbation, which suggests that the biological effects of high salt concentrations arise from modulation of peptide-membrane interactions.

We report a systematic evaluation of phenylalanine-to-pentafluorophenylalanine (Phe --> F5-Phe) mutants for the 35-residue chicken villin headpiece subdomain (c-VHP), the hydrophobic core of which features a cluster of three Phe side chains (residues 6, 10, and 17). Phe --> F5-Phe mutations are interesting because aryl-perfluoroaryl interactions of optimal geometry are intrinsically more favorable than aryl-aryl interactions and because perfluoroaryl units are more hydrophobic than are analogous aryl units. One mutant, Phe-10 --> F5-Phe, provides enhanced tertiary structural stability relative to the native sequence. The other six mutants analyzed caused a decrease in stability.

We report the first thermodynamic analysis of parallel beta-sheet formation in a model system that folds in aqueous solution. NMR chemical shifts were used to determine beta-sheet population, and van't Hoff anaysis provided thermodynamic parameters. Our approach relies upon the d-prolyl-1,1-dimethyl-1,2-diaminoethane unit to promote parallel beta-sheet formation between attached peptide strands. The development of a macrocyclic reference molecule to provide chemical shift data for the fully folded state was crucial to the quantitative anaylsis.

The coiled-coil, which consists of two or more interwoven amphiphilic alpha-helices, is formed by sequences that have a characteristic heptad repeat (abcdefg) where a and d are hydrophobic residues. Most efforts to elucidate the origins of coiled-coil pairing selectivity have focused on electrostatic interactions among side chains that flank the core (positions e and g) and on polar side chains that occur occasionally at core positions. We have used phage display to explore another source of coiled-coil specificity: steric matching among nonpolar side chains in the core. We introduced a destabilizing Leu-->Ala mutation into the core of one helix in a known heterodimer and then screened a phage-based library of potential partner helices in search of compensating mutations. We identified a new heterodimer pair (30 residues/helix) that is comparable in stability to the GCN4-p1 homodimer (33 residues/helix). Furthermore, the Leu-->Ala mutant shows specificity for its phage-derived partner over the original partner despite their similar sequences. These results show that a phage-based approach can provide unique insights on coiled-coil pairing preferences that should facilitate both the analysis of natural sequences and the development of specific dimerization motifs that are orthogonal to one another.

[structure: see text] We describe a series of beta-peptide hexamers that allow us to explore relationships between sequence and hairpin folding. Different reverse turn segments are compared at the central two positions, and the outer residues allow a variety of interstrand side chain-side chain pairings. NMR analysis in methanol demonstrates that several reverse turn and side chain pairing arrangements are compatible with antiparallel beta-peptide sheet structure; however, none of the beta-peptides folds in water.

Protegrins are short, cationic peptides that display potent, broad-spectrum antimicrobial activity. PG-1, the first of the five natural analogues discovered, forms a rigid antiparallel two-stranded beta-sheet that is stabilized by two disulfide bonds. The two strands of the sheet are linked by a short two-residue loop segment. Removal of the disulfide bridges (e.g., in Cys --> Ala analogues) is known to cause marked loss of antimicrobial activity. We have used basic principles of beta-hairpin design to develop linear analogues of PG-1 that lack cysteine but nevertheless display PG-1-like activity. Our most potent reengineered molecules contain three essential design features: (i) the four cysteine residues of PG-1 are replaced by residues that have high propensity for beta-strand conformation, (ii) D-proline is placed at the i + 1 position of the reverse turn to promote a type II' beta-turn, and (iii) amino functionality is incorporated at the gamma-carbon of the D-proline residue to mimic the charge distribution of the natural beta-hairpin. Structural studies revealed that the antimicrobial potency of the non-disulfide-bonded peptides can be correlated to the stability of the beta-hairpin conformations they adopt in aqueous solution. The presence of 150 mM NaCl was found to have little effect on the antimicrobial activity of PG-1, but one of our linear analogues loses some potency under these high salt conditions. Despite this discrepancy in salt sensitivity, NMR and CD data indicate that neither PG-1 nor our linear analogue experiences a significant decrease in beta-hairpin conformational stability in the presence of 150 mM NaCl. Thus, salt inactivation is not due to destabilization of the beta-hairpin conformation. Furthermore, our results show that beta-sheet design principles can be used to replace conformation-stabilizing disulfide bridges with noncovalent conformation-stabilizing features.

Foldamers are non-natural oligomers that adopt stable conformations reminiscent of those found in proteins. To evaluate the potential of foldameric subunits for catalysis, semisynthetic enzymes containing foldamer fragments constructed from α- and β-amino acid residues were designed and characterized. Systematic variation of the α→β substitution pattern and types of β-residue afforded highly proficient hybrid catalysts, thus demonstrating the feasibility of expanding the enzyme-engineering toolkit with non-natural backbones. [ABSTRACT FROM AUTHOR]

The understanding of integral membrane protein (IMP) structure and function is hampered by the difficulty of handling these proteins. Aqueous solubilization, necessary for many types of biophysical analysis, generally requires a detergent to shield the large lipophilic surfaces of native IMPs. Many proteins remain difficult to study owing to a lack of suitable detergents. We introduce a class of amphiphiles, each built around a central quaternary carbon atom derived from neopentyl glycol, with hydrophilic groups derived from maltose. Representatives of this maltose-neopentyl glycol (MNG) amphiphile family show favorable behavior relative to conventional detergents, as manifested in multiple membrane protein systems, leading to enhanced structural stability and successful crystallization. MNG amphiphiles are promising tools for membrane protein science because of the ease with which they may be prepared and the facility with which their structures may be varied. [ABSTRACT FROM AUTHOR]

Studies the length-dependent stability and strand length limits in antiparallel beta-sheet secondary structure. Factors that determine native conformations of proteins; Possibility of an intrinsic limit to strand length for most sequences in antiparallel beta-sheet secondary structure; Use of designed peptides for elucidating protein secondary structural preferences.

Describes the characteristics of the protein tertiary structure, focusing on the attribute of cooperativity. Occurrence of cooperativity; Classification of cooperativity; What the site-specific conformational data from nuclear magnetic resonance (NMR) spectroscopy (NOESY) allowed.

We report structure-activity trends among helix-forming β-amino acid oligomers that are intended to mimic α-helical host-defense peptides. Parallel synthesis of two small, focused β-peptide libraries allowed us to identity relatively short (11-residue) β-peptides that display antimicrobial activity. These β-peptides exhibit selectivity for bacteria relative to human red blood cells. A large hydrophobic helical surface is necessary for antimicrobial activity. Longer analogues (16 residues) of the most active library members were prepared and evaluated. Some of these longer β-peptides showed very good antimicrobial activity, but none was more active than a previously reported β-peptide [Porter, E. A.; Wang, X.; Lee, H.-S.; Weisblum, B.; Gellman, S. H. Nature 2000, 404, 565]. The extensive literature on α-helical host-defense peptides and related α-peptides indicates that such molecules are seldom active at concentrations below 1 μg/mL, and our results suggest that amphiphilic helical β-peptides are subject to a comparable limit. [ABSTRACT FROM AUTHOR]

The formation and deposition of amyloids is associated with many diseases. β-Sheet secondary structure is a common feature of amyloids, but the packing of sheets against one another is distinctive relative to soluble proteins. Standard methods that rely on perturbing a polypeptide's sequence and evaluating impact on folding can be problematic for amyloid aggregates because a single sequence can adopt multiple conformations and diverse packing arrangements. We describe initial steps toward a minimum-sized, soluble model system for the amyloid state that supports comparisons among sequence variants. Critical to this goal is development of a new linking strategy to enable intersheet association mediated by side chain interactions, which is characteristic of the amyloid state. The linker design we identified should ultimately support exploration of relationships between sequence and amyloid state stability for specific strand-association modes. [ABSTRACT FROM AUTHOR]

Studies the environment-independent 14-Helix formation of short Beta-peptides. Balance between shape control and functional diversity; Analysis of pertinent topics and relevant issues; Implications on biochemistry and nanotechnology.

Peptidic oligomers that contain both α- and β-amino acid residues, in regular patterns throughout the backbone, are emerging as structural mimics of α-helix-forming conventional peptides (composed exclusively of α-amino acid residues). Here we describe a comprehensive evaluation of diverse α/β-peptide homologues of the Bim BH3 domain in terms of their ability to bind to the BH3-recognition sites on two partner proteins, Bcl-xL and Mcl-1. These proteins are members of the anti-apoptotic Bcl-2 family, and both bind tightly to the Bim BH3 domain itself. All α/β-peptide homologues retain the side-chain sequence of the Bim BH3 domain, but each homologue contains periodic α-residue → β³-residue substitutions. Previous work has shown that the ααβαααβ pattern, which aligns the β³-residues in a 'stripe' along one side of the helix, can support functional α-helix mimicry, and the results reported here strengthen this conclusion. The present study provides the first evaluation of functional mimicry by ααβ and αααβ patterns, which cause the β³-residues to spiral around the helix periphery. We find that the αααβ pattern can support effective mimicry of the Bim BH3 domain, as manifested by the crystal structure of an α/β-peptide bound to Bcl-xL, affinity for a variety of Bcl-2 family proteins, and induction of apoptotic signaling in mouse embryonic fibroblast extracts. The best αααβ homologue shows substantial protection from proteolytic degradation relative to the Bim BH3 α-peptide. [ABSTRACT FROM AUTHOR]

Poly-β-peptides are attractive for biomedical applications because the backbone is similar enough to that of proteins for biocompatibility, but the backbone is sufficiently unnatural that these polymers evade proteolytic degradation. Prior investigations of poly-β-peptides have been hindered by two principal limitations: (1) most known examples are insoluble, and (2) the range of accessible side chain functionality has been quite limited (mostly simple hydrocarbon units). The present study describes innovations in poly-β-peptide synthesis that enable the preparation of diversely functionalized examples and provide the basis for broad exploration of the properties and applications of these nylon-3 materials. We describe several β-lactams with a protected amino group in their side chain that readily undergo ring-opening polymerization (ROP). These monomers are available in large quantities via N-chlorosulfonylisocyanate (CSI) cycloaddition reactions with functionalized alkenes; previously CSI reactions have been limited to alkenes with hydrocarbon substituents. Postpolymerization deprotection of the amino groups leads to water-soluble poly-β-peptides. In addition, we introduce a simple co-initiation strategy that allows placement of a wide variety of functional - groups at the N-termini of poly-β-peptide chains. ROP involving the new β-lactams and co-initiation strategy exhibits characteristics of a controlled polymerization and enables the preparation of amphiphilic block copolymers. We have recently shown that cationic copoly-β-peptides made available by these innovations mimic the selective antibacterial activity of host-defense peptides; the results described here provide the foundation for further exploration of this valuable activity and for the pursuit of other biological applications such as DNA/siRNA delivery and tissue engineering. [ABSTRACT FROM AUTHOR]

The predictable relationship between β-amino acid sequence and folding has inspired several biological applications of β-peptides. For many such applications, it would be desirable to prepare and screen β-peptide libraries. However, standard peptide synthesis protocols are not efficient enough to support a library approach for many types of β-peptides. We recently optimized the solid-phase synthesis of β-peptides using microwave irradiation, and we have now adapted this approach to synthesis on polystyrene macrobeads. We rapidly prepared a high-quality β-peptide combinatorial library via a split-and-mix strategy. This library was screened in search of β-peptide antagonists of the p53-MDM2 protein—protein interaction. [ABSTRACT FROM AUTHOR]

The article describes simple diacid linkers that promote parallel beta-sheet folding between peptide strands attached via their N-termini. These new linkers represent valuable tools for analysis of sequence-stability relationships in parallel beta-sheet secondary structure. A new linker for connecting peptide strands via their N-termini is introduced.

The article focuses on the stabilization and destabilization effects of Phenylalanines with regards to the folding patterns of small proteins. The stabilization is attributed to the increased hydrophobicity of flurocarbons and its interaction with hydrocarbons. Destabilization effects reflect with repulsions arising from the increased in side chain volume and conformational rearrangements. Fluoroaryl side chains can stabilize but it is not an effect of Phenylalanine mutations.

The article presents information on a research related to the antifungal activity of β-peptides. Treatment of fungal infections represents a profound medical challenge. Two β-peptide sequences have been evaluated to gauge the antifungal activities of amphiphilic host-defense peptides in the National Committee for Clinical Laboratory Standards assay.

The article focuses on hydrophobic core repacking in a coiled-coil dimer through phage display. The coiled-coil, in which two &b.alpha;-helices associate to bury hydrophobic surfaces against one another, is a very common protein dimerization motif that plays a critical role in transcriptional regulation and other biological processes. Elucidation of the factors responsible for dimerization affinity and specificity should improve the understanding of natural coiled-coil function and enhance the ability to design selective self-assembling systems. The article introduces a destabilizing mutation into one partner of a designed coiled-coil pair, and then uses phage-display to determine whether mutations in the other helix could compensate for this destabilizing alteration.