A rapid library screen for tailoring beta-peptide structure and function

Recently we described a beta-decapeptide (beta53-1) that folds into a 14-helix in aqueous solution, binds the oncoprotein hDM2 with submicromolar affinity, and inhibits the interaction of hDM2 with a peptide derived from the activation domain of p53 (p53AD). The solution structure of beta53-1 in CD3OH revealed an unexpected C-terminal unwinding that staggers the side chains comprising the hDM2 recognition epitope to better mimic those of p53AD. The structure-function relationship implied by this distortion suggested that a library of beta53-1 analogues possessing diversity along a nonrecognition face might contain molecules possessing greater affinity for hDM2. Here we describe (1) beta-peptide synthesis protocols that produce high quality one-bead-one-beta-peptide libraries suitable for on-bead screening without purification, (2) a versatile, scalable on-bead screen, and (3) a simple tandem mass spectrometry (MS/MS) decoding method. Using this procedure, we identified beta53-1 analogues with improved structural and functional properties.     

Helical beta-peptide inhibitors of the p53-hDM2 interaction

hDM2 is recognized in vivo by a short alpha-helix within the p53 trans-activation domain (p53AD). Disruption of the p53.hDM2 interaction is an important goal for cancer therapy. A functional epitope comprised of three residues on one face of the p53AD helix (F19, W23, and L26) contributes heavily to the binding free energy. We hypothesized that the p53AD functional epitope would be recapitulated if the side chains of F19, W23, and L26 were presented at successive positions three residues apart on a stabilized beta3-peptide 14-helix. Here, we report a set of beta3-peptides that possess significant 14-helix structure in water; one recognizes a cleft on the surface of hDM2 with nanomolar affinity. The strategy for beta3-peptide design that we describe is general and may have advantages over one in which individual or multiple beta-amino acid substitutions are introduced into a functional alpha-peptide, because it is based on homology at the level of secondary structure, not primary sequence.     

Relationship between salt-bridge identity and 14-helix stability of beta3-peptides in aqueous buffer

[STRUCTURE: SEE TEXT] We report a systematic analysis of the relationship between salt bridge composition and 14-helix structure within a family of model beta-peptides in aqueous buffer. We find an inverse relationship between side-chain length and the extent of 14-helix structure as judged by CD. Introduction of a stabilizing salt bridge pair within a previously reported beta-peptide ligand for hDM2 led to changes in structure that were detectable by NMR.     

Relationship between side chain structure and 14-helix stability of beta3-peptides in water

Folded polymers are used in Nature for virtually every vital process. Nonnatural folded polymers, or foldamers, have the potential for similar versatility, and the design and refinement of such molecules is of considerable current interest. Here we report a complete and systematic analysis of the relationship between side chain structure and the 14-helicity of a well-studied class of foldamers, beta(3)-peptides, in water. Our experimental results (1) verify the importance of macrodipole stabilization for maintaining 14-helix structure, (2) provide comprehensive evidence that beta(3)-amino acids branched at the first side chain carbon are 14-helix-stabilizing, (3) suggest a novel role for side chain hydrogen bonding as an additional stabilizing force in beta(3)-peptides containing beta(3)-homoserine or beta(3)-homothreonine, and (4) demonstrate that diverse functionality can be incorporated into a stable 14-helix. Gas- and solution-phase calculations and Monte Carlo simulations recapitulate the experimental trends only in the context of oligomers, yielding insight into the mechanisms behind 14-helix folding. The 14-helix propensities of beta(3)-amino acids differ starkly from the alpha-helix propensities of analogous alpha-amino acids. This contrast informs current models for alpha-helix folding, and suggests that 14-helix folding is governed by different biophysical forces than is alpha-helix folding. The ability to modulate 14-helix structure through side chain choice will assist rational design of 14-helical beta-peptide ligands for macromolecular targets.     

Crystallographic characterization of helical secondary structures in alpha/beta-peptides with 1:1 residue alternation

Oligomers that contain both alpha- and beta-amino acid residues in a 1:1 alternating pattern have recently been shown by several groups to adopt helical secondary structures in solution. The beta-residue substitution pattern has a profound effect on the type of helix formed and the stability of the helical conformation. On the basis of two-dimensional NMR data, we have previously proposed that beta-residues with a five-membered ring constraint promote two different types of alpha/beta-peptide helix. The "11-helix" contains i, i+3 CO...H-N hydrogen bonds between backbone amide groups; these hydrogen bonds occur in 11-atom rings. The alpha/beta-peptide "14/15-helix" contains i, i+4 CO...H-N hydrogen bonds, which occur in alternating 14- and 15-atom rings. Here we provide crystallographic data for 14 alpha/beta-peptides that form the 11-helix and/or the 14/15-helix. These results were obtained for a series of oligomers containing beta-residues derived from ( S,S)- trans-2-aminocyclopentanecarboxylic acid (ACPC) and alpha-residues derived from alpha-aminoisobutyric acid (Aib) or l-alanine (Ala). The crystallized alpha/beta-peptides range in length from 4 to 10 residues. Nine of the alpha/beta-peptides display the 11-helix in the solid state, three display the 14/15-helix, and two display conformations that contain both i, i+3 and i, i+4 CO...H-N hydrogen bonds, but not bifurcated hydrogen bonds. Only 3 of the 14 crystal structures presented here have been previously described. These results suggest that longer alpha/beta-peptides prefer the 14/15-helix over the 11-helix, a conclusion that is consistent with previously reported NMR data obtained in solution.     

Helix macrodipole control of beta 3 peptide 14-helix stability in water

beta-Peptides have attracted considerable attention by virtue of their ability to populate helical secondary structures in methanol, even in the absence of stabilizing tertiary interactions. Recent efforts in beta-peptide design have produced few beta3-peptides that form stable 14-helices in water; those that do require stabilizing intramolecular salt bridges on two of three helical faces and therefore possess limited utility as tools in biological research. Here we show that favorable interactions with the 14-helix macrodipole significantly stabilize the 14-helix in water, alleviating the need for multiple salt bridges on two of three helical faces. We also report the previously unrecognized stabilization of 14-helix structure by gamma-branched beta3-amino acids. The most structured molecules we describe are highly heterogeneous at the primary sequence level, containing seven different beta3-amino acids within an 11-residue sequence. These results represent the essential first step toward the design of well-folded 14-helices that explore the interactions between beta3-peptides and biological macromolecules in vitro and in vivo.     

Three-dimensional organization of helices: design principles for nucleobase-functionalized beta-peptides

The construction and molecular recognition of various three-dimensional biomimetic structures is based on the predictable de novo design of artificial molecules. In this regard beta-peptides are especially interesting, since stable secondary structures are obtained already with short sequences; one of them is the 14-helix in which every third residue has the same orientation. The covalent functionalization of every third 14-helix side chain with nucleobases was used for a reversible organization of two helices based on nucleobase pairing. A series of beta-peptides with various nucleobase sequences was synthesized and the stability of double strand formation was investigated. As few as four nucleobases are sufficient for considerable duplex stability. The stability of base pairing was examined by temperature-dependent UV spectroscopy and the formation of the 14-helix was confirmed by circular dichroism (CD) spectroscopy. The preferred strand orientation of complementary-nucleobase-modified beta-peptide helices was investigated as well as the influence of helix content on the duplex stability. The preorganization of a 14-helix in regard to double-strand recognition was tuned by the sequential order of polar beta-amino acids or by the amount of 2-aminocyclohexanecarboxylic acid units incorporated, which are known to facilitate 14-helix formation, respectively.     

Design, synthesis and structural investigations of a beta-peptide forming a 314-helix stabilized by electrostatic interactions

Two different strategies have been employed for the synthesis of Fmoc-protected beta(3)-homoarginine; the Arndt-Eistert homologation of alpha-arginine and the guanidinylation of beta(3)-homoornithine. Solid-phase beta-peptide synthesis was used for the preparation of beta-heptapeptide 1, which was designed to form a helix stabilized by electrostatic interactions through positively (beta(3)hArg) and negatively charged (beta(3)hGlu) amino acid residues. CD measurements and corresponding NMR investigations in MeOH and aqueous solutions do indeed show that the beta-peptidic 3(14)-helix can be stabilized by salt-bridge formation.     

Accommodation of alpha-substituted residues in the beta-peptide 12-helix: expanding the range of substitution patterns available to a foldamer scaffold

Beta-amino acid oligomers composed exclusively of homochiral trans-2-aminocyclopentanecarboxylic acid (ACPC) residues and/or related pyrrolidine-based residues are known to favor a specific helical secondary structure that is defined by 12-membered ring C=O(i)- -H-N(i+3) hydrogen bonds ("12-helix"). The 12-helix is structurally similar to the familiar alpha-helix and therefore represents a source of potential alpha-helix-mimics. The 12-helix will be most useful in this regard if this conformational scaffold can be employed to arrange specific sets of protein-like side chains in space. Here we examine whether the 12-helix tolerates insertion of acyclic beta-amino acid residues bearing a substituent in the alpha-position ("beta(2)-residues"). Seventeen homologous beta-peptide heptamers have been prepared in which one to four beta(2)-residues reside among ACPC and/or pyrrolidine residues. Circular dichroism comparisons suggest that beta(2)-residues have a lower 12-helical propensity than do residues preorganized by a five-membered ring, as expected, but that beta-peptides containing beta(2)-residues at one or two of the seven positions retain a significant preference for 12-helix formation. These results indicate that a limited number of beta(2)-residues can be used to introduce side chains at specific positions along the surface of a 12-helix.     

Inhibiting HIV fusion with a beta-peptide foldamer

Linear peptides derived from the HIV gp41 C-terminus (C-peptides), such as the 36-residue Fuzeon, are potent HIV fusion inhibitors. These molecules bind to the N-peptide region of gp41 and inhibit an intramolecular protein-protein interaction that powers fusion of the viral and host cell membranes. The N-peptide region contains a surface pocket that is occupied in the post-fusion state by three alpha-helical residues found near the gp41 C-terminus: Trp628, Trp631, and Ile635-the WWI epitope. Here, we describe a set of beta3-decapeptides (betaWWI-1-4) in which the WWI epitope is presented on one face of a short 14-helix stabilized by macrodipole neutralization and side chain-side chain salt bridges. betaWWI-1-4 bind in vitro to IZN17, a validated gp41 model, and inhibit syncytia formation in cell culture. Molecules lacking a complete WWI functional epitope neither bind IZN17 nor inhibit syncytia formation. These results provide evidence that short beta-peptide 14-helices can inhibit an intramolecular protein-protein interaction in vivo. Molecules related to betaWWI-1-4 could represent starting points for the development of highly potent inhibitors or antigens effective against HIV or other viruses, including SARS, Ebola, HRSV, and influenza, that employ common fusion mechanisms.     

(alpha/beta+alpha)-peptide antagonists of BH3 domain/Bcl-x(L) recognition: toward general strategies for foldamer-based inhibition of protein-protein interactions

The development of molecules that bind to specific protein surface sites and inhibit protein-protein interactions is a fundamental challenge in molecular recognition. New strategies for approaching this challenge could have important long-term ramifications in biology and medicine. We are exploring the concept that unnatural oligomers with well-defined conformations ("foldamers") can mimic protein secondary structural elements and thereby block specific protein-protein interactions. Here, we describe the identification and analysis of helical peptide-based foldamers that bind to a specific cleft on the anti-apoptotic protein Bcl-xL by mimicking an alpha-helical BH3 domain. Initial studies, employing a fluorescence polarization (FP) competition assay, revealed that among several alpha/beta- and beta-peptide foldamer backbones only alpha/beta-peptides intended to adopt 14/15-helical secondary structure display significant binding to Bcl-xL. The most tightly binding Bcl-xL ligands are chimeric oligomers in which an N-terminal alpha/beta-peptide segment is fused to a C-terminal alpha-peptide segment ((alpha/beta + alpha)-peptides)). Sequence-affinity relationships were probed via standard and nonstandard techniques (alanine scanning and hydrophile scanning, respectively), and the results allowed us to construct a computational model of the ligand/Bcl-xL complex. Analytical ultracentrifugation with a high-affinity (alpha/beta + alpha)-peptide established 1:1 ligand:Bcl-xL stoichiometry under FP assay conditions. Binding selectivity studies with the most potent (alpha/beta + alpha)-peptide, conducted via surface plasmon resonance measurements, revealed that this ligand binds tightly to Bcl-w as well as to Bcl-xL, while binding to Bcl-2 is somewhat weaker. No binding could be detected with Mcl-1. We show that our most potent (alpha/beta + alpha)-peptide can induce cytochrome C release from mitochondria, an early step in apoptosis, in cell lysates, and that this activity is dependent upon inhibition of protein-protein interactions involving Bcl-xL.     

Interpreting NMR data for beta-peptides using molecular dynamics simulations

NMR is one of the most used techniques to resolve structure of proteins and peptides in solution. However, inconsistencies may occur due to the fact that a polypeptide may adopt more than one conformation. Since the NOE distance bounds and (3)J-values used in such structure determination represent a nonlinear average over the total ensemble of conformers, imposition of NOE or (3)J-value restraints to obtain one unique conformation is not an appropriate procedure in such cases. Here, we show that unrestrained MD simulation of a solute in solution using a high-quality force field yields a conformational ensemble that is largely compatible with the experimental NMR data on the solute. Four 100 ns MD simulations of two forms of a nine-residue beta-peptide in methanol at two temperatures produced conformational ensembles that were used to interpret the NMR data on this molecule and resolve inconsistencies between the experimental NOEs. The protected and unprotected forms of the beta-peptide adopt predominantly a 12/10-helix in agreement with the qualitative interpretation of the NMR data. However, a particular NOE was not compatible with this helix indicating the presence of other conformations. The simulations showed that 3(14)()-helical structures were present in the ensemble of the unprotected form and that their presence correlates with the fulfillment of the particular NOE. Additionally, all inter-hydrogen distances were calculated to compare NOEs predicted by the simulations to the ones observed experimentally. The MD conformational ensembles allowed for a detailed and consistent interpretation of the experimental data and showed the small but specific conformational differences between the protected and unprotected forms of the peptide.     

Hydrolysis of the amyloid beta-peptide (A beta) 1-40 between Asp23-Val24 produces non-aggregating fragments. An electrospray mass spectrometric study

The aggregation of full-length (residues 1-40) amyloid beta-peptide (A beta) and fragments corresponding to residues 1-23 and 24-40 was studied by electrospray mass spectrometry, using gramicidin as a non-aggregating reference. Following a lag period, A beta(1-40) at 140 microM concentration aggregates with apparent first-order kinetics. Under acidic conditions A beta(1-40) undergoes spontaneous cleavage between Asp23-Val24 and to a lesser extent also at two other Asp-X motifs. Incubation in acidic H(2)18O showed incorporation of 18O in fragment A beta(1-23), confirming that the Asp23-Val24 peptide bond had been hydrolyzed. Incubation of synthetic A beta(1-23) and A beta(24-40) peptides with A beta(1-40) showed that A beta(24-40) remained in solution for several months, that A beta(1-23) partly disappeared from solution, whereas A beta(1-40) completely disappeared. Further, treatment of sedimentable aggregates formed after co-incubation of the three peptides with hexafluoro-2-propanol or formic acid recovered the intensity of A beta(1-40). These data support previous studies showing that the region of A beta encompassing residues 16-24 is necessary for aggregation into amyloid fibrils.     

Molecular biosensing system based on intrinsically disordered proteins

Intrinsically disordered proteins (IDPs) that undergo structural transition upon binding their target molecules are becoming increasingly known. IDPs, because of their binding specificity and induced folding properties, can serve as biological recognition elements for sensing applications. In this paper, BRCA1, an IDP, was utilized as the biological recognition element to detect tumor suppressor protein p53 through the BRCA1/p53 binding interaction to serve as a proof-of-concept for the use of IDPs as recognition elements. The binding resulted in a disordered-to-ordered BRCA1 conformational change, as seen in our circular dichroism (CD) measurements. This conformational change in BRCA1 (residues 219-498) was utilized in the detection of p53 (residues 311-393) via both intrinsic and extrinsic fluorescent probes. Intrinsic tryptophan residues within the BRCA1 sequence detected p53 (311-393) with a detection limit of 0.559 nM (0.112 pmol). Two environmentally sensitive fluorophores, tetramethylrhodamine-5-maleimide (TMR) and 6-((5-dimethylaminonaphthalene-1-sulfonyl)amino)hexanoic acid, succinimidyl ester (dansyl-X, SE) were conjugated to BRCA1 (219-498). Dansyl-X, SE-conjugated BRCA1 (219-498) detected p53 (311-393) with a detection limit of 1.50 nM (0.300 pmol). The sensitivities for TMR and dansyl-X, SE-conjugated BRCA1 for the detection of p53 were nearly threefold and twofold higher, respectively, than the sensitivity reported using intrinsic BRCA1 tryptophan fluorescence. CD measurements did not reveal a disruption of p53/dye-conjugated BRCA1 binding, thus validating the applicability of environmentally sensitive fluorophores as transduction moieties to detect molecules which bind to IDPs and induce a structural change.     

Protein secondary structure conformations and associated hydrophobicity scales

We have developed conformational preference functions and a hierarchy of algorithms that can evaluate the success of each hydrophobicity scale in predicting protein secondary conformation. The results of such evaluation are shown for fiftyfive different scales with respect to their ability to predict -helix, -sheet and coil structure in three testing sets of proteins: five integral membrane proteins, twelve -class and sixteen -class soluble proteins. Our scale of conformational parameters is the best predictor of secondary structure segments in membrane proteins and -class proteins. The success rate and correlation coefficient for -helix conformation in membrane proteins are 76% and 0.46 respectively, which is superior to the performance measures attained with other prediction schemes. Evaluation of solution hydrophobicity scales, often used to predict transmembrane segments in membrane proteins, indicated absence of correlation in prediction of helix segments and experimental results for the conformation of membrane proteins. Such scales have better performance (correlation coefficient around 0.30) in predicting sheet conformation in the -class proteins.     

Dipole-induced self-assembly of helical beta-peptides

In this work, the interactions between beta-peptides are investigated for helix-forming peptides using molecular simulation. The role of electrostatic interactions in the self-assembly of these peptides is studied by calculating the dipole moment of various 14-helical beta-peptides using molecular dynamics simulations. The stability of a beta-peptide that is known to form a liquid crystalline phase is determined by calculating the potential of mean force using the expanded ensemble density of states method. This peptide is found to form a mechanically stable 14-helix in an implicit solvent model. The interaction between two of these peptides is examined by calculating the potential of mean force between the two peptides in implicit solvent. The peptides are shown to favorably associate in an end-to-end manner, driven largely by dipolar interactions. In order to understand the possible structures that form when many peptides interact in solution, a coarse-grained model is developed. Brownian dynamics simulations of the coarse-grained model at intermediate concentrations (1-50 mM) are performed, and the aggregation behavior is understood by calculating the diffusivity and the radial distribution function. An analysis of the resultant structures reveals that the coarse-grained model of the peptide leads to the formation of spherical clusters.     

Positional screening and NMR structure determination of side-chain-to-side-chain cyclized β3-peptides

Many β-peptides fold in a 14-helical secondary structure in organic solvents, but similar 14-helix formation in water requires additional stabilizing elements. Especially the 14-helix stabilization of short β-peptides in aqueous solution is critical, due to the limited freedom for incorporating stabilizing elements. Here we show how a single lactam bridge, connecting two β-amino acid side-chains, can lead to high 14-helix character in short β(3)-peptides in water. A comparative study, using CD and NMR spectroscopy and structure calculations, revealed the strong 14-helix inducing power of a side-chain-to-side-chain cyclization and its optimal position on the β(3)-peptide scaffold with respect to pH and ionic strength effects. The lactam bridge is ideally incorporated in the N-terminal region of the β(3)-peptide, where it limits the conformational flexibility of the peptide backbone. The lactam bridge induces a 14-helical conformation in methanol and water to a similar extent. Based on the presented first high resolution NMR 3D structure of a lactam bridged β(3)-peptide, the fold shows a large degree of high order, both in the backbone and in the side-chains, leading to a highly compact and stable folded structure.     

Co-operative intra-protein structural response due to protein–protein complexation revealed through thermodynamic quantification: study of MDM2-p53 binding

The p53 protein activation protects the organism from propagation of cells with damaged DNA having oncogenic mutations. In normal cells, activity of p53 is controlled by interaction with MDM2. The well understood p53-MDM2 interaction facilitates design of ligands that could potentially disrupt or prevent the complexation owing to its emergence as an important objective for cancer therapy. However, thermodynamic quantification of the p53-peptide induced structural changes of the MDM2-protein remains an area to be explored. This study attempts to understand the conformational free energy and entropy costs due to this complex formation from the histograms of dihedral angles generated from molecular dynamics simulations. Residue-specific quantification illustrates that, hydrophobic residues of the protein contribute maximum to the conformational thermodynamic changes. Thermodynamic quantification of structural changes of the protein unfold the fact that, p53 binding provides a source of inter-element cooperativity among the protein secondary structural elements, where the highest affected structural elements (α2 and α4) found at the binding site of the protein affects faraway structural elements (β1 and Loop1) of the protein. The communication perhaps involves water mediated hydrogen bonded network formation. Further, we infer that in inhibitory F19A mutation of P53, though Phe19 is important in the recognition process, it has less prominent contribution in the stability of the complex. Collectively, this study provides vivid microscopic understanding of the interaction within the protein complex along with exploring mutation sites, which will contribute further to engineer the protein function and binding affinity.     

Efficient synthesis of a beta-peptide combinatorial library with microwave irradiation

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

Solid-phase synthesis and CD spectroscopic investigations of novel beta-peptides from L-aspartic acid and beta-amino-L-alanine

[structure: see text] A solid-phase synthesis method for the preparation of novel beta3- and beta2-peptides derived from l-aspartic acid and beta-amino-l-alanine, respectively, is described. The methodology allows independent buildup of the beta-peptide backbone and the introduction of sequential side chain substitutions. Representative peptides from the two classes, an amino-substituted beta3-hexapeptide and an acyl-substituted beta2-hexapeptide, have been prepared, and their solution conformation is studied by circular dichroism (CD) spectroscopy.