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.     

Solution structure of a beta-peptide ligand for hDM2

We recently reported a beta-peptide foldamer, beta53-1, that folds into a 14-helix in aqueous solution, binds the oncoprotein hDM2 with submicromolar affinity, and potently inhibits the interaction of hDM2 with a peptide derived from the activation domain of p53 (p53AD). Here, we present the solution structure of beta53-1 in methanol. Details of the structure illustrate fundamental and novel elements of beta-peptide folding and recognition. These elements include the detailed arrangement of a complex, 14-helix-stabilizing salt bridge on one helical face, and a unique "wedge into cleft" packing interaction along a second. The structure also reveals how a subtle distortion in the beta53-1 14-helix geometry alters the presentation of its recognition epitope, rendering it particularly well suited for alpha-helix mimicry. The solution structure of beta53-1 demonstrates that well folded beta-peptide oligomers can effectively present an extended, highly variable surface that could be used as a general platform for targeting critical protein-protein interfaces.     

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.     

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.     

Modulating rhodopsin receptor activation by altering the pKa of the retinal Schiff base

The visual pigment rhodopsin is a seven-transmembrane (7-TM) G protein-coupled receptor (GPCR). Activation of rhodopsin involves two pH-dependent steps: proton uptake at a conserved cytoplasmic motif between TM helices 3 and 6, and disruption of a salt bridge between a protonated Schiff base (PSB) and its carboxylate counterion in the transmembrane core of the receptor. Formation of an artificial pigment with a retinal chromophore fluorinated at C14 decreases the intrinsic pKa of the PSB and thereby destabilizes this salt bridge. Using Fourier transform infrared difference and UV-visible spectroscopy, we characterized the pH-dependent equilibrium between the active photoproduct Meta II and its inactive precursor, Meta I, in the 14-fluoro (14-F) analogue pigment. The 14-F chromophore decreases the enthalpy change of the Meta I-to-Meta II transition and shifts the Meta I/Meta II equilibrium toward Meta II. Combining C14 fluorination with deletion of the retinal beta-ionone ring to form a 14-F acyclic artificial pigment uncouples disruption of the Schiff base salt bridge from transition to Meta II and in particular from the cytoplasmic proton uptake reaction, as confirmed by combining the 14-F acyclic chromophore with the E134Q mutant. The 14-F acyclic analogue formed a stable Meta I state with a deprotonated Schiff base and an at least partially protonated protein counterion. The combination of retinal modification and site-directed mutagenesis reveals that disruption of the protonated Schiff base salt bridge is the most important step thermodynamically in the transition from Meta I to Meta II. This finding is particularly important since deprotonation of the retinal PSB is known to precede the transition to the active state in rhodopsin activation and is consistent with models of agonist-dependent activation of other GPCRs.     

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.     

Structure-activity studies of 14-helical antimicrobial beta-peptides: probing the relationship between conformational stability and antimicrobial potency

Antimicrobial alpha-helical alpha-peptides are part of the host-defense mechanism of multicellular organisms and could find therapeutic use against bacteria that are resistant to conventional antibiotics. Recent work from Hamuro et al. has shown that oligomers of beta-amino acids ("beta-peptides") that can adopt an amphiphilic helix defined by 14-membered ring hydrogen bonds ("14-helix") are active against Escherichia coli [Hamuro, Y.; Schneider, J. P.; DeGrado, W. F. J. Am. Chem. Soc. 1999, 121, 12200-12201]. We have created two series of cationic 9- and 10-residue amphiphilic beta-peptides to probe the effect of 14-helix stability on antimicrobial and hemolytic activity. 14-Helix stability within these series is modulated by varying the proportions of rigid trans-2-aminocyclohexanecarboxylic acid (ACHC) residues and flexible acyclic residues. We have previously shown that a high proportion of ACHC residues in short beta-peptides encourages 14-helical structure in aqueous solution [Appella, D. H.; Barchi, J. J.; Durell, S. R.; Gellman, S. H. J. Am. Chem. Soc. 1999, 121, 2309-2310]. Circular dichroism of the beta-peptides described here reveals a broad range of 14-helix population in aqueous buffer, but this variation in helical propensity does not lead to significant changes in antibiotic activity against a set of four bacteria. Several of the 9-mers display antibiotic activity comparable to that of a synthetic magainin derivative. Among these 9-mers, hemolytic activity increases slightly with increasing 14-helical propensity, but all of the 9-mers are less hemolytic than the magainin derivative. Previous studies with conventional peptides (alpha-amino acid residues) have provided conflicting evidence on the relationship between helical propensity and antimicrobial activity. This uncertainty has arisen because alpha-helix stability can be varied to only a limited extent among linear alpha-peptides without modifying parameters important for antimicrobial activity (e.g., net charge or hydrophobicity); a much greater range of helical stability is accessible with beta-peptides. For example, it is very rare for a linear alpha-peptide to display significant alpha-helix formation in aqueous solution and manifest antibacterial activity, while the linear beta-peptides described here range from fully unfolded to very highly folded in aqueous solution. This study shows that beta-peptides can be unique tools for analyzing relationships between conformational stability and biological activity.     

Aib-rich peptides containing lactam-bridged side chains as models of the 3(10)-helix.

Aib-rich side chain lactam-bridged oligomers with n =1, 2, 3, were designed and synthesized as putative models of the 3(10)-helix. These peptides were conformationally characterized in aqueous solution containing SDS micelles by CD, NMR, and computer simulations. The lactam bridge between the side chains of L-Glu and L-Lys in (i) and (i+3) positions was introduced in order to enhance the conformational preference toward the right-handed 3(10)-helix. The NMR results clearly indicate that there is an increase of 3(10)-helix formation upon chain elongation. In the dimer and trimer (n = 2 and n = 3, respectively, in the structure reported above) the observed NOE connectivities are compatible with the 3(10)-helical arrangement, confirmed by the temperature coefficients of the amide proton resonances which suggest the presence of a hydrogen-bonded structure. The phi and psi dihedral angles of the structures obtained by molecular dynamics calculations are also compatible with the 3(10)-helix. Identification of the hydrogen-bond pattern indicate that C=O(i)- - -HN(i+3) hydrogen bonds, typical of the 3(10)-helical conformation, are highly probable in all low-energy structures. The CD spectra of these Aib-rich lactam-bridged oligopeptides, obtained in the same solvent system used for NMR experiments, provide important insight into the spectroscopic characteristics of the 3(10)-helix.     

Formation of a stable 14-helix in short oligomers of furanoid cis-beta-sugar-amino acid

Oligomers of a new class of sugar amino acids (SAA) using a xylofuranoic acid has been shown to generate a robust 14-helix. The design involved the use of xylofuranose with a cis arrangement between the amine and carboxyl groups to promote the adoption of a 14-helix instead of a mixed 12/10-helix observed in a sugar oligomer using a ribofuranoic acid and beta-Ala. The observation of a stable right-handed 14-helix in a cis-SAA is unprecedented.     

Amyloid-β peptide structure in aqueous solution varies with fragment size

Various fragment sizes of the amyloid-β (Aβ) peptide have been utilized to mimic the properties of the full-length Aβ peptide in solution. Among these smaller fragments, Aβ16 and Aβ28 have been investigated extensively. In this work, we report the structural and thermodynamic properties of the Aβ16, Aβ28, and Aβ42 peptides in an aqueous solution environment. We performed replica exchange molecular dynamics simulations along with thermodynamic calculations for investigating the conformational free energies, secondary and tertiary structures of the Aβ16, Aβ28, and Aβ42 peptides. The results show that the thermodynamic properties vary from each other for these peptides. Furthermore, the secondary structures in the Asp1-Lys16 and Asp1-Lys28 regions of Aβ42 cannot be completely captured by the Aβ16 and Aβ28 fragments. For example, the β-sheet structures in the N-terminal region of Aβ16 and Aβ28 are either not present or the abundance is significantly decreased in Aβ42. The α-helix and β-sheet abundances in Aβ28 and Aβ42 show trends--to some extent--with the potential of mean forces but no such trend could be obtained for Aβ16. Interestingly, Arg5 forms salt bridges with large abundances in all three peptides. The formation of a salt bridge between Asp23-Lys28 is more preferred over the Glu22-Lys28 salt bridge in Aβ28 but this trend is vice versa for Aβ42. This study shows that the Asp1-Lys16 and Asp1-Lys28 regions of the full length Aβ42 peptide cannot be completely mimicked by studying the Aβ16 and Aβ28 peptides.     

Synthesis and structural characterization of homochiral homo-oligomers of parent cis- and trans-furanoid-β-amino acids

The synthesis of homochiral homo-oligomers of cis- and trans-3-aminotetrahydrofuran-2-carboxylic acids (parent cis- and trans-furanoid-β-amino acids, referred to as "cis-/trans-FAA") has been carried out to understand their secondary structures and their dependence on the ring heteroatom. The oligomers of two diastereomers have been shown to have a distinct left-handed helicity. The cis-FAA homo-oligomers show a 14-helix structure, in contrast to the homo-oligomers of cis-ACPC, which adopt a sheet like structure. The trans-FAA homo-oligomers were found to adopt a 12-helix structure, the same trend found in trans-ACPC homo-oligomers. With the help of ab initio calculations, the structural features of cis-ACPC and cis-FAA hexamers were compared. We believe that the more compact packing of the cis-FAA hexapeptide should be due to a more favorable interaction between the ring and the backbone amide hydrogen.     

Structural rearrangement of β-lactoglobulin at different oil-water interfaces and its effect on emulsion stability

Understanding the factors that control protein structure and stability at the oil-water interface continues to be a major focus to optimize the formulation of protein-stabilized emulsions. In this study, a combination of synchrotron radiation circular dichroism spectroscopy, front-face fluorescence spectroscopy, and dual polarization interferometry (DPI) was used to characterize the conformation and geometric structure of β-lactoglobulin (β-Lg) upon adsorption to two oil-water interfaces: a hexadecane-water interface and a tricaprylin-water interface. The results show that, upon adsorption to both oil-water interfaces, β-Lg went through a β-sheet to α-helix transition with a corresponding loss of its globular tertiary structure. The degree of conformational change was also a function of the oil phase polarity. The hexadecane oil induced a much higher degree of non-native α-helix compared to the tricaprylin oil. In contrast to the β-Lg conformation in solution, the non-native α-helical-rich conformation of β-Lg at the interface was resistant to further conformational change upon heating. DPI measurements suggest that β-Lg formed a thin dense layer at emulsion droplet surfaces. The effects of high temperature and the presence of salt on these β-Lg emulsions were then investigated by monitoring changes in the ζ-potential and particle size. In the absence of salt, high electrostatic repulsion meant β-Lg-stabilized emulsions were resistant to heating to 90 °C. Adding salt (120 mM NaCl) before or after heating led to emulsion flocculation due to the screening of the electrostatic repulsion between colloidal particles. This study has provided insight into the structural properties of proteins adsorbed at the oil-water interface and has implications in the formulation and production of emulsions stabilized by globular proteins.     

羧脒盐桥介导的单重态能量传递直接观察

设计构建了以羧脒盐桥联接的萘和蒽超分子组装体系,NA-(脒基-羧基)-An以及相应的模型体系.稳态和时间分辨荧光光谱研究表明,置于羧脒盐桥两端的萘和蒽基团之间发生了从萘到蒽的单重态能量传递,NA-(脒-羧)-An超分子体系中单重态能量传递效率和速率常数分别大于0.99和9.9×10⁹s^-1.推测羧脒盐桥介导了体系中的单重态能量传递过程,单重态能量传递‘通过键’以电子交换机制进行.     

Distinctive circular dichroism signature for 14-helix-bundle formation by beta-peptides

We identify a distinctive circular dichroism (CD) signature for self-assembled 14-helical beta-peptides. Our data show that self-assembly leads to a mimimum at 205 nm, which is distinct from the well-known minimum at 214 nm for a monomeric 14-helix. The onset of assembly is indicated by [theta]205/[theta]214>0.7. Our results will facilitate rapid screening for self-assembling beta-peptides and raise the possibility that far-UV CD will be useful for detecting higher-order structure for other well-folded oligoamide backbones.     

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.     

Carbon-13 NMR chemical shift and electronic structure of polypeptide as studied by tight-binding MO theory: poly (β-benzyl L-aspartate) with the right-handle α-helix and left-handed α-helix forms

An attempt was made to calculate ¹³C NMR chemical shifts of poly(β-benzyl L-aspartate) having the right-handed α-helix (α_R-helix) and left-handed α-helix (α_L-helix) forms by a tight-binding MO sum-over-states theory within the extended Hückel framework, in order to examine whether or not the conformation-dependent ¹³C chemical shifts previously determined by the cross polarization-magic angle spinning technique are reproduced by a change of electronic structure of the polymer. It is found that the relative displacements of the observed C_α, C_β and carbonyl ¹³C chemical shifts between the α_R- and α_L-helices are reproduced qualitatively by the calculation.     

Understanding electron transfer across negatively-charged Aib oligopeptides

The physicochemical effects modulating the conformational behavior and the rate of intramolecular dissociative electron transfer in phthalimide-Aibn-peroxide peptides (n = 0-3) have been studied by an integrated density functional/continuum solvent model. We found that three different orientations of the phthalimide ring are possible, labeled Phihel, PhiC7, and PhipII. In the condensed phase, they are very close in energy when the system is neutral and short. When the peptide chain length increases and the system is negatively charged, Phihel becomes instead the most stable conformer. Our calculations confirm that the 3(10)-helix is the most stable secondary structure for the peptide bridge. However, upon charge injection in the phthalimide end of the phthalimide-Aib3-peroxide, the peptide bridge can adopt an alpha-helix conformation as well. The study of the dependence of the frontier orbitals on the length and on the conformation of the peptide bridge (in agreement with experimental indications) suggests that for n = 3 the process could be influenced by a 3(10) --> alpha-helix conformational transition of the peptide chain.     

Influence of a Hydrophobic Environment on the Structure of Arginine—Carboxylate Salt Bridge

The exact structure of an arginine-carboxylate salt bridge in different chemical environments remains a controversial problem.In the present work,the zwitterionic and neutral forms of arginine-carboxylate salt bridge were studied by the B3LYP/6-311G(d,p)//PM3 method.It turns out that the neutral forms are more stable than the zwitterionic coumterparts in gas phase.However,whnen bound by α-cyclodextrin,the zwitterionic forms become more stable than the corresponding neutral ones.It is suggested that the hydrophobic environment provided by the cyclodextrin cavity leads to such behavior.Therefore,the salt bridge still could be in a zwitterionic form in the hydrophobic interior of the real proteins.     

Reversible α-helix formation controlled by a hydrogen bond surrogate

Strategically placed covalent linkages have been shown to stabilize helical conformations in short peptide sequences. Here we report the synthesis of a stabilized α-helix that utilizes an internal disulfide linkage. Structural analysis indicates that the dynamic nature of the disulfide bridge allows for the reversible formation of an α-helix through oxidation and reduction reactions.     

Specific inclusion mode of guest compounds in the amylose complex analyzed by solid state NMR spectroscopy

The inclusion compound formation between linear amylose of molecular weight 102500 (AS100) and p-aminobenzoic acid (PA) during the sealed-heating process was investigated by powder X-ray diffractometry, infrared spectroscopy and solid state NMR spectroscopy. Sealed-heating of AS100 and PA at 100 degrees C for 6 h provided an inclusion compound with 6(1)-helix structure, while a 7(1)-helix structure was found when sealed-heating was carried out at 150 degrees C for 1 h. The formation of an inclusion compound was not observed when sealed-heating was performed at 50 degrees C for 6 h. The 7(1)-helix inclusion compound maintained its structure even during storage at high temperature while the 6(1)-helix inclusion compound decomposed and returned to the original V(a)-amylose upon heating to 180 degrees C. Quantitative determination revealed that one PA molecule could be included per one helical turn of AS100 for both 6(1)-helix and 7(1)-helix inclusion compounds. Solid state NMR spectroscopy suggested that PA molecules were included in the amylose helix core in the 7(1)-helix inclusion compound, while in the case of 6(1)-helix inclusion compound, PA molecules were accommodated in the interstices between amylose helices. Moreover, the inclusion compound formation by sealed-heating of AS100 was also observed when using PA analogues as guest compounds. The binding ratio of AS100 and PA analogues varied depending on the size of guest molecules.