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Glyoxylic acid and glycine are widely considered to have been important prebiotic building blocks. Several mechanistic routes have been previously examined for conversion of glyoxylic acid to glycine. Here we provide evidence for a new mechanistic path. Glycine is spontaneously formed from glyoxylic acid in ammonium‐rich aqueous solutions at neutral pH; oxamic acid is generated as well. Hydride transfer from the glyoxylate‐derived hemiaminal to the corresponding iminium ion appears to underlie this transformation. This proposed mechanism parallels the well‐known Cannizzaro reaction mechanism, which leads us to suggest the designation "aza‐Cannizzaro reaction." This discovery offers a new perspective on prebiotic nitrogen incorporation because glycine can be a source of nitrogen for more complex molecules, including other α‐amino acids. [ABSTRACT FROM AUTHOR]

Peptide engineering efforts have delivered drugs for diverse human diseases. Side chain alteration is among the most common approaches to designing new peptides for specific applications. The peptide backbone can be modified as well, but this strategy has received relatively little attention. Here we show that new and favorable contacts between a His side chain on a target protein and an aromatic side chain on a synthetic peptide ligand can be engineered by rational and coordinated side chain modification and backbone extension. Side chain modification alone was unsuccessful. Binding measurements, high‐resolution structural studies and pharmacological outcomes all support the synergy between backbone and side chain modification in engineered ligands of the parathyroid hormone receptor‐1, which is targeted by osteoporosis drugs. These results should motivate other structure‐based designs featuring coordinated side chain modification and backbone extension to enhance the engagement of peptide ligands with target proteins. [ABSTRACT FROM AUTHOR]

Peptide engineering efforts have delivered drugs for diverse human diseases. Side chain alteration is among the most common approaches to designing new peptides for specific applications. The peptide backbone can be modified as well, but this strategy has received relatively little attention. Here we show that new and favorable contacts between a His side chain on a target protein and an aromatic side chain on a synthetic peptide ligand can be engineered by rational and coordinated side chain modification and backbone extension. Side chain modification alone was unsuccessful. Binding measurements, high‐resolution structural studies and pharmacological outcomes all support the synergy between backbone and side chain modification in engineered ligands of the parathyroid hormone receptor‐1, which is targeted by osteoporosis drugs. These results should motivate other structure‐based designs featuring coordinated side chain modification and backbone extension to enhance the engagement of peptide ligands with target proteins. [ABSTRACT FROM AUTHOR]

The intrinsic pathway of apoptosis is regulated by the Bcl-2 family of proteins. Inhibition of the anti-apoptotic members represents a strategy to induce apoptotic cell death in cancer cells. We have measured the membrane binding properties of a series of peptides, including modified α/β-peptides, designed to exhibit enhanced membrane permeability to allow cell entry and improved access for engagement of Bcl-2 family members. The peptide cargo is based on the pro-apoptotic protein Bim, which interacts with all anti-apoptotic proteins to initiate apoptosis. The α/β-peptides contained cyclic β-amino acid residues designed to increase their stability and membrane permeability. Dual polarisation interferometry was used to study the binding of each peptide to two different model membrane systems designed to mimic either the plasma membrane or the outer mitochondrial membrane. The impact of each peptide on the model membrane structure was also investigated, and the results demonstrated that the modified peptides had increased affinity for the mitochondrial membrane and significantly altered the structure of the bilayer. The results also showed that the presence of an RRR motif significantly enhanced the ability of the peptides to bind to and insert into the mitochondrial membrane mimic, and provide insights into the role of selective membrane targeting of peptides. The membrane binding of two peptides designed to exhibit enhanced membrane permeability to allow cell entry and engagement of Bcl-2 family members shows that an RRR motif significantly enhances peptide binding and insertion into a mitochondrial membrane mimic and provides insights into selective membrane targeting of peptides. [ABSTRACT FROM AUTHOR]

Location matters for GPCR signaling: Parathyroid hormone (PTH) receptor (PTHR) is a G protein–coupled receptor (GPCR) that controls vitamin D, Ca2+, and bone homeostasis by stimulating cAMP production at the plasma membrane and at endosomes after β-arrestin–dependent internalization. White et al. generated a synthetic PTH derivative (PTH7d) that inhibited β-arrestin coupling to PTHR, thereby leading to sustained cAMP production at the cell surface. Comparison of cellular and organismal responses to wild-type PTH, PTH7d, or a PTH analog that elicits sustained endosomal signaling demonstrated that the biological outcome was determined by the subcellular location of PTHR activity. These findings suggest that biological responses elicited by other GPCRs that are active at intracellular compartments may show a similar dependence on the location of signaling. The parathyroid hormone (PTH) type 1 receptor (PTHR) is a class B G protein–coupled receptor (GPCR) that regulates mineral ion, vitamin D, and bone homeostasis. Activation of the PTHR by PTH induces both transient cell surface and sustained endosomal cAMP production. To address whether the spatial (location) or temporal (duration) dimension of PTHR-induced cAMP encodes distinct biological outcomes, we engineered a biased PTHR ligand (PTH7d) that elicits cAMP production at the plasma membrane but not at endosomes. PTH7d stabilized a unique active PTHR conformation that mediated sustained cAMP signaling at the plasma membrane due to impaired β-arrestin coupling to the receptor. Experiments in cells and mice revealed that sustained cAMP production by cell surface PTHR failed to mimic the pharmacological effects of sustained endosomal cAMP production on the abundance of the rate-limiting hydroxylase catalyzing the formation of active vitamin D, as well as increases in circulating active vitamin D and Ca2+ and in bone formation in mice. Thus, similar amounts of cAMP generated by PTHR for similar lengths of time in different cellular locations, plasma membrane and endosomes, mediate distinct physiological responses. These results unveil subcellular signaling location as a means to achieve specificity in PTHR-mediated biological outcomes and raise the prospect of rational drug design based upon spatiotemporal manipulation of GPCR signaling. [ABSTRACT FROM AUTHOR]

cAMP production upon activation of Gs by G protein-coupled receptors has classically been considered to be plasma membranedelimited, but a shift in this paradigm has occurred in recent years with the identification of several receptors that continue to signal from early endosomes after internalization. The molecular mechanisms regulating this aspect of signaling remain incompletely understood. Here, we investigated the role of Gq/11 activation by the parathyroid hormone (PTH) type 1 receptor (PTHR) in mediating endosomal cAMP responses. Inhibition of Gq/11 signaling by FR900359 markedly reduced the duration of PTH-induced cAMP production, and this effect was mimicked in cells lacking endogenous Gaq/11. We determined that modulation of cAMP generation by Gq/11 occurs at the level of the heterotrimeric G protein via liberation of cell surface Gß? subunits, which, in turn, act in a phosphoinositide-3 kinase-dependent manner to promote the assembly of PTHR- ßarrestin-Gß? signaling complexes that mediate endosomal cAMP responses. These results unveil insights into the spatiotemporal regulation of Gs-dependent cAMP signaling. [ABSTRACT FROM AUTHOR]

Helical oligomers of β-peptides represent a particularly promising type of building block for directed assembly of organic nanostructures because the helical secondary structure can be designed to be very stable and because control of the β-amino acid sequence can lead to precise patterning of chemical functional groups over the helix surfaces. In this paper, we report the use of small angle x-ray scattering measurements (SAXS) to characterize nanostructures formed by the directed assembly of β-peptide A with sequence H2N-β3hTyr-β3hLys-β3hPhe-ACHC-β3hPhe-ACHC-β3hPhe-β3hLys-ACHC-ACHC-β3hPhe-β3hLys-CONH2. Whereas prior cryo-TEM studies have revealed the presence of nanofibers in aqueous solutions of β-peptide A, SAXS measurements from the nanofibers were not well-fit by a form factor model describing solid nanofibers. An improved fit to the scattering data at high q was obtained by using a form factor model describing a cylinder with a hollow center and radial polydispersity. When combined with a structure factor calculated from the polymer reference interaction site model (PRISM) theory, the scattered intensity of x-rays measured over the entire q range was well described by the model. Analysis of our SAXS data suggests a model in which individual β-peptides assemble to form long cylindrical nanofibers with a hollow core radius of 15 Å (polydispersity of 21%) and a shell thickness of 20 Å. This model is supported by negative stain transmission electron microscopy. [ABSTRACT FROM AUTHOR]

In this work, the interactions between β-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 β-peptides using molecular dynamics simulations. The stability of a β-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. [ABSTRACT FROM AUTHOR]

Synthesis of the C−C bonds of ketones relies upon one high‐availability reagent (carboxylic acids) and one low‐availability reagent (organometallic reagents or alkyl iodides). We demonstrate here a ketone synthesis that couples two different carboxylic acid esters, N‐hydroxyphthalimide esters and S‐2‐pyridyl thioesters, to form aryl alkyl and dialkyl ketones in high yields. The keys to this approach are the use of a nickel catalyst with an electron‐poor bipyridine or terpyridine ligand, a THF/DMA mixed solvent system, and ZnCl2 to enhance the reactivity of the NHP ester. The resulting reaction can be used to form ketones that have previously been difficult to access, such as hindered tertiary/tertiary ketones with strained rings and ketones with α‐heteroatoms. The conditions can be employed in the coupling of complex fragments, including a 20‐mer peptide fragment analog of Exendin(9–39) on solid support. [ABSTRACT FROM AUTHOR]

Synthesis of the C−C bonds of ketones relies upon one high‐availability reagent (carboxylic acids) and one low‐availability reagent (organometallic reagents or alkyl iodides). We demonstrate here a ketone synthesis that couples two different carboxylic acid esters, N‐hydroxyphthalimide esters and S‐2‐pyridyl thioesters, to form aryl alkyl and dialkyl ketones in high yields. The keys to this approach are the use of a nickel catalyst with an electron‐poor bipyridine or terpyridine ligand, a THF/DMA mixed solvent system, and ZnCl2 to enhance the reactivity of the NHP ester. The resulting reaction can be used to form ketones that have previously been difficult to access, such as hindered tertiary/tertiary ketones with strained rings and ketones with α‐heteroatoms. The conditions can be employed in the coupling of complex fragments, including a 20‐mer peptide fragment analog of Exendin(9–39) on solid support. [ABSTRACT FROM AUTHOR]

Human parathyroid hormone (PTH) and N-terminal fragments thereof activate two receptors, hPTHR1 and hPTHR2, which share ~51% sequence similarity. A peptide comprising the first 34 residues of PTH is fully active at both receptors and is used to treat osteoporosis. We have used this system to explore the hypothesis that backbone modification of a promiscuous peptidic agonist can provide novel receptor-selective agonists. We tested this hypothesis by preparing a set of variants of PTH(1-34)-NH2 that contained a single β-amino-acid residue replacement at each of the first eight positions. These homologs, each containing one additional backbone methylene unit relative to PTH(1-34)-NH2 itself, displayed a wide range of potencies in cell-based assays for PTHR1 or PTHR2 activation. The β-scan series allowed us to identify two homologs, each containing two α→β replacements, that were highly selective, one for PTHR1 and the other for PTHR2. These findings suggest that backbone modification of peptides may provide a general strategy for achieving activation selectivity among polypeptide-modulated receptors, and that success requires consideration of both β² - and β³-residues, which differ in terms of side-chain location. [ABSTRACT FROM AUTHOR]

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Oligomers containing α‐ and β‐amino acid residues (α/β‐peptides) have been shown to mimic the α‐helical conformation of conventional peptides when the unnatural residues are derived from β3‐amino acids or cyclic β‐amino acids, but the impact of incorporating β2 residues has received little attention. The effects of β2 residues on the conformation and recognition behavior of α/β‐peptides that mimic an isolated α‐helix were investigated. This effort has focused on 26‐mers based on the Bim BH3 domain; a set of isomers with identical α/β backbones that differ only in the placement of certain side chains along the backbone (β3 vs. β2 substitution) was compared. Circular dichroism data suggest that β2 residues can be helix‐destabilizing relative to β3 residues, although the size of this effect seems to depend on side chain identity. Binding data show that β3→β2 substitution at sites that contact a partner protein, Bcl‐xL, can influence affinity in a way that transcends effects on helicity. What a difference a side chain makes: Isomeric β3‐to‐β2‐amino acid substitution within a helical α/β‐peptide significantly influences the peptide's properties. β2 residues can be helix‐destabilizing but can also increase the peptide's affinity for a protein binding partner. [ABSTRACT FROM AUTHOR]

Gas-phase single-conformation spectroscopy is used to study Ac-Gln-Gln-NHBn in order to probe the interplay between sidechain hydrogen bonding and backbone conformational preferences. This small, amide-rich peptide offers many possibilities for backbone-backbone, sidechain-backbone, and sidechain-sidechain interactions. The major conformer observed experimentally features a type-I β-turn with a canonical 10-membered ring C=O-H−N hydrogen bond between backbone amide groups. In addition, the C=O group of each Gln sidechain participates in a seven-membered ring hydrogen bond with the backbone NH of the same residue. Thus, sidechain hydrogen-bonding potential is satisfied in a manner that is consistent with and stabilizes the β-turn secondary structure. This turn-forming propensity may be relevant to pathogenic amyloid formation by polyglutamine segments in human proteins. [ABSTRACT FROM AUTHOR]

Gas-phase single-conformation spectroscopy is used to study Ac-Gln-Gln-NHBn in order to probe the interplay between sidechain hydrogen bonding and backbone conformational preferences. This small, amide-rich peptide offers many possibilities for backbone-backbone, sidechain-backbone, and sidechain-sidechain interactions. The major conformer observed experimentally features a type-I β-turn with a canonical 10-membered ring C=O-H−N hydrogen bond between backbone amide groups. In addition, the C=O group of each Gln sidechain participates in a seven-membered ring hydrogen bond with the backbone NH of the same residue. Thus, sidechain hydrogen-bonding potential is satisfied in a manner that is consistent with and stabilizes the β-turn secondary structure. This turn-forming propensity may be relevant to pathogenic amyloid formation by polyglutamine segments in human proteins. [ABSTRACT FROM AUTHOR]

The conformational preferences of a series of short, aromatic-capped, glutamine-containing peptides have been studied under jet-cooled conditions in the gas phase. This work seeks a bottom-up understanding of the role played by glutamine residues in directing peptide structures that lead to neurodegenerative diseases. Resonant ion-dip infrared (RIDIR) spectroscopy is used to record single-conformation infrared spectra in the NH stretch, amide I and amide II regions. Comparison of the experimental spectra with the predictions of calculations carried out at the DFT M05-2X/6-31+G(d) level of theory lead to firm assignments for the H-bonding architectures of a total of eight conformers of four molecules, including three in Z-Gln-OH, one in Z-Gln-NHMe, three in Ac-Gln-NHBn, and one in Ac-Ala-Gln-NHBn. The Gln side chain engages actively in forming H-bonds with nearest-neighbor amide groups, forming C8 H-bonds to the C-terminal side, C9 H-bonds to the N-terminal side, and an amide-stacked geometry, all with an extended (C5) peptide backbone about the Gln residue. The Gln side chain also stabilizes an inverse γ-turn in the peptide backbone by forming a pair of H-bonds that bridge the γ-turn and stabilize it. Finally, the entire conformer population of Ac-Ala-Gln-NHBn is funneled into a single structure that incorporates the peptide backbone in a type I β-turn, stabilized by the Gln side chain forming a C7 H-bond to the central amide group in the β-turn not otherwise involved in a hydrogen bond. This β-turn backbone structure is nearly identical to that observed in a series of X-(AQ)-Y β-turns in the protein data bank, demonstrating that the gas-phase structure is robust to perturbations imposed by the crystalline protein environment. [ABSTRACT FROM AUTHOR]

We have examined whether parallel β-sheet secondary structure becomes more stable as the number of β-strands increases, via comparisons among peptides designed to adopt two- or three-stranded parallel β-sheet conformations in aqueous solution. Our three-strand design is the first experimental model of a triple-stranded parallel β-sheet. Analysis of the designed peptides by nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy supports the hypothesis that increasing the number of β-strands, from two to three, increases the stability of the parallel β-sheet. We present the first experimental evidence for cooperativity in the folding of a triple-stranded parallel β-sheet, and we show how minimal model systems may enable experimental documentation of characteristic properties, such as CD spectra, of parallel β-sheets. [ABSTRACT FROM AUTHOR]

We have examined whether parallel β‐sheet secondary structure becomes more stable as the number of β‐strands increases, via comparisons among peptides designed to adopt two‐ or three‐stranded parallel β‐sheet conformations in aqueous solution. Our three‐strand design is the first experimental model of a triple‐stranded parallel β‐sheet. Analysis of the designed peptides by nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy supports the hypothesis that increasing the number of β‐strands, from two to three, increases the stability of the parallel β‐sheet. We present the first experimental evidence for cooperativity in the folding of a triple‐stranded parallel β‐sheet, and we show how minimal model systems may enable experimental documentation of characteristic properties, such as CD spectra, of parallel β‐sheets.More strands, more stable: The first experimental model of a triple‐stranded parallel β‐sheet was designed. Analysis by NMR and circular dichroism spectroscopy offers the first experimental evidence that increasing the number of β‐strands from two to three increases the stability of the parallel β‐sheet. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The structure of water near non-polar molecular fragments or surfaces mediates the hydrophobic interactions that underlie a broad range of interfacial, colloidal and biophysical phenomena. Substantial progress over the past decade has improved our understanding of hydrophobic interactions in simple model systems, but most biologically and technologically relevant structures contain non-polar domains in close proximity to polar and charged functional groups. Theories and simulations exploring such nanometre-scale chemical heterogeneity find it can have an important effect, but the influence of this heterogeneity on hydrophobic interactions has not been tested experimentally. Here we report chemical force microscopy measurements on alkyl-functionalized surfaces that reveal a dramatic change in the surfaces' hydrophobic interaction strengths on co-immobilization of amine or guanidine groups. Protonation of amine groups doubles the strength of hydrophobic interactions, and guanidinium groups eliminate measurable hydrophobic interactions in all pH ranges investigated. We see these divergent effects of proximally immobilized cations also in single-molecule measurements on conformationally stable β-peptides with non-polar subunits located one nanometre from either amine- or guanidine-bearing subunits. Our results demonstrate the importance of nanometre-scale chemical heterogeneity, with hydrophobicity not an intrinsic property of any given non-polar domain but strongly modulated by functional groups located as far away as one nanometre. The judicious placing of charged groups near hydrophobic domains thus provides a strategy for tuning hydrophobic driving forces to optimize molecular recognition or self-assembly processes. [ABSTRACT FROM AUTHOR]

Minimum inhibitory concentrations (MICs) against E. coli were measured for three nylon-3 polymers using Luria-Bertani broth (LB), brain-heart infusion broth (BHI), and a chemically defined complete medium (EZRDM). The polymers differ in the ratio of hydrophobic to cationic subunits. The cationic homopolymer is inert against E. coli in BHI and LB, but becomes highly potent in EZRDM. A mixed hydrophobic/cationic polymer with a hydrophobic t-butylbenzoyl group at its N-terminus is effective in BHI, but becomes more effective in EZRDM. Supplementation of EZRDM with the tryptic digest of casein (often found in LB) recapitulates the LB and BHI behavior. Additional evidence suggests that polyanionic peptides present in LB and BHI may form electrostatic complexes with cationic polymers, decreasing activity by diminishing binding to the anionic lipopolysaccharide layer of E. coli. In contrast, two natural antimicrobial peptides show no medium effects. Thus, the use of a chemically defined medium helps to reveal factors that influence antimicrobial potency of cationic polymers and functional differences between these polymers and evolved antimicrobial peptides. [ABSTRACT FROM AUTHOR]

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]

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]

Membrane proteins are inherently amphipathic and undergo dynamic conformational changes for proper function within native membranes. Maintaining the functional structures of these biomacromolecules in aqueous media is necessary for structural studies but difficult to achieve with currently available tools, thus necessitating the development of novel agents with favorable properties. This study introduces several new glucose-neopentyl glycol (GNG) amphiphiles and reveals some agents that display favorable behaviors for the solubilization and stabilization of a large, multi-subunit membrane protein assembly. Furthermore, a detergent structure-property relationship that could serve as a useful guideline for the design of novel amphiphiles is discussed. [ABSTRACT FROM AUTHOR]

Amphipathic agents called detergents serve as membrane-mimetic systems to maintain the native structures of membrane proteins during their manipulation. However, membrane proteins solubilized in conventional detergents tend to undergo denaturation and aggregation, necessitating the development of novel amphipathic agents with enhanced properties. Here we describe several new amphiphiles that contain an N-oxide group as the hydrophilic portion. The new amphiphiles have been evaluated for the ability to solubilize and stabilize a fragile multi-subunit assembly from biological membranes. We found that cholate-based agents were promising in supporting retention of the native protein quaternary structure, while deoxycholate-based amphiphiles were highly efficient in extracting/solubilizing the intact superassembly from the native membrane. Monitoring superassembly solubilization and stabilization as a function of variation in amphiphile structure led us to propose that a non-hydrocarbon moiety such as an amide, ether, or a hydroxy group present in the lipophilic regions can manifest distinctive effects in the context of membrane protein manipulation. [ABSTRACT FROM AUTHOR]

The human Pinl WW domain is a small autonomously folding protein that has been useful as a model system for biophysical studies of β-sheet folding. This domain has resisted previous attempts at crystallization for X-ray diffraction studies, perhaps because of intrinsic conformational flexibility that interferes with the formation of a crystal lattice. Here, the crystal structure of the human Pinl WW domain has been obtained via racemic crystallization in the presence of smallmolecule additives. Both enantiomers of a 36-residue variant of the Pinl WW domain were synthesized chemically, and the L- and D-polypeptides were combined to afford diffracting crystals. The structural data revealed packing interactions of small carboxylic acids, either achiral citrate or A D,L mixture of malic acid, with a mobile loop region of the WW-domain fold. These interactions with solution additives may explain our success in crystallization of this protein racemate. Moleculardynamics simulations starting from the structure of the Pinl WW domain suggest that the crystal structure closely resembles the conformation of this domain in solution. The structural data presented here should provide a basis for further studies of this important model system. [ABSTRACT FROM AUTHOR]

Integral membrane proteins play central roles in controlling the flow of information and molecules across membranes. Our understanding of membrane protein structures and functions, however, is seriously limited, mainly due to difficulties in handling and analysing these proteins in aqueous solution. The use of a detergent or other amphipathic agents is required to overcome the intrinsic incompatibility between the large lipophilic surfaces displayed by the membrane proteins in their native forms and the polar solvent molecules. Here, we introduce new tripod amphiphiles displaying favourable behaviours toward several membrane protein systems, leading to an enhanced protein solubilisation and stabilisation compared to both conventional detergents and previously described tripod amphiphiles. [ABSTRACT FROM AUTHOR]

(1 R,2 S)-2-Aminocyclohexanecarboxylic acid ( cis-ACHC) is a preorganized β-amino acid. cis-ACHC favors two conformations that feature gauche conformations about the Cα-Cβ bond with torsion angles of opposite signs. The diastereomeric β-amino acid trans-ACHC has been widely studied as a foldamer building block, but cis-ACHC has received less attention in this regard. We examined the conformational behaviour of three types of oligomer: (1) homooligomers of cis-ACHC, (2) β-peptides in which cis-ACHC and β3h-Ala alternate, and (3) 1:1 α/β-peptides in which cis-ACHC and Ala alternate. Two-dimensional NMR experiments suggest that all three types of oligomer adopt extended conformations rather than folded conformations in solution. Two crystal structures of oligomers that contain cis-ACHC residues, a cis-ACHC dimer and an α/β-peptide tetramer, show extended conformations in which the cis-ACHC residues contain six-membered-ring C=O ···H-N hydrogen bonds. [ABSTRACT FROM AUTHOR]

The introduction of non-natural modules could provide unprecedented control over folding/unfolding behavior, conformational stability, and biological function of proteins. Success requires the interrogation of candidate modules in natural contexts. Here, expressed protein ligation is used to replace a reverse turn in bovine pancreatic ribonuclease (RNase A) with a synthetic β-dipeptide: β2-homoalanine-β3-homoalanine. This segment is known to adopt an unnatural reverse-turn conformation that contains a 10-membered ring hydrogen bond, but one with a donor-acceptor pattern opposite to that in the 10-membered rings of natural reverse turns. The RNase A variant has intact enzymatic activity, but unfolds more quickly and has diminished conformational stability relative to native RNase A. These data indicate that hydrogen-bonding pattern merits careful consideration in the selection of beneficial reverse-turn surrogates. [ABSTRACT FROM AUTHOR]

The ability to design biomaterials that interact with biological environments in a predictable manner necessitates an improved understanding of how surface chemistry influences events such as protein adsorption and cell adhesion. In this work, we examined mechanisms governing the interactions between 3T3 fibroblasts and nylon-3 polymers, which have a protein-like polyamide backbone and are highly amenable to tuning of chemical and physical properties. Protein adsorption and cell adhesion to a library of nylon-3 polymers were characterized and analyzed by partial least squares regression. This analysis revealed that specific chemical features of the nylon-3 polymers correlated with the extent of protein adsorption, which, in turn, correlated with cell adhesion in a serum-containing environment. In contrast, in a serum-free environment, cell adhesion could be predicted solely from chemical properties. Enzymatic treatments of 3T3 cells before plating indicated that proteins bound to the cell surface mediated cell-nylon-3 polymer interactions under serum-free conditions, with additional analysis suggesting that cell-associated fibronectin played a dominant role in adhesion in the absence of serum. The mechanistic insight gained from these studies can be used to inform the design of new polymer structures in addition to providing a basis for continued development of nylon-3 copolymers for tissue engineering applications. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A 100A:2750-2759, 2012. [ABSTRACT FROM AUTHOR]

G protein-coupled receptors (GPCRs) are responsible for the majority of cellular responses to hormones and neurotransmitters as well as the senses of sight, olfaction and taste. The paradigm of GPCR signalling is the activation of a heterotrimeric GTP binding protein (G protein) by an agonist-occupied receptor. The ?2 adrenergic receptor (?2AR) activation of Gs, the stimulatory G protein for adenylyl cyclase, has long been a model system for GPCR signalling. Here we present the crystal structure of the active state ternary complex composed of agonist-occupied monomeric ?2AR and nucleotide-free Gs heterotrimer. The principal interactions between the ?2AR and Gs involve the amino- and carboxy-terminal ?-helices of Gs, with conformational changes propagating to the nucleotide-binding pocket. The largest conformational changes in the ?2AR include a 14?Å outward movement at the cytoplasmic end of transmembrane segment 6 (TM6) and an ?-helical extension of the cytoplasmic end of TM5. The most surprising observation is a major displacement of the ?-helical domain of G?s relative to the Ras-like GTPase domain. This crystal structure represents the first high-resolution view of transmembrane signalling by a GPCR. [ABSTRACT FROM AUTHOR]