One-pot total chemical synthesis of human α-synuclein.

Post-translational modifications (PTMs) regulate key aspects of the physiological and pathogenic properties of Parkinson's disease-associated presynaptic protein α-synuclein. We herein describe a one-pot total chemical synthesis that should enable site-specific introduction of single or multiple PTMs or small molecule probes essentially at any site within the protein.

Amyloidogenic protein-membrane interactions: mechanistic insight from model systems.

The toxicity of amyloid-forming proteins is correlated with their interactions with cell membranes. Binding events between amyloidogenic proteins and membranes result in mutually disruptive structural perturbations, which are associated with toxicity. Membrane surfaces promote the conversion of amyloid-forming proteins into toxic aggregates, and amyloidogenic proteins, in turn, compromise the structural integrity of the cell membrane. Recent studies with artificial model membranes have highlighted the striking resemblance of the mechanisms of membrane permeabilization of amyloid-forming proteins to those of pore-forming toxins and antimicrobial peptides.

Colorful methods to detect ion channels and pores: intravesicular chromogenic probes that respond to pH, pM and covalent capture.

The activity of synthetic pores, ion channels, transporters and carriers is usually determined with fluorescent probes in vesicles or by conductance measurements in planar lipid bilayers. Elaborating on more colorful alternatives, we here introduce 5(6)-carboxynaphthofluorescein (CNF) as an intravesicular pH probe for the colorimetric detection of activity, selectivity and cooperativity of ion channels such as gramicidin A. We further report that intravesicular pyrocatechol violet (PV), together with extravesicular Cu2+, extravesicular 4-carboxyphenylboronic acid (CBA) or intravesicular 4-(benzyl-N-glutamate)boronic acid (BGBA) can detect the activity of synthetic pores or cell-penetrating peptide (CPP) sensors. Their response to analytes such as dodecylphosphate, hyaluronan or IP6 are reported as high-contrast color changes from yellow to blue, from yellow to red, or from red to green.

Interaction energies and dynamics of acid-base pairs isolated in cavitands.

The use of capsules and cavitands in physical organic chemistry is briefly reviewed, and their application to the study of salt bridges is introduced. Carboxylate/ammonium ion pairs are generated within an environment that more or less surrounds the functional groups within a synthetic fixed introverted solvent sphere. This is provided by cavitands that fold around amines and present them with a carboxylic acid function. Both organic and water-soluble versions were prepared, and their equilibrium affinities with quinuclidine bases were determined by NMR methods. The association constants range from approximately 10(3) M(-1) in water to more than 10(5) M(-1) in organic solvents. Studies of nitrogen inversion and tumbling of [2.2.2]-diazabicyclooctane within the introverted acids also illustrate the strength of the acid-base interactions. The barriers to in-out exchange of several amine guests were determined to be in the range from 15 to 24 kcal mol(-1). Some parallels with enzymes are drawn: the receptor folds around the guest species; presents them with inwardly directed functionality; and provides a generally hydrophobic environment and a periphery of secondary amide bonds.

A cavitand stabilizes the Meisenheimer complex of SNAr reactions.

A deep cavitand binds amine nucleophiles and accelerates their subsequent S(N)Ar reactions by solvating the intermediate Meisenheimer complex.

A synthetic mimic of protein inner space: buried polar interactions in a deep water-soluble host.

A deep water-soluble cavitand was functionalized with a carboxylic acid directed toward the hydrophobic interior of the host. The buried salt-bridge interaction formed with a quinuclidium cationic guest was determined to be worth -3 kcal/mol using a free energy cycle. The strength of the interaction correlates well with buried salt bridges in proteins, indicating that the cavitand interior mimics the hydrophobic inner space of proteins.

The recognition of nucleotides with model beta-hairpin receptors: investigation of critical contacts and nucleotide selectivity.

[reaction: see text] We have investigated the factors that contribute to binding of ATP by a designed 12-residue beta-hairpin peptide, WKWK, and have determined its selectivity for binding to the naturally occurring nucleotide triphosphates. We have previously shown that WKWK creates an ATP binding pocket on one face of the beta-hairpin consisting of two Trp and two Lys residues. Mutation of the two Lys residues on the binding face of the beta-hairpin resulted in a lower affinity, indicating that each is involved in ATP binding and that each residue contributes approximately -1.5 kcal/mol to the energy of complexation. Replacement of either Trp residue of the ATP binding pocket with Phe or Leu destabilizes the complex formed with ATP by approximately 1 kcal/mol, indicating that both Trp residues participate in interactions with ATP. For binding to the nucleotide triphosphates, the order of binding affinity was shown to follow dTTP > GTP > ATP > CTP, with differences in binding energies spanning as much as 1.6 kcal/mol. NMR analysis demonstrates that both aromatic interactions with the Trp side chains and CH-pi interactions between the ribose protons and the Trp residues may contribute significantly to binding. The results from our model system provide useful thermodynamic information regarding protein-nucleic acid interactions that occur at the surface of a beta-sheet.

Minimalist protein design: a beta-hairpin peptide that binds ssDNA.

A 28-residue beta-hairpin dimer (WKWK)2 with two Trp and two Lys residues on one face of each beta-sheet was shown to form a complex with single-stranded oligonucleotides at low micromolar concentrations. Each beta-hairpin of the dimer contains a cross-strand Trp-Trp pair in a diagonal orientation which has previously been shown to create a cleft for the intercalation of aromatic guests such as adenine (J. Am. Chem. Soc. 2003, 125, 9580). The beta-hairpin dimer binds 5-residue ssDNA sequences 5'-AAAAA-3', 5'-TTTTT-3', and 5'-CCCCC-3' in water with dissociation constants in the range of 12-30 muM. A weak energetic preference for binding to sequence 5'-AAAAA-3' was observed, which is believed to result from stronger stacking interactions between Trp and the adenine base. The interaction of 5'-AAAAA-3' with the Lys and Trp residues of the peptide was evident by NMR, and a 1:1 association was demonstrated. The recognition of an 11-residue ssDNA sequence occurred with a dissociation constant of 3 muM under near-physiological ionic strength and pH, demonstrating that the beta-hairpin dimer binds ssDNA as strongly as many naturally occurring proteins. The salt dependence of the interaction of the 11-residue oligonucleotide with the peptide dimer indicates that Trp-nucleobase stacking interactions contribute about -4 kcal/mol to recognition, which is much greater than the contribution of nonionic interactions in unstructured peptides containing Trp. Moreover, recognition of the ssDNA demonstrated reduced salt dependence relative to the corresponding duplex, resulting in selectivity for ssDNA under high salt conditions. Peptide (WKWK)2 is a relevant mimic of OB-fold (oligonucleotide/oligosaccharide-binding) proteins which bind ssDNA on the surface of a beta-sheet.

A designed beta-hairpin peptide for molecular recognition of ATP in water.

A designed 12-residue beta-hairpin peptide with a diagonal tryptophan (Trp) pair was shown to bind ATP in water through a combination of aromatic and electrostatic interactions. The affinity for ATP was 5800 M-1 (DeltaG approximately -5.0 kcal/mol), a remarkable affinity for a short, structured peptide in water, consisting of entirely natural amino acid residues. Proton NMR measurements indicate that the adenine ring of the nucleotide is intercalated between the diagonal tryptophans in the bound state. Delineation of the contributions to ATP binding to the hairpin suggest that aromatic interactions contribute approximately -1.8 kcal/mol, while individual electrostatic interactions involving the ATP phosphates and positively charged side chains of the hairpin contribute approximately -1 kcal/mol each. The designed beta-hairpin receptor presents a novel minimalist system to investigate the energetic contributions to protein-nucleic acid recognition through the surface of a beta-sheet.

Contribution of aromatic interactions to alpha-helix stability.

The influence of natural and unnatural i, i + 4 aromatic side chain-side chain interactions on alpha-helix stability was determined in Ala-Lys host peptides by circular dichroism (CD). All interactions investigated provided some stability to the helix; however, phenylalanine-phenylalanine (F-F) and phenylalanine-pentafluorophenylalanine (F-f5F) interactions resulted in the greatest enhancement in helicity, doubling the helical content over i, i + 5 control peptides at internal positions. Quantification of these interactions using AGADIR multistate helix-coil algorithm revealed that the F-F and F-f5F interaction energies are equivalent at internal positions in the sequence (deltaGF-F = deltaGF-f5F = -0.27 kcal/mol), despite the differences in their expected geometries. As the strength of a face-to-face stacked phenyl-pentafluorophenyl interaction should surpass an edge-to-face or offset-stacked phenyl-phenyl interaction, we believe this result reflects the inability of the side chains in F-f5F to attain a fully stacked geometry within the context of an alpha-helix. Positioning the interactions at the C-terminus led to much stronger interactions (deltaGF-F = -0.8 kcal/mol; deltaGF-f5F = -0.55 kcal/mol) likely because of favorable chi(1) rotameric preferences for aromatic residues at C-capping regions of alpha-helices, suggesting that aromatic side chain-side chain interactions are an effective alpha-helix C-capping method.