Crystal Structure and NMR of an α,δ-Peptide Foldamer Helix Shows Side-Chains are Well Placed for Bifunctional Catalysis: Application as a Minimalist Aldolase Mimic.

We report the first NMR and X-ray diffraction (XRD) structures of an unusual 13/11-helix (alternating i, i+1 {NH-O=C} and i, i+3 {C=O-H-N} H-bonds) formed by a heteromeric 1 : 1 sequence of α- and δ-amino acids, and demonstrate the application of this framework towards catalysis. Whilst intramolecular hydrogen bonds (IMHBs) are the clear driver of helix formation in this system, we also observe an apolar interaction between the ethyl residue of one δ-amino acid and the cyclohexyl group of the next δ-residue in the sequence that seems to stabilize one type of helix over another. To the best of our knowledge this type of additional stabilization leading to a specific helical preference has not been observed before. Critically, the helix type realized places the α-residue functionalities in positions proximal enough to engage in bifunctional catalysis as demonstrated in the application of our system as a minimalist aldolase mimic.

Crystal Structure and NMR of an α,δ-Peptide Foldamer Helix Shows Side-Chains are Well Placed for Bifunctional Catalysis: Application as a Minimalist Aldolase Mimic.

We report the first NMR and X-ray diffraction (XRD) structures of an unusual 13/11-helix (alternating i, i+1 {NH-O=C} and i, i+3 {C=O-H-N} H-bonds) formed by a heteromeric 1 : 1 sequence of α- and δ-amino acids, and demonstrate the application of this framework towards catalysis. Whilst intramolecular hydrogen bonds (IMHBs) are the clear driver of helix formation in this system, we also observe an apolar interaction between the ethyl residue of one δ-amino acid and the cyclohexyl group of the next δ-residue in the sequence that seems to stabilize one type of helix over another. To the best of our knowledge this type of additional stabilization leading to a specific helical preference has not been observed before. Critically, the helix type realized places the α-residue functionalities in positions proximal enough to engage in bifunctional catalysis as demonstrated in the application of our system as a minimalist aldolase mimic.

Organocatalytic Access to a cis-Cyclopentyl-γ-amino Acid: An Intriguing Model of Selectivity and Formation of a Stable 10/12-Helix from the Corresponding γ/α-Peptide.

In this study, we have developed a highly enantioselective organocatalytic route to the (1S,2R)-2-(aminomethyl)cyclopentane-1-carboxylic acid monomer precursor, which has a cis-configuration between the C- and N-termini around the cyclopentane core. Kinetic measurements show that the product distribution changes over time due to epimerization of the C1 center. Computations suggest the cis-selectivity is a result of selective C-C bond formation, while subsequent steps appear to influence the selectivity at higher temperature. The resulting γ-amino acid residue was incorporated into a novel γ/α-peptide, which forms a well-ordered 10/12-helix with alternate H-bond directionality in spite of the smallest value of the ζ-angle yet observed for a helix of this type. This highly defined structure is also a result of the narrow range of potential ζ-angles in our monomer. In contrast, the larger range of potential ζ-values observed for the corresponding trans-system can be fulfilled by several competing helical structures.

Modulating the Structural Properties of α,γ-Hybrid Peptides by α-Amino Acid Residues: Uniform 12-Helix Versus "Mixed" 12/10-Helix.

The most important natural α- and 310 -helices are stabilized by unidirectional intramolecular hydrogen bonds along the helical cylinder. In contrast, we report here on 12/10-helical conformations with alternately changing hydrogen-bond directionality in sequences of α,γ-hybrid peptides P1-P5 [P1: Boc-Ala-Aic-Ala-Aic-COOH; P2: Boc-Leu-Aic-Leu-Aic-OEt; P3: Boc-Leu-Aic-Leu-Aic-Leu-Aic-Aib-OMe; P4: Boc-Ala-Aic-Ala-Aic-Ala-Aic-Ala-OMe; P5: Boc-Leu-Aic-Leu-Aic-Leu-Aic-Leu-Aic-Aib-OMe; Aic=4-aminoisocaproic acid, Aib=2-aminoisobutyric acid] composed of natural α-amino acids and the achiral γ4,4 -dimethyl substituted γ-amino acid Aic in solution and in single crystals. The helical conformations are stabilized by alternating i→i+3 and i→i-1 intramolecular hydrogen bonds. The experimental data are supported by ab initio MO calculations. Surprisingly, replacing the natural α-amino acids of the sequence by the achiral dialkyl amino acid Ac6 c [P6: Boc-Ac6 c-Aic-Ac6 c-Aic-Ac6 c-Aic-Ac6 c-Aic-Ac6 c-CONHMe; Ac6 c = 1-aminocyclohexane-1-carboxylic acid] led to a 12-helix with unidirectional hydrogen bonds showing an entirely different backbone conformation. The results presented here emphasize the influence of the structure of the α-amino acid residues in dictating the helix types in α,γ-hybrid peptide foldamers and demonstrate the consequences for folding of small structural variations in the monomers.

Structural Dimorphism of Achiral α,γ-Hybrid Peptide Foldamers: Coexistence of 12- and 15/17-Helices.

Here, novel 12-helices in α,γ-hybrid peptides composed of achiral α-aminoisobutyric acid (Aib) and 4-aminoisocaproic acid (Aic, doubly homologated Aib) monomers in 1:1 alternation are reported. The 12-helices were indicated by solution and crystal structural analyses of tetra- and heptapeptides. Surprisingly, single crystals of the longer nonapeptide displayed two different helix types: the novel 12-helix and an unprecedented 15/17-helix. Quantum chemical calculations on both helix types in a series of continuously lengthened Aib/Aic-hybrid peptides confirm that the 12-helix is more stable than the 15/17-helix in shorter peptides, whereas the 15/17-helix is more stable in longer sequences. Thus, the coexistence of both helix types can be expected within a definite range of sequence lengths. The novel 15/17- and 12-helices in α,γ-hybrid peptides with 5→1 and 4→1 hydrogen-bonding patterns, respectively, can be viewed as backbone-expanded analogues of native α- and 310 -helices.

Solvent-Directed Switch of a Left-Handed 10/12-Helix into a Right-Handed 12/10-Helix in Mixed ß-Peptides.

Present study describes the synthesis and conformational analysis of ß-peptides from C-linked carbo-ß-amino acids [ß-Caa(l)] with a d-lyxo furanoside side chain and ß-hGly in 1:1 alternation. NMR and CD investigations on peptides with an (S)-ß-Caa(l) monomer at the N-terminus revealed a right-handed 10/12-mixed helix. An unprecedented solvent-directed "switch" both in helical pattern and handedness was observed when the sequence begins with a ß-hGly residue instead of a (S)-ß-Caa(l) constituent. NMR studies on these peptides in chloroform indicated a left-handed 10/12-helix, while the CD spectrum in methanol inferred a right-handed secondary structure. The NMR data for these peptides in CD3OH showed the presence of a right-handed 12/10-helix. NMR investigations in acetonitrile indicated the coexistence of both helix types. Quantum chemical studies predicted a small energy difference of 0.3 kcal/mol between the two helix types, which may explain the possibility of solvent influence. Examples for a solvent-directed switch of both the H-bonding pattern and the handedness of foldamer helices are rare so far. A comparable solvent effect was not found in the corresponding peptides with (R)-ß-Caa(l) residues, where right-handed 12/10-helices are predominating.

Synthesis of C-linked carbo-ß2-amino acids and ß2-peptides: design of new motifs for left-handed 12/10- and 10/12-mixed helices.

C-linked carbo-ß(2)-amino acids (ß(2)-Caa), a new class of ß-amino acid with a carbohydrate side chain having d-xylo configuration, were prepared from d-glucose. The main idea behind the design of the new ß-amino acids was to move the steric strain of the bulky carbohydrate side chain from the Cß- to the Cα-carbon atom and to explore its influence on the folding propensities in peptides with alternating (R)- and (S)-ß(2)-Caas. The tetra- and hexapeptides derived were studied employing NMR (in CDCl(3)), CD, and molecular dynamics simulations. The ß(2)-peptides of the present study form left-handed 12/10- and 10/12-mixed helices independent of the order of the alternating chiral amino acids in the sequence and result in a new motif. These results differ from earlier findings on ß(3)-peptides of the same design, containing a carbohydrate side chain with d-xylo configuration, which form exclusively right-handed 12/10-mixed helices. Quantum chemical calculations employing ab initio MO theory suggest the side chain chirality as an important factor for the observed definite left- or right-handedness of the helices in the ß(2)- and ß(3)-peptides.

Influence of lithium cations on prolyl peptide bonds.

The influence of lithium cations on the cis/trans isomerization of prolyl peptide bonds was investigated in a quantitative manner in trifluoroethanol (TFE) and acetonitrile, employing NMR techniques. The focus was on various environmental and structural aspects, such as lithium cation and water concentrations, the type of the partner amino acid in the prolyl peptide bond, and the peptide sequence length. Comparison of the thermodynamic parameters of the isomerization in LiCl/TFE and TFE shows a lithium cation concentration dependence of the cis/trans ratio, which saturates at cation concentrations >200 mM. A pronounced increase in the cis isomer content in the presence of lithium cations occurs with the exception of peptides with Gly-Pro and Asp-Pro moieties. The cation effect appears already at the dipeptide level. The salt concentration can considerably be reduced in solvents with a lower number of nucleophilic centers like acetonitrile. The lithium cation effect decreases with small amounts of water and disappears at a water concentration of about 5%. The isomerization kinetics under the influence of lithium cations suggests a weak cation interaction with the carbonyl oxygen of the peptide bond.

NH exchange in point mutants of human ubiquitin.

Several point mutants of human ubiquitin (Ub(T9V), Ub(F45W), Ub(F45G), and Ub(A46S)) were prepared by recombinant techniques. The NH exchange rate constants were measured by the NMR diffusion and the MEXICO methods and compared with those in the wild type to examine the influence of structural changes and to improve the understanding of this important reaction in studies of protein folding and denaturation. The observed changes follow qualitatively the polarity and steric alterations caused by the introduced amino acids. Attempts to reproduce quantitatively the observed changes by modeling studies and molecular dynamics simulations were not satisfactory.

Conversion of a decarboxylating to a non-decarboxylating glutaryl-coenzyme A dehydrogenase by site-directed mutagenesis.

Glutaryl-coenzyme A (CoA) dehydrogenases (GDHs) are acyl-CoA dehydrogenases, which usually dehydrogenate and decarboxylate the substrate to crotonyl-CoA. In some anaerobic bacteria, non-decarboxylating GDHs exist that release glutaconyl-CoA (2,3-dehydroglutaryl-CoA) without decarboxylation. The differing mechanisms of decarboxylating and non-decarboxylating GDHs were investigated by site-directed mutagenesis of the gene coding for the crotonyl-CoA-forming GDH from Geobacter metallireducens. Exchange of single amino acids involved in substrate carboxylate binding impaired the decarboxylation step, resulting in relative glutaconyl-CoA:crotonyl-CoA formation rates of 1:1 (S97A) or 13:1 (Y370A). The total amount of glutaconyl-CoA formed was maximal in the Y370V+S97A double mutant. The results obtained indicate that an invariant deprotonated Tyr plays a crucial role for optimizing the leaving group potential of CO(2) in decarboxylating GDHs.

How many hydrogen-bonded α-turns are possible?

The formation of α-turns is a possibility to reverse the direction of peptide sequences via five amino acids. In this paper, a systematic conformational analysis was performed to find the possible isolated α-turns with a hydrogen bond between the first and fifth amino acid employing the methods of ab initio MO theory in vacuum (HF/6-31G*, B3LYP/6-311 + G*) and in solution (CPCM/HF/6-31G*). Only few α-turn structures with glycine and alanine backbones fulfill the geometry criteria for the i←(i + 4) hydrogen bond satisfactorily. The most stable representatives agree with structures found in the Protein Data Bank. There is a general tendency to form additional hydrogen bonds for smaller pseudocycles corresponding to ß- and γ-turns with better hydrogen bond geometries. Sometimes, this competition weakens or even destroys the i←(i + 4) hydrogen bond leading to very stable double ß-turn structures. This is also the reason why an "ideal" α-turn with three central amino acids having the perfect backbone angle values of an α-helix could not be localized. There are numerous hints for stable α-turns with a distance between the C(α)-atoms of the first and fifth amino acid smaller than 6-7 Å, but without an i←(i + 4) hydrogen bond.

Double helix formation in alpha-peptides: a theoretical study.

A complete overview on all possible hydrogen bonding patterns of double helices with antiparallel and parallel strand orientation in alpha-peptide sequences is provided on the basis of ab initio molecular orbital theory. The most stable representatives belong to the group of antiparallel helices. The study on side chain influence shows that these double helices can only be realized if the strands are composed of L- and D-amino acids in alternate order. The stability of the double helices is compared with that of competing single-stranded helices. The data contribute to an understanding of secondary structure formation in peptides and provide a basis for a rational design of membrane channels.

Unusual evolution of a catalytic core element in CCA-adding enzymes.

CCA-adding enzymes are polymerases existing in two distinct enzyme classes that both synthesize the C-C-A triplet at tRNA 3'-ends. Class II enzymes (found in bacteria and eukaryotes) carry a flexible loop in their catalytic core required for switching the specificity of the nucleotide binding pocket from CTP- to ATP-recognition. Despite this important function, the loop sequence varies strongly between individual class II CCA-adding enzymes. To investigate whether this loop operates as a discrete functional entity or whether it depends on the sequence context of the enzyme, we introduced reciprocal loop replacements in several enzymes. Surprisingly, many of these replacements are incompatible with enzymatic activity and inhibit ATP-incorporation. A phylogenetic analysis revealed the existence of conserved loop families. Loop replacements within families did not interfere with enzymatic activity, indicating that the loop function depends on a sequence context specific for individual enzyme families. Accordingly, modeling experiments suggest specific interactions of loop positions with important elements of the protein, forming a lever-like structure. Hence, although being part of the enzyme's catalytic core, the loop region follows an extraordinary evolutionary path, independent of other highly conserved catalytic core elements, but depending on specific sequence features in the context of the individual enzymes.

Helix formation in beta/delta-hybrid peptides: correspondence between helices of different peptide foldamer classes.

An overview on all hydrogen-bonded helix types, which can be expected in the novel class of beta/delta-hybrid peptides, is provided on the basis of ab initio molecular orbital theory. The results show a considerable potential of different folding patterns, which makes this novel class of peptide foldamers interesting for peptide and protein design. Thus, this study may stimulate the synthesis of such hybrid peptides. The large number of helix types found for beta/delta-hybrid peptides together with the many helices experimentally found and theoretically predicted in the other classes of peptide and hybrid peptide foldamers provide a good basis to establish general correspondence rules for their mutual exchange in peptide sequences. This is done by introduction of the concept of pattern and shape correspondence.

4-Aminoethylamino-emodin--a novel potent inhibitor of GSK-3beta--acts as an insulin-sensitizer avoiding downstream effects of activated beta-catenin.

Glycogen synthase kinase-3beta (GSK-3beta) is a key target and effector of downstream insulin signalling. Using comparative protein kinase assays and molecular docking studies we characterize the emodin-derivative 4-[N-2-(aminoethyl)-amino]-emodin (L4) as a sensitive and potent inhibitor of GSK-3beta with peculiar features. Compound L4 shows a low cytotoxic potential compared to other GSK-3beta inhibitors determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide assay and cellular ATP levels. Physiologically, L4 acts as an insulin-sensitizing agent that is able to enhance hepatocellular glycogen and fatty acid biosynthesis. These functions are particularly stimulated in the presence of elevated concentrations of glucose and in synergy with the hormone action at moderate but not high insulin levels. In contrast to other low molecular weight GSK-3beta inhibitors (SB216763 and LiCl) or Wnt-3alpha-conditioned medium, however, L4 does not induce reporter and target genes of activated beta-catenin such as TOPflash, Axin2 and glutamine synthetase. Moreover, when present together with SB216763 or LiCl, L4 counteracts expression of TOPflash or induction of glutamine synthetase by these inhibitors. Because L4 slightly activates beta-catenin on its own, these results suggest that a downstream molecular step essential for activation of gene transcription by beta-catenin is also inhibited by L4. It is concluded that L4 represents a potent insulin-sensitizing agent favouring physiological effects of insulin mediated by GSK-3beta inhibition but avoiding hazardous effects such as activation of beta-catenin-dependent gene expression which may lead to aberrant induction of cell proliferation and cancer.

Hybrid helices: motifs for secondary structure scaffolds in foldamers.

The concept of "hybrid helices" as a new motif for foldamers is presented. Hybrid helices can be realized by a combination of two or more different types of homologous and hybrid peptides, for example, beta-peptides and alpha/beta- and alpha/gamma-hybrid peptides, within the same oligomer. The different helix types of the various peptide foldamer classes are maintained and form a regular helix along the sequence of the oligomer. The transition from one helix type to another was found to be rather smooth with high compatibility of the different helix types. Such hybrid helices represent novel motifs of secondary structure scaffolds. They open up the possibility to change the direction of helix propagation in a subtle manner. Hybrid helices enrich the arsenal of defined foldamer structures for a structural and functional mimicry of native peptides and proteins.

Theoretical and experimental studies on alpha/epsilon-hybrid peptides: design of a 14/12-helix from peptides with alternating (S)-C-linked carbo-epsilon-amino acid [(S)-epsilon-Caa((x))] and L-ala.

An (S)-C-linked carbo-epsilon-amino acid [(S)-epsilon-Caa((x))] was prepared from the known (S)-delta-Caa. This monomer was utilized together with l-Ala to give novel alpha/epsilon-hybrid peptides in 1:1 alternation. Conformational analysis on penta- and hexapeptides by NMR (in CDCl(3)), CD, and MD studies led to the identification of robust 14/12-mixed helices. This is in agreement with the data from a theoretical conformational analysis on the basis of ab initio MO theory providing a complete overview on all formally possible hydrogen-bonded helix patterns of alpha/epsilon-hybrid peptides with 1:1 backbone alternation. The "new motif" of a mixed 14/12-helix was predicted as most stable in vacuum. Obviously, the formation of ordered secondary structures is also possible in peptide foldamers with amino acid constituents of considerable backbone lengths. Thus, alpha/epsilon-hybrid peptides expand the domain of foldamers and allow the introduction of desired functionalities via the alpha-amino acid constituents.

Novel foldamer structural architecture from cofacial aromatic building blocks.

This communication demonstrates the utility of peri-substituted 1,8-diphenylnaphthalene as an effective building block for the construction of novel conformationally ordered synthetic oligomers displaying cofacial structural features.

Sterically controlled naphthalene homo-oligoamides with novel structural architectures.

Herein we report novel naphthalene homo-oligoamides, derived from 4-amino-3-methoxy-naphthalene-2-carboxylic acid and 4-amino-1-methoxy-naphthalene-2-carboxylic acid as monomer building blocks, that display an anti-periplanar arrangement of the naphthyl rings, primarily induced by steric interactions between adjacent groups and functionalities.

Nanofibers from oxazolidi-2-one containing hybrid foldamers: what is the right molecular size?

A series of oligomers of the type Boc-(L-Phe-D-Oxd)(n)-OBn (Boc = tert-butoxycarbonyl; Oxd = 4-methyl-5-carboxy oxazolidin-2-one; Bn = benzyl) were prepared for n = 2-5. The shortest oligomer, Boc-(L-Phe-D-Oxd)(2)-OBn, aggregates and forms a fiber-like material with an anti-parallel beta-sheet structure in which the oligopeptide units are connected to each other by only one intermolecular hydrogen bond. The longer oligomers exhibit structural heterogeneity. They start to organize into secondary structures by the formation of intramolecular hydrogen bonds at the pentamer level. Microscopy and diffraction of the oligomers indicated a crystalline character for only the shorter ones.