Propensity for local folding induced by the urea fragment in short-chain oligomers

Examination of local folding and H-bonding patterns in model compounds can be extremely informative to gain insight into the propensity of longer-chain oligomers to adopt specific folding patterns (i.e. foldamers) based on remote interactions. Using a combination of experimental techniques (i.e. X-ray diffraction, FT-IR absorption and NMR spectroscopy) and theoretical calculations at the density functional theory (DFT) level, we have examined the local folding patterns induced by the urea fragment in short-chain aza analogues of beta- and gamma-amino acid derivatives. We found that the urea-turn, a robust C(8) conformation based on 1<--3 H-bond interaction, is largely populated in model ureidopeptides (I-IV) obtained by replacing the alpha-carbon of a beta-amino acid by a nitrogen. This H-bonding scheme is likely to compete with remote H-bond interactions, thus preventing the formation of secondary structures based on remote intrastrand interactions in longer oligomers. In related oligomers obtained by the addition of a methylene in the main chain (V-VIII), nearest-neighbour H-bonded interactions are unfavourable i.e. the corresponding C9 folding pattern is hardly populated. In this series, folding based on remote intrastrand interactions becomes possible for longer oligomers. We present spectroscopic evidence that tetraurea VIII is likely to be the smallest unit capable of reproducing the H-bonded motif found in 2.5-helical N,N'-linked oligoureas.     

Aromatic-aromatic interactions in proteins: beyond the dimer

Aromatic residues are key widespread elements of protein structures and have been shown to be important for structure stability, folding, protein-protein recognition, and ligand binding. The interactions of pairs of aromatic residues (aromatic dimers) have been extensively studied in protein structures. Isolated aromatic molecules tend to form higher order clusters, like trimers, tetramers, and pentamers, that adopt particular well-defined structures. Taking this into account, we have surveyed protein structures deposited in the Protein Data Bank in order to find clusters of aromatic residues in proteins larger than dimers and characterized them. Our results show that larger clusters are found in one of every two unique proteins crystallized so far, that the clusters are built adopting the same trimer motifs found for benzene clusters in vacuum, and that they are clearly nonlocal brining primary structure distant sites together. We extensively analyze the trimers and tetramers conformations and found two main cluster types: a symmetric cluster and an extended ladder. Finally, using calmodulin as a test case, we show aromatic clsuters possible role in folding and protein-protein interactions. All together, our study highlights the relevance of aromatic clusters beyond the dimer in protein function, stability, and ligand recognition.     

蛋白质结构的“限域下最低能量结构片段”假说与蛋白质进化的“石器时代”

蛋白质折叠问题被称为第二遗传密码,至今未破译;蛋白质序列的天书仍然是"句读之不知,惑之不解"。在最近工作的基础上,我们提出了蛋白质结构的"限域下最低能量结构片段"假说。这一假说指出,蛋白质中存在一些关键的长程强相互作用位点,这些位点相当于标点符号,将蛋白质序列的天书变成可读的句子(多肽片段)。这些片段的天然结构是在这些强长程相互作用位点限域下的能量最低状态。完整的蛋白质结构由这些"限域下最低能量结构片段"拼合而成,而蛋白质整体结构并不一定是全局性的能量最低状态。在蛋白质折叠过程中,局部片段的天然结构倾向性为强长程相互作用的形成提供主要基于焓效应的驱动力,而天然强长程相互作用的形成为局部片段的天然结构提供主要基于熵效应的稳定性。在蛋白质进化早期,可能存在一个"石器时代",即依附不同界面(比如岩石)的限域作用而稳定的多肽片段先进化出来,后由这些片段逐步进化(包括拼合)而成蛋白质。     

Backbone-rigidified oligo(m-phenylene ethynylenes)

Oligo(m-phenylene ethynylenes) (oligo(m-PE)) with backbones rigidified by intramolecular hydrogen bonds were found to fold into well-defined conformations. The localized intramolecular hydrogen bond involves a donor and an acceptor from two adjacent benzene rings, respectively, which enforces globally folded conformations on these oligomers. Oligomers with two to seven residues have been synthesized and characterized. The persistence of the intramolecular hydrogen bonds and the corresponding curved conformations were established by ab initio and molecular mechanics calculations, 1D and 2D (1)H NMR spectroscopy, and UV spectroscopy. Pentamer 5, hexamer 6, and heptamer 7 adopt well-defined helical conformations. Such a backbone-based conformational programming should lead to molecules whose conformations are resilient toward structural variation of the side groups. These m-PE oligomers have provided a new approach for achieving folded unnatural oligomers under conditions that are otherwise unfavorable for previously described, solvent-driven folding of m-PE foldamers. Stably folded structures based on the design principle described here can be developed and may find important applications.     

Phenanthroline dicarboxamide-based helical foldamers: stable helical structures in methanol

A series of new aromatic oligoamides 2-5 based on 1,10-phenanthroline diacid and o-phenylenediamine have been synthesized through a convergent segment coupling strategy. These oligomers can fold into well-defined helical structures in solution through intramolecular hydrogen bonds and aromatic stacking interactions, which has been established by 1H NMR, fluorescence, and UV/vis spectra. In particular, it was found that the oligomers were more favorable to fold into stable helical structures in methanol than in chloroform and dichloromethane. The helical foldamers formed in the solid state have been characterized by single-crystal X-ray diffraction analysis. The results showed that the high curvature of the strands led to one and a half turns for both 2 and 21, three turns for 4, and nearly four turns for 5.     

Helical Folding of Conjugated Oligo(phenyleneethynylene): Chain‐Length Dependence,Solvent Effects,and Intermolecular Assembly

As a representative folding system that features a conjugated backbone, a series of monodispersed (o‐phenyleneethynylene)‐alt‐(p‐phenyleneethynylene) (PE) oligomers of varied chain length and different side chains were studied. Molecules with the same backbone but different side‐chain structures were shown to exhibit similar helical conformations in respectively suitable solvents. Specifically, oligomers with dodecyloxy side chains folded into the helical structure in apolar aliphatic solvents, whereas an analogous oligomer with tri(ethylene glycol) (Tg) side chains adopted the same conformation in polar solvents. The fact that the oligomers with the same backbone manifested a similar folded conformation independent of side chains and the nature of the solvent confirmed the concept that the driving force for folding was the intramolecular aromatic stacking and solvophobic interactions. Although all were capable of inducing folding, different solvents were shown to bestow slightly varied folding stability. The chain‐length dependence study revealed a nonlinear correlation between the folding stability with backbone chain length. A critical size of approximately 10 PE units was identified for the system, beyond which folding occurred. This observation corroborated the helical nature of the folded structure. Remarkably, based on the absorption and emission spectra, the effective conjugation length of the system extended more effectively under the folded state than under random conformations. Moreover, as evidenced by the optical spectra and dynamic light‐scattering studies, intermolecular association took place among the helical oligomers with Tg side chains in aqueous solution. The demonstrated ability of such a conjugated foldamer in self‐assembling into hierarchical supramolecular structures promises application potential for the system.     

Sequence-controlled oligomers fold into nanosolenoids and impart unusual optical properties

Controlled syntheses give unique block oligomers with alternating flexible ethylene glycol and rigid perylenetetracarboxylic diimide (PDI) units. The number of rigid units vary from n=1 to 10. PDI units were stitched together by using efficient phosphoramidite chemistry. The resulting oligomers undergo folding in most solvents, including chloroform. In their ground state, these folded oligomers were characterized by using Fourier transform ion cyclotron resonance mass spectrometry (FTICR‐MS), NMR spectroscopy, and electronic absorption spectroscopy. FTICR‐MS revealed the exact masses of these sequence‐controlled oligomers, which confirmed the chemical composition and validated the synthetic strategy. The NMR neighboring ring‐current effect (NRE) indicates the formation of cofacial π stacks; the stacked aromatic rings have nearly coaxial alignment akin to a nanosoleniod. Nanosolenoidal shielding in π stacks causes all aromatic protons to shift upfield, whereas NOE in a cyclic hetero‐chromophoric dimer supports a rotated, cofacial π‐stacking orientation separated by about 3.5 Å. Electron–phonon coupling is much stronger than excitonic coupling in these self‐folded PDI oligomers; thus, Franck–Condon factors dictate the observed spectral features in visible spectra. The absorbance spectrum exhibits weak hypochromism due to π stacking with increasing stacking units n. Finally, ab initio calculations support the experimental observations, indicating 3.5 Å cofacial spacing in which one molecule is rotated 30° from the eclipsed orientation and higher oligomers can adopt, without a compensating energy penalty, either the right/left‐handed helices or the 1,3‐eclipsed structures. Both theory and experiments validate the nano‐π‐solenoids and their novel photophysical properties.     

Cyclic modular beta-sheets

The development of peptide beta-hairpins is problematic, because folding depends on the amino acid sequence and changes to the sequence can significantly decrease folding. Robust beta-hairpins that can tolerate such changes are attractive tools for studying interactions involving protein beta-sheets and developing inhibitors of these interactions. This paper introduces a new class of peptide models of protein beta-sheets that addresses the problem of separating folding from the sequence. These model beta-sheets are macrocyclic peptides that fold in water to present a pentapeptide beta-strand along one edge; the other edge contains the tripeptide beta-strand mimic Hao [JACS 2000, 122, 7654] and two additional amino acids. The pentapeptide and Hao-containing peptide strands are connected by two delta-linked ornithine (deltaOrn) turns [JACS 2003, 125, 876]. Each deltaOrn turn contains a free alpha-amino group that permits the linking of individual modules to form divalent beta-sheets. These "cyclic modular beta-sheets" are synthesized by standard solid-phase peptide synthesis of a linear precursor followed by solution-phase cyclization. Eight cyclic modular beta-sheets 1a-1h containing sequences based on beta-amyloid and macrophage inflammatory protein 2 were synthesized and characterized by 1H NMR. Linked cyclic modular beta-sheet 2, which contains two modules of 1b, was also synthesized and characterized. 1H NMR studies show downfield alpha-proton chemical shifts, deltaOrn delta-proton magnetic anisotropy, and NOE cross-peaks that establish all compounds but 1c and 1g to be moderately or well folded into a conformation that resembles a beta-sheet. Pulsed-field gradient NMR diffusion experiments show little or no self-association at low (相似文献   

Improving foldamer synthesis through protecting group induced unfolding of aromatic oligoamides

The hydrogen bond rigidified backbones of aromatic oligoamides are temporarily interrupted by replacing the amide hydrogens with the acid-labile 2,4-dimethoxybenzyl (DMB) group, which allows the efficient preparation of long folding oligomers that, upon removal of the DMB groups, fold into multiturn helices. [structure: see text]     

Synthesis and circular dichroism spectroscopic investigations of oligomeric beta-peptoids with alpha-chiral side chains

Biomimetic oligomers are of large interest both as targets for combinatorial and parallel synthetic efforts and as foldamers. For example, shorter peptoid derivatives of beta-peptides, i.e., oligo-N-substituted beta-Ala, have been described as potential lead structures. Herein, we describe a solid-phase synthetic route to beta-peptoids with alpha-chiral aromatic N-substituents up to 11 residues long. Furthermore, the folding propensities of these oligomers were investigated by circular dichroism (CD) spectroscopy.     

Light‐Controlled Conformational Switch of an Aromatic Oligoamide Foldamer

An aromatic oligoamide sequence composed of a light‐responsive diazaanthracene‐based aromatic β‐sheet flanked by two variable diameter helical segments was prepared. Structural investigations revealed that such oligomers adopt two distinct conformations: a canonical symmetrical conformation with the two helices stacked above and below the sheet, and an unanticipated unsymmetrical conformation in which one helix has flipped to directly stack with the first helix. Photoirradiation of the foldamer led to the quantitative, and thermally reversible, formation of a single photoproduct resulting from the [4+4] cycloaddition of two diazaanthracenes within the aromatic β‐sheet. NMR and crystallographic studies revealed a parallel arrangement of the diazaanthracene photoproduct and a complete conversion into a symmetrical conformation requiring a rearrangement of all unsymmetrical conformers. These results highlight the potential of foldamers, with structures more complex than isolated helices, for the design of photoswitches showing nontrivial nanometer scale shape changes.     

Structural and spectroscopic studies of peptoid oligomers with alpha-chiral aliphatic side chains

Substantial progress has been made in the synthesis and characterization of various oligomeric molecules capable of autonomous folding to well-defined, repetitive secondary structures. It is now possible to investigate sequence-structure relationships and the driving forces for folding in these systems. Here, we present detailed analysis by X-ray crystallography, NMR, and circular dichroism (CD) of the helical structures formed by N-substituted glycine (or "peptoid") oligomers with alpha-chiral, aliphatic side chains. The X-ray crystal structure of a N-(1-cyclohexylethyl)glycine pentamer, the first reported for any peptoid, shows a helix with cis-amide bonds, approximately 3 residues per turn, and a pitch of approximately 6.7 A. The backbone dihedral angles of this pentamer are similar to those of a polyproline type I peptide helix, in agreement with prior modeling predictions. This crystal structure likely represents the major solution conformers, since the CD spectra of analogous peptoid hexamers, dodecamers, and pentadecamers, composed entirely of either (S)-N-(1-cyclohexylethyl)glycine or (S)-N-(sec-butyl)glycine monomers, also have features similar to those of the polyproline type I helix. Furthermore, this crystal structure is similar to a solution NMR structure previously described for a peptoid pentamer comprised of chiral, aromatic side chains, which suggests that peptoids containing either aromatic or aliphatic alpha-chiral side chains adopt fundamentally similar helical structures in solution, despite distinct CD spectra. The elucidation of detailed structural information for peptoid helices with alpha-chiral aliphatic side chains will facilitate the mimicry of biomolecules, such as transmembrane protein domains, in a distinctly stable form.     

Selection of G-quadruplex folding topology with LNA-modified human telomeric sequences in K+ solution

G-rich nucleic acid oligomers can form G-quadruplexes built by G-tetrads stacked upon each other. Depending on the nucleotide sequence, G-quadruplexes fold mainly with two topologies: parallel, in which all G-tracts are oriented parallel to each other, or antiparallel, in which one or more G-tracts are oriented antiparallel to the other G-tracts. In the former topology, all glycosidic bond angles conform to anti conformations, while in the latter topology they adopt both syn and anti conformations. It is of interest to understand the molecular forces that govern G-quadruplex folding. Here, we approach this problem by examining the impact of LNA (locked nucleic acid) modifications on the folding topology of the dimeric model system of the human telomere sequence. In solution, this DNA G-quadruplex forms a mixture of G-quadruplexes with antiparallel and parallel topologies. Using CD and NMR spectroscopies, we show that LNA incorporations can modulate this equilibrium in a rational manner and we establish a relationship between incorporation of LNA nucleotides in syn and/or anti positions and the shift of the equilibrium to obtain exclusively the parallel G-quadruplex. The change in topology is driven by a combination of the C3'-endo puckering of LNA nucleotides and their preference for the anti glycosidic conformation. In addition, the parallel LNA-modified G-quadruplexes are thermally stabilised by about 11 °C relative to their DNA counterparts.     

Helical aromatic oligoamides: reliable, readily predictable folding from the combination of rigidified structural motifs

Factors responsible for the folding of aromatic oligoamides with backbones rigidified by local three-center H-bonds were investigated. The stability of the three-center H-bonds was quantified by the half-lives of amide proton-deuterium exchange reactions, which show that the three-center H-bonds were largely intact at room temperature in the oligomer examined. This result is consistent with our current and previous 2D NMR studies. The overall helical conformation of nonamer 1 was found by variable-temperature NOESY studies to be dynamic. As temperature rose, the end-to-end NOEs rapidly disappeared, while the amide side chain NOEs were still readily detectable, corresponding to the "breath" and stretching of the helix by slightly twisting the local H-bonded rings. Based on the simple repetition of the same structural motif and local conformational preference, undecamer 2 was found to fold into well-defined helical conformation. The predictability of the folding of these backbone-rigidified aromatic oligoamides was demonstrated by a simple modeling method using structural parameters from oligomers with known crystal structures. The reliability and generality of the modeling methods were shown by the excellent agreement between the modeled structures corresponding to 1 and 2 and data from NOESY studies.     

Expanding the realm of ultrafast protein folding: gpW, a midsize natural single-domain with alpha+beta topology that folds downhill

All ultrafast folding proteins known to date are either very small in size (less than 45 residues), have an alpha-helix bundle topology, or have been artificially engineered. In fact, many of them share two or even all three features. Here we show that gpW, a natural 62-residue alpha+beta protein expected to fold slowly in a two-state fashion, folds in microseconds (i.e., from tau = 33 micros at 310 K to tau = 1.7 micros at 355 K). Thermodynamic analyses of gpW reveal probe dependent thermal denaturation, complex coupling between two denaturing agents, and differential scanning calorimetry (DSC) thermogram characteristic of folding over a negligible thermodynamic folding barrier. The free energy surface analysis of gpW folding kinetics also produces a marginal folding barrier of about thermal energy ( RT) at the denaturation midpoint. From these results we conclude that gpW folds in the downhill regime and is close to the global downhill limit. This protein seems to be poised toward downhill folding by a loosely packed hydrophobic core with low aromatic content, large stabilizing contributions from local interactions, and abundance of positive charges on the native surface. These special features, together with a complex functional role in bacteriophage lambda assembly, suggest that gpW has been engineered to fold downhill by natural selection.     

典型蛋白质折叠中阳离子-π相互作用的特异性

蛋白质中的阳离子-π相互作用是带正电荷的氨基酸(Lys、Arg)和芳香族氨基酸(Phe、Tyr、Trp)之间的一种作用力.对α/β类蛋白中两种典型折叠类型(单绕和双绕)的研究表明:(1)单绕结构中阳离子-π相互作用的分布密度大约是双绕结构中的2.6倍;(2)在单绕结构中,样本所含氨基酸残基数量与样本中阳离子-π的数量有明显的相关性,在双绕结构中没有发现类似的相关性;(3)Lys、Arg与Tyr在单绕中比在双绕中更容易形成阳离子-π相互作用;(4)Arg-Tyr组合在单绕中出现的几率较大,Arg-Phe组合在双绕中出现的几率较大;(5)阳离子-π相互作用在65%的单绕样本中形成阵列或分布在结构的首尾间.     

Conformational Modularity of an Abiotic Secondary‐Structure Motif in Aqueous Solution

We have investigated an abiotic secondary structure based on the stacking of alternating electron‐rich (1,5‐dialkoxynaphthalene (Dan)) and electron‐deficient (1,4,5,8‐naphthalene‐tetracarboxylic diimide (Ndi)=benzo[lmn][3,8]phenanthroline‐1,3,6,8(2H,7H)‐tetrone) aromatic units. Previously, the specifics of conformational behavior were uncovered in the minimal folding unit, namely the dimer, consisting of one Dan and one Ndi unit linked through various amino acid residues. Here is reported the investigation of a series of larger oligomers (trimers and tetramers) composed of selected dimer units. We determined that some of the larger oligomers displayed conformational modularity, that is, the persistence of subunit‐conformational propensities when those subunits were used as components of larger structures. Conformational modularity can be viewed as a desirable property of folding molecules because it simplifies not only the design of larger, more complex oligomers, but also the structural analyses of such species.     

Folding a nonbiological polymer into a compact multihelical structure

The only molecules that are currently known to fold into unique three-dimensional conformations and perform sophisticated functions are biological polymers - proteins and some RNA molecules. Our aim is to create a nonbiological sequence-specific polymer that folds in aqueous solution. Toward that end, we synthesized sequence-specific 30mer, 45mer, and 60mer peptoid oligomers (N-substituted glycine polymers) consisting of 15mer units we chained together by disulfide and oxime linkages to mimic the helical bundle structures commonly found in proteins. Because these 15mer sequences were previously shown to form defined helical structures that aggregate together at submillimolar concentrations, we expected that by covalently linking multiple 15mers together, they might fold as helical bundles. To probe whether they folded, we used fluorescence resonance energy transfer (FRET) reporter groups. We found that certain constructs fold up with a hydrophobic core and have cooperative folding transitions. Such molecules may ultimately provide a platform for designing specific functions resembling those of proteins.     

Generalizing the Aromatic δ-Amino Acid Foldamer Helix

A series of aromatic oligoamide foldamer sequences containing different proportions of three δ-amino acids derived from quinoline, pyridine, and benzene and possessing varying flexibility, for example due to methylene bridges, were synthesized. Crystallographic structures of two key sequences and ¹H NMR data in water concur to show that a canonical aromatic helix fold prevails in almost all cases and that helix stability critically depends on the ratio between rigid and flexible units. Notwithstanding subtle variations of curvature, i. e. the numbers of units per turn, the aromatic δ-peptide helix is therefore shown to be general and tolerant of a great number of sp³ centers. We also demonstrate canonical helical folding upon alternating two monomers that do not promote folding when taken separately: folding occurs with two methylenes between every other unit, not with one methylene between every unit. These findings highlight that a fine-tuning of helix handedness inversion kinetics, curvature, and side chain positioning in aromatic δ-peptidic foldamers can be realized by systematically combining different yet compatible δ-amino acids.     

Actinomycin D binds to single stranded DNA oligomers which contain double GTC triplets

The present work carried out a study on the interactions between Actinomycin D (ActD) and some single-stranded DNA oligomers, which contain double GTC triplets separated by TTT sequence. The interactions of drugs with DNA oligomers were investigated by UV, circular dichroism (CD) spectroscopy and isothermal titration calorimetry (ITC). The results indicate that ActD binds to the single stranded DNA oligomers in the fold back binding model as supported by added A/T base at DNA strand terminal which facilitates the formation of hairpin. The apparent binding constant K_b, the apparent binding molar enthalpy ΔH⁰ and other thermodynamic data were obtained. The binding affinities are sequence dependent and related to the length of DNA strand. And the higher molar binding enthalpy indicates that the binding process is enthalpy driven.