Incorporating beta-turns and a turn mimetic out of context in loop 1 of the WW domain affords cooperatively folded beta-sheets.

To probe the conformational requirements of loop 1 in the Pin1 WW domain, the residues at the i + 2 and i + 3 positions of a beta-turn within this loop were replaced by dPro-Gly and Asn-Gly, which are known to prefer the conformations required at the i + 1 and i + 2 positions of type II' and type I' beta-turns. Conformational specificity or lack thereof was further examined by incorporating into the i + 2 and i + 3 positions a non-alpha-amino acid-based beta-turn mimetic (4-(2'-aminoethyl)-6-dibenzofuran propionic acid residue, 1), which was designed to replace the i + 1 and i + 2 positions of beta-turns. All these Pin WW variants are monomeric and folded as discerned by analytical ultracentrifugation, NMR, and CD. They exhibit cooperative two-state transitions and display thermodynamic stability within 0.5 kcal/mol of the wild-type WW domain, demonstrating that the acquisition of native structure and stability does not require a specific sequence and, by extension, conformation within loop 1. However, it could be that these loop 1 mutations alter the kinetics of antiparallel beta-sheet folding, which will be addressed by subsequent kinetic studies.     

Folding pathways of human telomeric type-1 and type-2 G-quadruplex structures

We have investigated new folding pathways of human telomeric type-1 and type-2 G-quadruplex conformations via intermediate hairpin and triplex structures. The stabilization energies calculated by ab initio methods evidenced the formation of a hairpin structure with Hoogsteen GG base pairs. Further calculations revealed that the G-triplet is more stable than the hairpin conformation and equally stable when compared to the G-tetrad. This indicated the possibility of a triplex intermediate. The overall folding is facilitated by K(+) association in each step, as it decreases the electrostatic repulsion. The K(+) binding site was identified by molecular dynamics simulations. We then focused on the syn/anti arrangement and found that the anti conformation of deoxyguanosine is more stable than the syn conformation, which indicated that folding would increase the number of anti conformations. The K(+) binding to a hairpin near the second lateral TTA loop was found to be preferable, considering entropic effects. Stacking of G-tetrads with the same conformation (anti/anti or syn/syn) is more stable than mixed stacking (anti/syn and vice versa). These results suggest the formation of type-1 and type-2 G-quadruplex structures with the possibility of hairpin and triplex intermediates.     

Folding mechanisms of individual beta-hairpins in a Go model of Pin1 WW domain by all-atom molecular dynamics simulations

This paper examines the folding mechanism of an individual beta-hairpin in the presence of other hairpins by using an off-lattice model of a small triple-stranded antiparallel beta-sheet protein, Pin1 WW domain. The turn zipper model and the hydrophobic collapse model originally developed for a single beta-hairpin in literature is confirmed to be useful in describing beta-hairpins in model Pin1 WW domain. We find that the mechanism for folding a specific hairpin is independent of whether it folds first or second, but the formation process are significantly dependent on temperature. More specifically, beta1-beta2 hairpin folds via the turn zipper model at a low temperature and the hydrophobic collapse model at a high temperature, while the folding of beta2-beta3 hairpin follows the turn zipper model at both temperatures. The change in folding mechanisms is interpreted by the interplay between contact stability (enthalpy) and loop lengths (entropy), the effect of which is temperature dependent.     

RNA architecture dictates the conformations of a bound peptide.

BACKGROUND: The biological function of several viral and bacteriophage proteins, and their arginine-rich subdomains, involves RNA-mediated interactions. It has been shown recently that bound peptides adopt either beta-hairpin or alpha-helical conformations in viral and phage peptide-RNA complexes. We have compared the structures of the arginine-rich peptide domain of HIV-1 Rev bound to two RNA aptamers to determine whether RNA architecture can dictate the conformations of a bound peptide. RESULTS: The core-binding segment of the HIV-1 Rev peptide class II RNA aptamer complex spans the two-base bulge and hairpin loop of the bound RNA and the carboxy-terminal segment of the bound peptide. The bound peptide is anchored in place by backbone and sidechain intermolecular hydrogen bonding and van der Waals stacking interactions. One of the bulge bases participates in U*(A*U) base triple formation, whereas the other is looped out and flaps over the bound peptide in the complex. The seven-residue hairpin loop is closed by a sheared G*A mismatch pair with several pyrimidines looped out of the hairpin fold. CONCLUSIONS: Our structural studies establish that RNA architecture dictates whether the same HIV-1 Rev peptide folds into an extended or alpha-helical conformation on complex formation. Arginine-rich peptides can therefore adapt distinct secondary folds to complement the tertiary folds of their RNA targets. This contrasts with protein-RNA complexes in which elements of RNA secondary structure adapt to fit within the tertiary folds of their protein targets.     

The shape-shifting quasispecies of RNA: one sequence, many functional folds

E Unus pluribum, or "Of One, Many", may be at the root of decoding the RNA sequence-structure-function relationship. RNAs embody the large majority of genes in higher eukaryotes and fold in a sequence-directed fashion into three-dimensional structures that perform functions conserved across all cellular life forms, ranging from regulating to executing gene expression. While it is the most important determinant of the RNA structure, the nucleotide sequence is generally not sufficient to specify a unique set of secondary and tertiary interactions due to the highly frustrated nature of RNA folding. This frustration results in folding heterogeneity, a common phenomenon wherein a chemically homogeneous population of RNA molecules folds into multiple stable structures. Often, these alternative conformations constitute misfolds, lacking the biological activity of the natively folded RNA. Intriguingly, a number of RNAs have recently been described as capable of adopting multiple distinct conformations that all perform, or contribute to, the same function. Characteristically, these conformations interconvert slowly on the experimental timescale, suggesting that they should be regarded as distinct native states. We discuss how rugged folding free energy landscapes give rise to multiple native states in the Tetrahymena Group I intron ribozyme, hairpin ribozyme, sarcin-ricin loop, ribosome, and an in vitro selected aptamer. We further describe the varying degrees to which folding heterogeneity impacts function in these RNAs, and compare and contrast this impact with that of heterogeneities found in protein folding. Embracing that one sequence can give rise to multiple native folds, we hypothesize that this phenomenon imparts adaptive advantages on any functionally evolving RNA quasispecies.     

Folding of small proteins using a single continuous potential

Extensive Monte Carlo folding simulations for four proteins of various structural classes are carried out, using a single continuous potential (united-residue force field). In all cases, collapse occurs at a very early stage, and proteins fold into their nativelike conformations at appropriate temperatures. We also observe that glassy transitions occur at low temperatures. The simulation results demonstrate that the folding mechanism is controlled not only by thermodynamic factors but also by kinetic factors: The way a protein folds into its native structure is also determined by the convergence point of early folding trajectories, which cannot be obtained by the free energy surface.     

Construction of double-stranded metallosupramolecular polymers with a controlled helicity by combination of salt bridges and metal coordination

We describe the construction of the first double-stranded metallosupramolecular helical polymers. We designed and synthesized a supramolecular duplex comprised of complementary m-terphenyl-based strands bearing a chiral amidine or achiral carboxylic acid together with two pyridine groups at the four ends. Supramolecular polymerization of the duplex with cis-PtPh2(DMSO)2 in 1,1,2,2-tetrachloroethane produced the double-stranded metallosupramolecular polymer with a controlled helicity of which the two complementary metallostrands are intertwined through the amidinium-carboxylate salt bridges. The structures and hydrodynamic dimensions of the metallosupramolecular polymers were characterized by 1H NMR, diffusion-ordered NMR, dynamic light scattering, absorption, and CD measurements. The polymeric structure was also visualized by atomic force microscopy.     

alpha-L-LNA (alpha-L-ribo configured locked nucleic acid) recognition of DNA: an NMR spectroscopic study

We have used NMR and CD spectroscopy to study and characterise two alpha-L-LNA:DNA duplexes, a nonamer that incorporates three alpha-L-LNA nucleotides and a decamer that incorporates four alpha-L-LNA nucleotides, in which alpha-L-LNA is alpha-L-ribo-configured locked nucleic acid. Both duplexes adopt right-handed helical conformations and form normal Watson-Crick base pairing with all nucleobases in the anti conformation. Deoxyribose conformations were determined from measurements of scalar coupling constants in the sugar rings, and for the decamer duplex, distance information was derived from 1H-1H NOE measurements. In general, the deoxyriboses in both of the alpha-L-LNA:DNA duplexes adopt S-type (B-type structure) sugar puckers, that is the inclusion of the modified alpha-L-LNA nucleotides does not perturb the local native B-like double-stranded DNA (dsDNA) structure. The CD spectra of the duplexes confirm these findings, as these display B-type characteristic features that allow us to characterise the overall duplex type as B-like. The 1H-1H NOE distances which were determined for the decamer duplex were employed in a simulated annealing protocol to generate a model structure for this duplex, thus allowing a more detailed inspection of the impact of the alpha-L-ribo-configured nucleotides. In this structure, it is evident that the malleable DNA backbone rearranges in the vicinity of the modified nucleotides in order to accommodate them and present their nucleobases in a geometry suitable for Watson-Crick base pairing.     

温控分子动力学研究微管蛋白活性肽链的折叠机制

运用温控和常温分子动力学方法, 研究了微管蛋白活性部位Pep1-28肽链的折叠机制, 总模拟时间为380.0 ns. 对于温控分子动力学, 逐渐降温可以清晰显示Pep1-28肽链的折叠途径, 发生明显折叠的温度约为550 K, 其折叠和去折叠可逆机制为U(>1200 K)←→I1(1200-1000 K)←→I2(800 K)←→I3(600 K)←→I4(450 K)←→F1(400 K)←→F2(300 K), 其中U为去折叠态构象, I1、I2、I3和I4是折叠过程中的四个重要的中间态构象, F1和F2是两个结构相近的折叠态构象. 对于常温(300 K)分子动力学, 其构象转变和折叠过程相当迅速, 很难观察到有效、稳定的中间态构象. 尤其引人注意的是, 其折叠态结构陷入了能量局域极小点, 与温控(300 K)的有较大差异, 两者能量差高达297.53 kJ·mol-1. 可见, 温控分子动力学方法不仅清晰地显示多肽和蛋白质折叠过程的重要中间态构象, 为折叠和去折叠机制提供直接、可靠的依据, 而且还有助于跨越较高的构象能垒, 促使多肽和蛋白质折叠以形成全局能量最低的稳定结构.     

Conformational analysis: crystallographic,NMR, and molecular mechanics studies of flexible sulfonic esters

Two novel X-ray structures of the sulfonic ester derivatives 2-(6-iodo-1,3-benzodioxol-5-yl)ethyl 4-nitrobenzenesulfonate, 3, and 2-(6-iodo-1,3-benzodioxol-5-yl)ethyl 4-methylbenzenesulfonate, 4, have been obtained in a study aimed at analyzing the structures and conformations of sulfonic ester derivatives that are routinely used in alkaloid syntheses. The crystal structure of 4 is highly unusual, containing four independent molecules that belong to two distinct conformational types: (1) a hairpin conformation (stabilized mainly by intramolecular pi-stacking) and (2) a stepped conformation (stabilized mainly by intermolecular pi-stacking). Compound 3, on the other hand, crystallizes exclusively as the hairpin conformer. New MM+ force field parameters for sulfonic esters have been developed using the X-ray data, empirical rules, and DFT calculations to estimate the bond dipole parameters. Grid searches of conformational space for 3 and 4 using MM methods show that there are several gas-phase conformations within 5 kcal/mol of the global minimum and that the lowest energy conformations (by approximately 4.6 kcal/mol) are of the hairpin type. Analysis of the MM conformational energies suggests that the dominant intramolecular interaction stabilizing the hairpin conformations of 3 and 4 is van der Waals attraction. Moreover, the lattice energies for packing the hairpin conformations of 3 and 4 are approximately 4 kcal/mol more favorable than for the stepped conformations. Various intermolecular interactions contribute to the complexity of the observed crystal structures of 3 and 4, including electrostatic attraction between O and I atoms in neighboring molecules. Langevin dynamics (LD) simulations at several temperatures (6.0 ns, friction coefficient = 2.5 ps(-1)) indicate that the conformational exchange rates are approximately 10(10)-10(11) s(-1) over the temperature range 213-400 K, accounting for the temperature-independent (1)H NMR spectra of 3 and 4.     

Measurement of the effect of monovalent cations on RNA hairpin stability

Using optical tweezers, we have measured the effect of monovalent cation concentration and species on the folding free energy of five large (49-124 nt) RNA hairpins, including HIV-1 TAR and molecules approximating A.U and G.C homopolymers. RNA secondary structure thermodynamics are accurately described by a model consisting of nearest-neighbor interactions and additive loop and bulge terms. Melting of small (<15 bp) duplexes and hairpins in 1 M NaCl has been used to determine the parameters of this model, which is now used extensively to predict structure and folding dynamics. Few systematic measurements have been made in other ionic conditions or for larger structures. By applying mechanical force, we measured the work required to fold and unfold single hairpins at room temperature over a range of cation concentrations from 50 to 1000 mM. Free energies were then determined using the Crooks fluctuation theorem. We observed the following: (1) In most cases, the nearest-neighbor model accurately predicted the free energy of folding at 1 M NaCl. (2) Free energy was proportional to the logarithm of salt concentration. (3) Substituting potassium ions for sodium slightly decreased hairpin stability. The TAR hairpin also misfolded nearly twice as often in KCl, indicating a differential kinetic response. (4) Monovalent cation concentration affects RNA stability in a sequence-dependent manner. G.C helices were unaffected by changing salt concentration, A.U helices were modestly affected, and the hairpin loop was very sensitive. Surprisingly, the U.C.U bulge of TAR was found to be equally stable in all conditions tested. We also report a new estimate for the elastic parameters of single-stranded RNA.     

NMR solution structure of an oligonucleotide hairpin with a 2'F-ANA/RNA stem: implications for RNase H specificity toward DNA/RNA hybrid duplexes.

The first structure of a 2'-deoxy-2'-fluoro-D-arabinose nucleic acid (2'F-ANA)/RNA duplex is presented. We report the structural characterization by NMR spectroscopy of a small hybrid hairpin, r(GGAC)d(TTCG)2'F-a(GTCC), containing a 2'F-ANA/RNA stem and a four-residue DNA loop. Complete (1)H, (13)C, (19)F, and (31)P resonance assignments, scalar coupling constants, and NOE constraints were obtained from homonuclear and heteronuclear 2D spectra. In the chimeric duplex, the RNA strand adopts a classic A-form structure having C3' endo sugar puckers. The 2'F-ANA strand is neither A-form nor B-form and contains O4' endo sugar puckers. This contrasts strongly with the dynamic sugar conformations previously observed in the DNA strands of DNA/RNA hybrid duplexes. Structural parameters for the duplex, such as minor groove width, x-displacement, and inclination, were intermediate between those of A-form and B-form duplexes and similar to those of DNA/RNA duplexes. These results rationalize the enhanced stability of 2'F-ANA/RNA duplexes and their ability to elicit RNase H activity. The results are relevant for the design of new antisense drugs based on sugar-modified nucleic acids.     

Site-specific relaxation kinetics of a tryptophan zipper hairpin peptide using temperature-jump IR spectroscopy and isotopic labeling

Two antiparallel beta-strands connected by a turn make beta-hairpins an ideal model system to analyze the interactions and dynamics of beta-sheets. Site-specific conformational dynamics were studied by temperature-jump IR spectroscopy and isotopic labeling in a model based on the tryptophan zipper peptide, Trpzip2, developed by Cochran et al. (Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 5578). The modified Trpzip2C peptides have nearly identical equilibrium spectral behavior as Trpzip2 showing that they also form well-characterized beta-hairpin conformations in aqueous solution. Selective introduction of 13C=O groups on opposite strands lead to distinguishable cross-strand coupling of the labeled residues as monitored in the amide I' band. These frequency patterns reflect theoretical predictions, and the coupled 13C=O band loses intensity with increase in temperature and unfolding of the hairpin. Thermal relaxation kinetics were analyzed for unlabeled and cross-strand isotopically labeled variants. T-jumps of approximately 10 degrees C induce relaxation times of a few microseconds that decrease with increase of the peptide temperature. Differences in kinetic behavior for the loss of beta-strand and gain of disordered structure can be used to distinguish localized structure dynamics by comparison of nonlabeled and labeled amide I' components. Analysis of the data supports multistate dynamic and equilibrium behavior, but because of this process it is not possible to clearly define a folding and unfolding rate. Nonetheless, site-specific relaxation kinetics could be seen to be consistent with a hydrophobic collapse hypothesis for hairpin folding.     

Effects of alternative side chain pairings and reverse turn sequences on antiparallel sheet structure in beta-peptide hairpins

[structure: see text] We describe a series of beta-peptide hexamers that allow us to explore relationships between sequence and hairpin folding. Different reverse turn segments are compared at the central two positions, and the outer residues allow a variety of interstrand side chain-side chain pairings. NMR analysis in methanol demonstrates that several reverse turn and side chain pairing arrangements are compatible with antiparallel beta-peptide sheet structure; however, none of the beta-peptides folds in water.     

Combinatorial Biomimetic Chemistry: Parallel Synthesis of a Small Library of β‐Hairpin Mimetics Based on Loop III from Human Platelet‐Derived Growth Factor B

Combinatorial diversity in hypervariable β‐hairpin loops is exploited by the immune system to select binding sites on antibodies for a wide variety of different protein antigens. In a first step towards mimicking this strategy in vitro, for the selection of novel protein ligands, an approach is described here for the parallel synthesis of small libraries of conformationally defined β‐hairpin protein epitope mimetics. Starting from a protruding hairpin loop in platelet‐derived growth factor B (PDGF‐B), 8 and 12 residues were first transplanted from the protein to a D ‐Pro‐L ‐Pro template, to afford the cyclic peptide‐loop mimetics 1 and 2 , respectively. NMR and MD studies in aqueous solution show that both mimetics populate conformations which closely mimic the β‐hairpin in the crystal structure of the native protein (Fig. 5). Based on 1 as a scaffold, a library of 24 mimetics was synthesized in which the four residues at the tip of the loop (VRKK) were held constant, and flanking residues at positions 1, 2, 7, and 8 in the hairpin were varied (Fig. 7). The library was prepared by parallel synthesis in a two‐stage solid‐phase assembly/solution‐phase cyclization process. The products were analyzed by MS, NMR, and CD. 2D‐NOESY revealed for most library members characteristic long‐range NOEs that show that the hairpin conformation is stably maintained. The results suggest that this approach may be useful for the synthesis of much larger libraries of peptide and protein mimetics based on a β‐hairpin scaffold.     

Length dependent folding kinetics of phenylacetylene oligomers: structural characterization of a kinetic trap

Using simulation to study the folding kinetics of 20-mer poly-phenylacetylene (pPA) oligomers, we find a long time scale trapped kinetic phase in the cumulative folding time distribution. This is demonstrated using molecular dynamics to simulate an ensemble of over 100 folding trajectories. The simulation data are fit to a four-state kinetic model which includes the typical folded and unfolded states, along with an intermediate state, and most surprisingly, a kinetically trapped state. Topologically diverse conformations reminiscent of alpha helices, beta turns, and sheets in proteins are observed, along with unique structures in the form of knots. The nonhelical conformations are implicated, on the basis of structural correlations to kinetic parameters, to contribute to the trapped kinetic behavior. The strong solvophobic forces which mediate the folding process and produce a stable helical folded state also serve to overstabilize the nonhelical conformations, ultimately trapping them. From our simulations, the folding time is predicted to be on the order of 2.5-12.5 mus in the presence of the trapped kinetic phase. The folding mechanism for these 20-mer chains is compared with the previously reported folding mechanism for the pPA 12-mer chains. A linear scaling relationship between the chain length and the mean first passage time is predicted in the absence of the trapped kinetic phase. We discuss the major implications of this discovery in the design of self-assembling nanostructures.     

Simulation of the pressure and temperature folding/unfolding equilibrium of a small RNA hairpin

We report molecular dynamics simulations of the equilibrium folding/unfolding thermodynamics of the RNA tetraloop in explicit solvent. A replica exchange molecular dynamics study of the r(CGUUGCCG) oligomer that forms a hairpin is performed for 226 ns per replica, using 52 replicas. We are able to show the unbiased folding of all replicas starting from extended conformations. The equilibrium pressure-temperature free energy of folding, DeltaG(P,T), is calculated from the averaged energy, pressure, and specific volume change upon folding of the oligomer as a function of T at constant volume. We find that this oligomer is destabilized by increasing hydrostatic pressure, similar to the behavior of globular proteins.     

Formation of (3+1) G-quadruplexes with a long loop by human telomeric DNA spanning five or more repeats

Structural studies of human telomeric repeats represent an active field of research with potential applications toward the development of specific telomeric quadruplex-targeting drugs for anticancer treatment. To date, high-definition structures were limited to DNA sequences containing up to four GGGTTA repeats. Here we investigate the formation of G-quadruplexes in sequences spanning five to seven human telomeric repeats using NMR, UV, and CD spectroscopy. A (3+1) G-quadruplex with a long propeller loop was isolated from a five-repeat sequence utilizing a guanine-to-inosine substitution. A simple approach of selective site-specific labeling of guanine residues was devised to rigorously determine the folding topology of the oligonucleotide. The same scaffold could be extrapolated to six- and seven-repeat sequences. Our results suggest that long human telomeric sequences consisting of five or more GGGTTA repeats could adopt (3+1) G-quadruplex structures harboring one or more repeat(s) within a single loop. We report on the formation of a Watson-Crick duplex within the long propeller loop upon addition of the complementary strand, demonstrating that the long loop could serve as a new recognition motif.     

Structure and electronic spectra of DNA mini-hairpins with G(n):C(n) stems

The solution structure of a synthetic DNA mini-hairpin possessing a stilbenediether linker and three G:C base pairs has been obtained using (1)H NMR spectral data and constrained torsion angle molecular dynamics. Notable features of this structure include a compact hairpin loop having a short stilbene-guanine plane-to-plane distance and approximate B-DNA geometry for the three base pairs. Comparison of the electronic spectra of mini-hairpins having one-to-four G:C base pairs and stilbenediether or hexamethyleneglycol linkers reveals the presence of features in the UV and CD spectra of the stilbene-linked hairpins that are not observed for the ethyleneglycol-linked hairpins. Investigation of the electronic structure of a stilbene-linked hairpin having a single G:C base pair by means of time-dependent density functional theory shows that the highest occupied molecular orbital, but not the lowest unoccupied molecular orbital, is delocalized over the stilbene and adjacent guanine. The calculated UV and CD spectra are highly dependent upon hairpin conformation, but reproduce the major features of the experimental spectra. These results illustrate the utility of an integrated experimental and theoretical approach to understanding the complex electronic spectra of pi-stacked chromophores.