Hairpin Conformation Research Articles

Helix and hairpin nucleation in short peptides using centrally positioned conformationally constrained dipeptide segments

The effect of incorporation of a centrally positioned Ac(6)c-Xxx segment where Xxx = (L)Val/(D)Val into a host oligopeptide composed of l-amino acid residues has been investigated. Studies of four designed octapeptides Boc-Leu-Phe-Val-Ac(6)c-Xxx-Leu-Phe-Val-OMe (Xxx = (D)Val 1, (L)Val 2) Boc-Leu-Val-Val-Ac(6)c-Xxx-Leu-Val-Val-OMe (Xxx = (D)Val 3, (L)Val 4) are reported. Diagnostic nuclear Overhouse effects characteristic of hairpin conformations are observed for Xxx = (D)Val peptides (1 and 3) while continuous helical conformation characterized by sequential N(i)H ↔ N(i+1)H NOEs are favored for Xxx = (L)Val peptides (2 and 4) in methanol solutions. Temperature co-efficient of NH chemical shifts are in agreement with distinctly different conformational preferences upon changing the configuration of the residue at position 5. Crystal structures of peptides 2 and 4 (Xxx = (L)Val) establish helical conformations in the solid state, in agreement with the structures deduced from NMR data. The results support the design principle that centrally positioned type I β-turns may be used to nucleate helices in short peptides, while type I'β-turns can facilitate folding into β-hairpins.

Structural characterization of the caveolin scaffolding domain in association with cholesterol-rich membranes.

Members of the caveolin protein family are implicated in the formation of caveolae and play important roles in a number of signaling pathways and in the regulation of various proteins. We employ complementary spectroscopic methods to study the structure of the caveolin scaffolding domain (CSD) in caveolin-1 fragments, while bound to cholesterol-rich membranes. This key domain is thought to be involved in multiple critical functions that include protein recognition, oligomerization, and cholesterol binding. In our membrane-bound peptides, residues within the flanking intramembrane domain (IMD) are found to adopt an α-helical structure, consistent with its commonly believed helical hairpin conformation. Intriguingly, in these same peptides, we observe a β-stranded conformation for residues in the CSD, contrasting with earlier reports, which commonly do not reflect β-structure. Our experimental data based on solid-state NMR, CD, and FTIR are found to be consistent with computational analyses of the secondary structure preference of the primary sequence. We discuss how our structural data of membrane binding Cav fragments may match certain general features of cholesterol-binding domains and could be consistent with the role for CSD in protein recognition and homo-oligomerization.

Structural insights into the neutralization mechanism of a higher primate antibody against dengue virus

The four serotypes of dengue virus (DENV-1 to -4) cause the most important emerging viral disease. Protein E, the principal viral envelope glycoprotein, mediates fusion of the viral and endosomal membranes during virus entry and is the target of neutralizing antibodies. However, the epitopes of strongly neutralizing human antibodies have not been described despite their importance to vaccine development. The chimpanzee Mab 5H2 potently neutralizes DENV-4 by binding to domain I of E. The crystal structure of Fab 5H2 bound to E from DENV-4 shows that antibody binding prevents formation of the fusogenic hairpin conformation of E, which together with in-vitro assays, demonstrates that 5H2 neutralizes by blocking membrane fusion in the endosome. Furthermore, we show that human sera from patients recovering from DENV-4 infection contain antibodies that bind to the 5H2 epitope region on domain I. This study, thus, provides new information and tools for effective vaccine design to prevent dengue disease.

X-ray structure of the arenavirus glycoprotein GP2 in its postfusion hairpin conformation

Arenaviruses are important agents of zoonotic disease worldwide. The virions expose a tripartite envelope glycoprotein complex at their surface, formed by the glycoprotein subunits GP1, GP2 and the stable signal peptide. This complex is responsible for binding to target cells and for the subsequent fusion of viral and host-cell membranes for entry. During this process, the acidic environment of the endosome triggers a fusogenic conformational change in the transmembrane GP2 subunit of the complex. We report here the crystal structure of the recombinant GP2 ectodomain of the lymphocytic choriomeningitis virus, the arenavirus type species, at 1.8-Å resolution. The structure shows the characteristic trimeric coiled coil present in class I viral fusion proteins, with a central stutter that allows a close structural alignment with most of the available structures of class I and III viral fusion proteins. The structure further shows a number of intrachain salt bridges stabilizing the postfusion hairpin conformation, one of which involves an aspartic acid that appears released from a critical interaction with the stable signal peptide upon low pH activation.

The domain I-domain III linker plays an important role in the fusogenic conformational change of the alphavirus membrane fusion protein.

The alphavirus Semliki Forest virus (SFV) infects cells through a low-pH-dependent membrane fusion reaction mediated by the virus fusion protein E1. Acidic pH initiates a series of E1 conformational changes that culminate in membrane fusion and include dissociation of the E1/E2 heterodimer, insertion of the E1 fusion loop into the target membrane, and refolding of E1 to a stable trimeric hairpin conformation. A highly conserved histidine (H3) on the E1 protein was previously shown to promote low-pH-dependent E1 refolding. An SFV mutant with an alanine substitution at this position (H3A) has a lower pH threshold and reduced efficiency of virus fusion and E1 trimer formation than wild-type SFV. Here we addressed the mechanism by which H3 promotes E1 refolding and membrane fusion. We identified E1 mutations that rescue the H3A defect. These revertants implicated a network of interactions that connect the domain I-domain III (DI-DIII) linker region with the E1 core trimer, including H3. In support of the importance of these interactions, mutation of residues in the network resulted in more acidic pH thresholds and reduced efficiencies of membrane fusion. In vitro studies of truncated E1 proteins demonstrated that the DI-DIII linker was required for production of a stable E1 core trimer on target membranes. Together, our results suggest a critical and previously unidentified role for the DI-DIII linker region during the low-pH-dependent refolding of E1 that drives membrane fusion.

Hairpin conformation of an 11-mer peptide

An 11-mer peptide taken from a subsequence of the human protein ubiquitin was synthesized. The peptide has been fully characterized by NMR spectroscopy using chemical shift analysis and by NOE measurements. The conformation was calculated using state of the art MD methods of protein chemistry. A hairpin conformation was found which is to a large part identical with the structure of this peptide fragment within the human ubiquitin. The surprising result that already an 11-mer peptide adopts a hairpin conformation in aqueous solution is discussed in terms of initials sites for protein folding.

Abstract 3888: Ionizing radiation destabilizes inverted ALU repeats linked to LOH

Abstract Ionizing radiation is a potent inducer of Loss of Heterozygosity (LOH), driven primarily by DNA double strand breaks. Such a process impacts both tumorigenesis through deletion of linked tumor suppressor genes and, similarly, the potential development of radiation resistant tumor clones during radiation therapy. The human mucoepidermoid cell line, H292, was exposed to either 4 or 8 Gy and surviving clones developed from two rounds of cloning analyzed for LOH using polymorphic microsatellite markers. A high level of LOH was observed within individual clones extending from 11q21 to 11q24 (6/49 after 4 Gy and 8/50 after 8Gy). More detailed analysis using a SNP screen on selected LOH positive clones indicated a sharp telomeric termination of LOH adjacent to a common SNP location. The telomeric extent of LOH observed co-mapped to a region of elevated recombination instability documented previously using linkage analysis. Further, SNP analysis showed a region of approximately 1 Mbp LOH that included the region of recombination instability identified through linkage analysis, in some clones isolated either with or without irradiation. The consistency of the telomeric boundary of LOH observed in the irradiated clones permitted a targeted analysis of DNA double strand breaks at this location, using LM-PCR, both before and after 4Gy irradiation of the same cell line. Using a six primer set screen covering a 9 kbp region at the telomeric extent of radiation-induced LOH, a single primer set showed an elevation in DNA breaks from 2-8h after irradiation. The timing and discrete distribution of such breaks indicated that these were not directly induced by the radiation itself. Cells that were unirradiated were not affected. Further inspection of the location demonstrating selective radiation-linked fragmentation showed the presence of an ALU inverted repeat, separated by approximately 1kbp, where the DNA breaks determined by LM-PCR were clustered within the 5’ ALU. Others have shown previously that inverted ALU repeats separated by <20bp are recombinationally unstable and are underrepresented within the human genome. It is proposed that, within the H292 cell line, otherwise stable ALU inverted repeats are capable of forming a transient hairpin conformation, perhaps facilitated by their own transcription via RNA polymerase III, which is unstable within a genome undergoing DNA damage and repair. The combination of such inverted ALU repeats and genotoxic exposure, such as during radiation therapy, may therefore destabilize specific regions of the genome and contribute to the development of aberrant cell clones. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3888. doi:10.1158/1538-7445.AM2011-3888

Electrochemical aptameric sensor based on the Klenow fragment polymerase reaction for cocaine detection

An electrochemical aptasensor based on Klenow fragment (KF) polymerase reaction that combines the aggregation of ferrocene-functionalized oligonucleotide has been developed successfully for cocaine detection. In the presence of cocaine, the recognition probe changed its hairpin conformation into the tripartite complex. The aptamer-cocaine complex gave a 3′-single-stranded tail sequence complementary to the surface-tethered capture probe. In KF polymerase reaction, the recognition probe served as a template for the extension of a capture probe. It requires a sample volume of 2 μL and is complete within 1 h. The ferrocene-appended oligonucleotide incorporated into the newly synthesized complementary probe leads to an electrochemical response. This sensitive detection of cocaine is due to a very low background signal and large signal enhancement up to 9-fold upon addition of analyte. It permits detection of as low as 200 μM cocaine. The simple and isothermal procedure does not require thermal cycling or special laboratory conditions, which makes it adaptable to low-cost and robust biosensing.

Effect of Pressure on the Stability of Short DNA Hairpins

DNA hairpins are models for more complex, naturally occurring loop-containing nucleic acid structures. They consist of two distinct structural domains: a double stranded stem and a single-stranded loop that connect the two strands of the stem. Previous studies of short DNA hairpins have revealed that the base sequence of the loop and stem significantly affect the relative thermodynamic stability of the hairpin and coil conformations. We have studied the effect of hydrostatic pressure on the heat-induced helix-coil transition of short DNA hairpins in order to gain knowledge about the role of hydration in the stability of the secondary structure adopted by these self-complementary DNA molecules. We report the effect of hydrostatic pressure on the helix-coil transition temperature (TM), monitored by absorption, for eleven 16-base DNA hairpins at different sodium chloride concentrations. The studied hairpins form by intramolecular folding of 16-base self-complementary DNA oligodeoxyribonucleotides. Each of the hairpins structures has a six-base pair duplex-forming stem linked by a four-base loop. We have varied only loop sequence and identity of the duplex base pair adjacent to the loop; the first four base pairs in the stem are the same for every molecule. Transition parameters based on a two-state denaturation process, such as ΔHvH and transition volume (ΔV), were calculated from the optical melting transitions. Experiments revealed the ΔV for denaturation of these molecules range from −2.35 to +6.74 cm3mol−1. We propose that the sequence dependence of the molar volume changes is attributable to differences in the hydration of these molecules.

Studying the Stabilizing Effects of the W-WTG Capping Motif for Short Beta Hairpins by Replica Exchange Molecular Dynamics

The folded/unfolded equilibrium and the stabilizing effects of the W-WTG capping of the short peptide Ac-WVSINGKKIWTG-NH2 were studied in explicit aqueous solution by a Replica Exchange Molecular Dynamics (REMD) simulation using the Amber99SB forcefield and TIP3P water model. This peptide contains the Trp-Trp capping motif for beta hairpins and sheets recently proposed by Andersen et al[1], and was reported remarkably stable experimentally. The replicas were started from an unbiased totally extended conformation and sampled the system for a total time of about 10 micro seconds at 40 different temperatures. Free energy landscapes of the system showed high preference for beta hairpin conformations at lower temperatures with populations greater than 80%, allowing us to postulate a native structure candidate for the system and to describe the thermodynamics of the system.This work has been supported by the National Science Foundation (Grant MCB-0543769).[1] Stabilizing capping motif for β-hairpins and sheets; Brandon L. Kier, Irene Shu, Lisa A. Eidenschink, and Niels H. Andersen; PNAS 2010 107 (23) 10466-10471;

Effect of Confinement on the Folding Dynamics of Amyloid-Beta (21-30) Protein: A Molecular Dynamics Study

Confinement is an ubiquitous concept in protein dynamics and studying its effect on protein stability and folding kinetics is necessary to understand protein folding in cellular environments. Confinement easily arises from the crowded in vivo environment that include other proteins and membranes, among others. Effects on the dynamics of the amyloid beta protein of Alzheimer's disease are know to be influenced by it proximity to lipid membranes and other proteins. Here we aim at understanding the relative effects of confinement on the dynamics and folding events of the amyloid beta protein fragment (21-30). For this, we use molecular dynamics in explicit solvent to explore the dynamics of this fragment in differently sized spherical confinement cavities. The different radiuses of the cavities are achieved by dividing the solvent into six different layers of immobile atoms. Starting from a simulation in bulk, each subsequent simulation considers one additional layer of confinement. In this way, the system is slowly taken from bulk to the more confined space. Specifically, starting from a size of 24A we decrease the radius of the confining spherical cavity to a minimum size of 14 A. We measure the change in the free energy of the stretched conformation of the amyloid beta (21-30) by varying the confined cavity size. In addition, we study how this confinement affects the stability of pre-formed beta-hairpin conformations by examining possible conformational changes as a function of time along with the lifetimes of these beta hairpin conformations relative to lifetimes observed in the bulk. Lastly, we present results on mutations of the amyloid beta peptide (21-30) that include the Dutch [Glu22Gln], Arctic [Glu22Gly] and Iowa [Asp23Asn] mutations.

Insights into the mechanism of HIV-1 envelope induced membrane fusion as revealed by its inhibitory peptides

HIV-1 fusion with its target cells is mediated by the glycoprotein 41 (gp41) transmembrane subunit of the viral envelope glycoprotein (ENV). The current models propose that gp41 undergoes several conformational changes between the apposing viral and cell membranes to facilitate fusion. In this review we focus on the progress that has been made in revealing the dynamic role of the N-terminal heptad repeat (NHR) and the C-terminal heptad repeat (CHR) regions within gp41 to the fusion process. The involvement of these regions in the formation of the gp41 pre-hairpin and hairpin conformations during an ongoing fusion event was mainly discovered by their derived inhibitory peptides. For example, the core structure within the hairpin conformation in a dynamic fusion event is suggested to be larger than its high resolution structure and its minimal boundaries were determined in situ. Also, inhibitory peptides helped reveal the dual contribution of the NHR to the fusion process. Finally, we will also discuss several developments in peptide design that has led to a deeper understanding of the mechanism of viral membrane fusion.

Structure, Interactions, and Antibacterial Activities of MSI-594 Derived Mutant Peptide MSI-594F5A in Lipopolysaccharide Micelles: Role of the Helical Hairpin Conformation in Outer-Membrane Permeabilization

Lipopolysaccharide (LPS) provides a well-organized permeability barrier at the outer membrane of Gram-negative bacteria. Host defense cationic antimicrobial peptides (AMPs) need to disrupt the outer membrane before gaining access to the inner cytoplasmic membrane or intracellular targets. Several AMPs are largely inactive against Gram-negative pathogens due to the restricted permeation through the LPS layer of the outer membrane. MSI-594 (GIGKFLKKAKKGIGAVLKVLTTG) is a highly active AMP with a broad-spectrum of activities against bacteria, fungi, and virus. In the context of LPS, MSI-594 assumes a hairpin helical structure dictated by packing interactions between two helical segments. Residue Phe5 of MSI-594 has been found to be engaged in important interhelical interactions. In order to understand plausible structural and functional inter-relationship of the helical hairpin structure of MSI-594 with outer membrane permeabilization, a mutant peptide, termed MSI-594F5A, containing a replacement of Phe5 with Ala has been prepared. We have compared antibacterial activities, outer and inner membrane permeabilizations, LPS binding affinity, perturbation of LPS micelles structures by MSI-594 and MSI-594F5A peptides. Our results demonstrated that the MSI-594F5A has lower activities against Gram-negative bacteria, due to limited permeabilization through the LPS layer, however, retains Gram-positive activity, akin to MSI-594. The atomic-resolution structure of MSI-594F5A has been determined in LPS micelles by NMR spectroscopy showing an amphipathic curved helix without any packing interactions. The 3D structures, interactions, and activities of MSI-594 and its mutant MSI-594F5A in LPS provide important mechanistic insights toward the requirements of LPS specific conformations and outer membrane permeabilization by broad-spectrum antimicrobial peptides.

Effects of pH and Temperature on the Structural and Thermodynamic Character of a-syn12 Peptide in Aqueous Solution

The structural and thermodynamic characters of α-syn12 peptide in aqueous solution at different pH and temperatures have been investigated through temperature replica exchange molecular dynamics (T-REMD) simulations with GROMOS 43A1 force field. The two independent T-REMD simulations were completed at pH = 7.0 and 10.0, respectively. Each replica was run for 300 ns. The structural and thermodynamic characters of α-syn12 peptide were studied based on the distributions of backbone dihedral angles, the free energy surface, and the stability of different type structure and the favorite conformations of the peptide. The results showed that the simulation at pH = 10.0 produced more sampling in α region than the simulation at pH = 7.0. The temperature changes from 283 K to 308 K result in negligible effects on the distributions of backbone dihedral angle. The β hairpin conformation with Turn9-6 and four hydrogen bonds (HB4-11, HB6-9, HB9-6 and HB11-4) is the lowest free energy state in the simulation at pH = 7.0. However, for the simulation at pH = 10.0, the lowest free energy state corresponds to a structure with Turn9-6 and two hydrogen bonds (HB6-10 and HB10-6) induced by an overly strong residue-residue interaction effect between lysine residues. For the simulation at pH = 7.0, the free energy change of the α-syn12 peptide from the unfolded state to the β hairpin state was in good agreement with the experiments and molecular dynamics simulation results for the other β-peptides, the β hairpin state of the α-syn12 peptide included the conformations that not only the Turn9-6 is formed, but also the terminus are closed together in space. However, the subtle balances between lysine-lysine interactions and lysine-solvent interaction are disrupted in the simulation at pH = 10.0, which induced the assembly of lysine residues, the β hairpin conformation is destabilized by the deprotonation of the Lys side chain. This study can help us to understand the conformation changes and the thermodynamic character of α-syn12 peptide at atomic level induced by changing pH and temperature, which is propitious to reveal the nosogenesis of Parkinson disease. In our knowledge, this is the first report to study the influence of pH and temperature on isolated α-syn12 peptide in water by T-REMD.

Conformational study of bovine lactoferricin in membrane-micking conditions by molecular dynamics simulation and circular dichroism

Lactoferricins are potent antimicrobial peptides released by pepsin cleavage of Lactoferrins. Bovine Lactoferricin (LfcinB) has higher activity than the intact bovine Lactoferrin, and is the most active among the other Lactoferricins of human, murine and caprine origin. In the intact protein the fragment corresponding to LfcinB is in an helical conformation, while in water LfcinB adopts an amphipathic β-hairpin structure. However, whether any of these structural motifs is the antibacterial active conformation, i.e., the one interacting with bacterial membrane components, remains to be seen. Here we present Circular Dichroism (CD) spectra and Molecular Dynamics (MD) simulations indicating that in membrane-mimicking solvents the LfcinB adopts an amphipathic β-hairpin structure similar to that observed in water, but differing in the dynamic behavior of the side-chains of the two tryptophan residues. In the membrane-mimicking solvent these side-chains show a high propensity to point towards the hydrophobic environment, rather than being in the hydrophobic core as seen in water, while the backbone preserves the hairpin conformation as found in water. These results suggest that the tryptophans might act as anchors pulling the stable, solvent-invariant hairpin structure into the membrane.

CGG repeats associated with fragile X chromosome form left-handed Z-DNA structure

This work is a continuation of our effort to determine the structure responsible for expansion of the (CGG)(n) motif that results in fragile X chromosome syndrome. In our previous report, we demonstrated that the structure adopted by an oligonucleotide with this repeat sequence is not a quadruplex as was suggested by others. Here we demonstrate that (CGG) runs adopt another anomalous arrangement-a left-handed Z-DNA structure. The Z-DNA formation was induced by high salt and millimolar concentrations of Ni(2+) ions and likelihood of its formation increased with increasing number of repeats. In an oligonucleotide in which the CGG runs were interrupted by AGG triplets, as is observed in genomes of healthy individuals, the hairpin conformation was stabilized and Z-DNA formation was hindered. We show here that methylation of the (CGG) runs markedly stabilized Z-DNA formation. We hypothesize that rather than in the expansion process the Z-DNA may be formed by long, expanded (CGG) stretches that become hypermethylated; this would inhibit transcription resulting in disease.

The effect of hydrogen bonding on oligoleucine structure in water: A molecular dynamics simulation study

Molecular dynamics simulations were conducted in order to improve our understanding of the forces that determine polyleucine chains conformations and govern polyleucine self-assembly in aqueous solutions. Simulations of 10 repeat unit oligoleucine in aqueous solution were performed using the optimized potential for liquid simulations (OPLS) – all atom force field using the canonical ensemble for a minimum of 1.3 ns. These simulations provided information on conformations, chain collapse and intermolecular aggregation. Simulations indicate that single isotactic oligoleucine chains in dilute solution assume tightly packed, regular hairpin conformations while atactic oligoleucine assumes a much less regular and less compact structure. The regular, compact collapsed isotactic chain exhibited a greater degree of intramolecular hydrogen bonding and an increased level of hydrophobic t-butyl functional group aggregation compared to the atactic chain. This occurs at the expense of reduced leucine–water hydrogen bonding.

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.

Mechanistic Studies of Hairpin to Duplex Conversion for Trinucleotide Repeat Sequences

The expansion of a trinucleotide repeat sequence, such as CAG/CTG, has been pinpointed as the molecular basis for a number of neurodegenerative disorders. It has been proposed that as part of the expansion process, these repetitive sequences adopt non-B conformations such as hairpins. However, the prevalence of these hairpins and their contributions to the DNA expansion have not been well defined. In this work, we utilized a molecular beacon strategy to examine the stability of the (CAG)(10) hairpin and also its behavior in the presence of the complementary (CTG)(10) hairpin. We find that the two hairpins represent kinetically trapped species that can coexist but irreversibly convert to duplex upon thermal induction. Furthermore, as monitored by fluorescence and optical analysis, modifications to the base composition of either the loop or stem region have a profound effect on the ability of the trinucleotide repeat hairpins to convert to duplex. Additionally, the rate of duplex formation is also reduced with these loop and stem-modified hairpins. These results demonstrate that the trinucleotide repeat hairpins can convert to duplex via two independent mechanisms as follows: the loop-loop interactions found in kissing hairpins or the stem-stem interactions of a cruciform.

Macrocyclic BACE inhibitors: Optimization of a micromolar hit to nanomolar leads

We have identified macrocyclic inhibitors of the aspartic protease BACE, implicated in the etiology of Alzheimer’s disease. An X-ray structure of screening hit 1 in the BACE active site revealed a hairpin conformation suggesting that constrained macrocyclic derivatives may also bind there. Several of the analogs we prepared were >100× more potent than 1, such as 7 (5 nM K i).