Folding and unfolding kinetics of DNA hairpins in flowing solution by multiparameter fluorescence correlation spectroscopy

Dynamic equilibrium between the folded and unfolded conformations of single stranded DNA hairpin molecules containing polythymine hairpin loops was investigated using simultaneous two-beam fluorescence cross-correlation spectroscopy and single beam autocorrelation spectroscopy. The hairpins were end-labeled with a fluorescent dye and a quencher, such that folding and unfolding of the DNA hairpin primary structure caused the dye fluorescence to fluctuate on the same characteristic time scale as the folding and unfolding reaction. These fluctuations were observed as the molecules flowed sequentially between two spatially offset, microscopic detection volumes. Cross-correlation analysis of fluorescence from the two detection volumes revealed the translational diffusion and flow properties of the hairpins, as well as the average molecular occupancy of the two volumes. Autocorrelation analysis of the fluorescence from the individual detection volumes revealed the kinetics of hairpin folding and unfolding, with the parameters relating to diffusion, flow, and molecular occupancy constrained to the values determined from the cross-correlation analysis. This allowed unambiguous characterization of the folding and unfolding kinetics, without the need to determine the hydrodynamic properties by analyzing a separate control sample. The analysis revealed nonexponential relaxation kinetics and DNA size-dependent folding times characteristic of dynamic heterogeneity in the DNA hairpin-forming mechanism.     

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.     

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 Dynamics of 10‐Residue β‐Hairpin Peptide Chignolin

Short peptides that fold into β‐hairpins are ideal model systems for investigating the mechanism of protein folding because their folding process shows dynamics typical of proteins. We performed folding, unfolding, and refolding molecular dynamics simulations (total of 2.7 μs) of the 10‐residue β‐hairpin peptide chignolin, which is the smallest β‐hairpin structure known to be stable in solution. Our results revealed the folding mechanism of chignolin, which comprises three steps. First, the folding begins with hydrophobic assembly. It brings the main chain together; subsequently, a nascent turn structure is formed. The second step is the conversion of the nascent turn into a tight turn structure along with interconversion of the hydrophobic packing and interstrand hydrogen bonds. Finally, the formation of the hydrogen‐bond network and the complete hydrophobic core as well as the arrangement of side‐chain–side‐chain interactions occur at approximately the same time. This three‐step mechanism appropriately interprets the folding process as involving a combination of previous inconsistent explanations of the folding mechanism of the β‐hairpin, that the first event of the folding is formation of hydrogen bonds and the second is that of the hydrophobic core, or vice versa.     

A three-state mechanism for DNA hairpin folding characterized by multiparameter fluorescence fluctuation spectroscopy

The folding of a dye-quencher labeled DNA hairpin molecule was investigated using fluorescence autocorrelation and cross-correlation spectroscopy (FCS) and photon counting histogram analysis (PCH). The autocorrelation and cross-correlation measurements revealed the flow and diffusion times of the DNA molecules through two spatially offset detection volumes, the relaxation time of the folding reaction, and the total concentration of DNA molecules participating in the reaction. The PCH measurements revealed the equilibrium distribution of DNA molecules in folded and unfolded conformations and the specific brightnesses of the fluorophore in each conformational state. These measurements were carried out over a range of NaCl concentrations, from those that favored the open form of the DNA hairpin to those that favored the closed form. DNA melting curves obtained from each sample were also analyzed for comparison. It was found that the reactant concentrations were depleted as the reaction progressed and that the equilibrium distributions measured by FCS and PCH deviated from those obtained from the melting curve analyses. These observations suggest a three-state mechanism for the DNA hairpin folding reaction that involves a stable intermediate form of the DNA hairpin. The reaction being probed by FCS and PCH is suggested to be a rapid equilibrium between open and intermediate conformations. Formation of the fully closed DNA hairpin is suggested to occur on a much longer time scale than the FCS and PCH measurement time. The closed form of the hairpin thus serves as a sink into which the reactants are depleted as the reaction progresses.     

Formation of a Ligand‐Assisted Complex of Two RNA Hairpin Loops

The hairpin structure is one of the most common secondary structures in RNA and holds a central position in the stream of RNA folding from a non‐structured RNA to structurally complex and functional ribonucleoproteins. Since the RNA secondary structure is strongly correlated to the function and can be modulated by the binding of small molecules, we have investigated the modulation of RNA folding by a ligand‐assisted formation of loop–loop complexes of two RNA hairpin loops. With a ligand (NCT6), designed based on the ligand binding to the G–G mismatches in double‐stranded DNA, we successfully demonstrated the formation of both inter‐ and intra‐molecular NCT6‐assisted complex of two RNA hairpin loops. NCT6 selectively bound to the two hairpin loops containing (CGG)₃ in the loop region. Native polyacrylamide gel electrophoresis analysis of two doubly‐labeled RNA hairpin loops clearly showed the formation of intermolecular NCT6‐assisted loop–loop complex. Förster resonance energy‐transfer studies of RNA constructs containing two hairpin loops, in which each hairpin was labeled with Alexa488 and Cy3 fluorophores, showed the conformational change of the RNA constructs upon binding of NCT6. These experimental data showed that NCT6 simultaneously bound to two hairpin RNAs at the loop region, and can induce the conformational change of the RNA molecule. These data strongly support that NCT6 functions as molecular glue for two hairpin RNAs.     

Probing the formation of stable tertiary structure in a model miniprotein at atomic resolution: determinants of stability of a helical hairpin

The minimal model system to study the basic principles of protein folding is the hairpin. The formation of beta-hairpins, which are the basic components of antiparallel beta-sheets, has been studied extensively in the past decade, but much less is known about helical hairpins. Here, we probe hairpin formation between a polyproline type-II helix and an alpha-helix as present in the natural miniprotein peptide YY (PYY). Both turn sequence and interactions of aromatic side chains from the C-terminal alpha-helix with the pockets formed by N-terminal Pro residues are shown by site-directed mutagenesis and solution NMR spectroscopy in different solvent systems to be important determinants of backbone dynamics and hairpin stability, suggesting a close analogy with some beta-hairpin structures. It is shown that multiple relatively weak contacts between the helices are necessary for the formation of the helical hairpin studied here, whereas the type-I beta-turn acts like a hinge, which through certain single amino acid substitutions is destabilized such that hairpin formation is completely abolished. Denaturation and renaturation of tertiary structure by temperature or cosolvents were probed by measuring changes of chemical shifts. Folding of PYY is both reversible and cooperative as inferred from the sigmoidal denaturation curves displayed by residues at the interface of the helical hairpin. Such miniproteins thus feature an important hallmark of globular proteins and should provide a convenient system to study basic aspects of helical hairpin folding that are complementary to those derived from studies of beta-hairpins.     

Contributions of a Surface Hydrophobic Cluster to the Folding and Structural Stability of Ubiquitin

The role of the small exterior hydrophobic cluster (SEHC) in the strand region of the N‐terminal β‐hairpin of ubiquitin on the structural stability and the folding/unfolding kinetics of the protein have been examined. We introduce a Phe→Ala substitution at residue 4 in the strand region of the N‐terminal β‐hairpin of the ubiquitin. A peptide with the same amino acid sequence as the first 21 residues of the mutated ubiquitin has also been synthesized. The F4A mutation unfolds the hairpin structure of the peptide segment without disruption of the turn. The same mutation does not seem to affect the overall structure, but the stability of the mutated full‐length protein decreases by approx. 2 kcal/mol. Kinetically, the entire hairpin structure is implicated in the transition state during folding of the wild type protein. The rate of refolding is retarded by the F4A mutation in ～80% of the protein molecules. The F4A substitution also increases the unfolding rate of the protein by 10 fold. Thus the hydrophobic side‐chain of Phe‐4 not only contributes to the stability of the hairpin, but also to the stability of the entire protein by forming a cluster together with the hydrophobic residues on the C‐terminal strand.     

Folding pathways and rates for the three-stranded beta-sheet peptide Beta3s using discrete path sampling

The discrete path sampling method was used to investigate the folding of a three-stranded antiparallel beta-sheet peptide, Beta3s, described by an empirical potential and implicit solvent model. After application of a coarse-graining scheme that groups together sets of minima in local equilibrium, the calculated folding time was in reasonable agreement with other simulations and consistent with the experimental upper bound. The folding mechanism exhibited by the most significant discrete paths involves early formation of the C-terminal hairpin followed by docking of the N-terminal strand.     

Millisecond time-scale folding and unfolding of DNA hairpins using rapid-mixing stopped-flow kinetics

We report stopped-flow kinetics experiments to study the folding and unfolding of 5 base-pair stem and 21 nucleotide polythymidine loop DNA hairpins over various concentrations of NaCl. The reactions occurred on a time scale of milliseconds, considerably longer than the microsecond time scale suggested by previous kinetics studies of similar-sized hairpins. In comparison to a recent fluorescence correlation spectroscopy study (J. Am. Chem. Soc. 2006, 128, 1240-1249), we suggest the microsecond time-scale reactions are due to intermediate states and the millisecond time-scale reactions reported here are due to the formation of the fully folded DNA hairpin. These results support our view that DNA hairpin folding occurs via a minimum three-state mechanism.     

Isomerization‐ and Temperature‐Jump‐Induced Dynamics of a Photoswitchable β‐Hairpin

Conformational changes in proteins and peptides can be initiated by diverse processes. This raises the question how the variation of initiation mechanisms is connected to differences in folding or unfolding processes. In this work structural dynamics of a photoswitchable β‐hairpin model peptide were initiated by two different mechanisms: temperature jump (T‐jump) and isomerization of a backbone element. In both experiments the structural changes were followed by time‐resolved IR spectroscopy in the nanosecond to microsecond range. When the photoisomerization of the azobenzene backbone switch initiated the folding reaction, pronounced absorption changes related to folding into the hairpin structure were found with a time constant of about 16 μs. In the T‐jump experiment kinetics with the same time constant were observed. For both initiation processes the reaction dynamics revealed the same strong dependence of the reaction time on temperature. The highly similar transients in the microsecond range show that the peptide dynamics induced by T‐jump and isomerization are both determined by the same mechanism and exclude a downhill‐folding process. Furthermore, the combination of the two techniques allows a detailed model for folding and unfolding to be presented: The isomerization‐induced folding process ends in a transition‐state reaction scheme, in which a high energetic barrier of 48 kJ mol^?1 separates unfolded and folded structures.     

Hairpin folding behavior of mixed α/β-peptides in aqueous solution

The invention of new strategies for the design of protein-mimetic oligomers that manifest the folding encoded in natural amino acid sequences is a significant challenge. In contrast to the α-helix, mimicry of protein β-sheets is less understood. We report here the aqueous folding behavior of a prototype α-peptide hairpin model sequence varied at cross-strand positions by incorporation of 16 different β-amino acid monomers. Our results provide a folding propensity scale for β-residues in a protein β-sheet context as well as high-resolution structures of several mixed-backbone α/β-peptide hairpins in water.     

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.     

Effects of turn stability on the kinetics of refolding of a hairpin in a beta-sheet

As part of our continuing study of the effects of the turn sequence on the conformational stability as well as the mechanism of folding of a beta-sheet structure, we have undertaken a parallel investigation of the solution structure, conformational stability, and kinetics of refolding of the beta-sheet VFIVDGOTYTEV(D)PGOKILQ. The latter peptide is an analogue of the original Gellman beta-sheet VFITS(D)PGKTYTEV(D)PGOKILQ, wherein the TS(D)PGK turn sequence in the first hairpin has been replaced by VDGO. Thermodynamics studies revealed comparable conformational stability of the two peptides. However, unlike the Gellman peptide, which showed extremely rapid refolding of the first hairpin, early kinetic events associated with the refolding of the corresponding hairpin in the VDGO mutant were found to be significantly slower. A detailed study of the conformation of the modified peptide suggested that hydrophobic interactions might be contributing to its stability. Accordingly, we surmise that the early kinetic events are sensitive to whether the formation of the hairpin is nucleated at the turn or by sequestering of the hydrophobic residues across the strand, before structural rearrangements to produce the nativelike topology. Nucleation of the hairpin at the turn is expected to be intrinsically rapid for a strong turn. However, if the process must involve collapse of hydrophobic side chains, the nucleation should be slower as solvent molecules must be displaced to sequester the hydrophobic residues. These findings reflect the contribution of different forces toward nucleation of hairpins in the mechanism of folding of beta-sheets.     

Probing the complete folding trajectory of a DNA hairpin using dual beam fluorescence fluctuation spectroscopy

The conformational fluctuations of dye-quencher labeled DNA hairpin molecules in aqueous solution were investigated using dual probe beam fluorescence fluctuation spectroscopy. The measurements revealed the flow and diffusion times of the DNA molecules through two spatially offset optical probe regions, the absolute and relative concentrations of each conformational substate of the DNA, and the kinetics of the DNA hairpin folding and unfolding reactions in the 1 micros to 10 ms time range. A DNA hairpin containing a 21-nucleotide polythymine loop and a 4-base pair stem exhibited double exponential relaxation kinetics, with time constants of 84 and 393 micros. This confirms that folding and melting of the DNA hairpin structure is not a two state process but proceeds by way of metastable intermediate states. The fast time constant corresponds to formation and unfolding of an intermediate, and the slow time constant is due to formation and disruption of the fully base-paired stem. This is consistent with a previous study of a similar DNA hairpin with a 5-base pair stem, in which the fast reaction was attributed to the fluctuations of an intermediate DNA conformation [J. Am. Chem. Soc. 2006, 128, 1240-1249]. In that case, reactions involving the native conformation could not be observed directly due to the limited observation time range of the fluorescence correlation spectroscopy experiment. The intermediate states of the DNA hairpins are suggested to be due to a collapsed ensemble of folded hairpins containing various partially folded or misfolded conformations.     

Filaggrin Peptides with β‐Hairpin Structure Bind Rheumatoid Arthritis Antibodies

In the early detection of rheumatoid arthritis (RA) synthetic filaggrin peptides serve as antigens for rheumatoid‐specific autoantibodies (anti‐citrullinated peptide antibody, ACPA) in ELISA tests. In this work we present a peptide that exhibits the binding epitope of ACPA in the form of a stable folding β‐hairpin. The homogeneity of the peptide folding was confirmed by NMR spectroscopy and might lead to the first proposed structure of the antibody‐bound conformation of the epitope.     

The beta-peptide hairpin in solution: conformational study of a beta-hexapeptide in methanol by NMR spectroscopy and MD simulation.

The structural and thermodynamic properties of a 6-residue beta-peptide that was designed to form a hairpin conformation have been studied by NMR spectroscopy and MD simulation in methanol solution. The predicted hairpin would be characterized by a 10-membered hydrogen-bonded turn involving residues 3 and 4, and two extended antiparallel strands. The interproton distances and backbone torsional dihedral angles derived from the NMR experiments at room temperature are in general terms compatible with the hairpin conformation. Two trajectories of system configurations from 100-ns molecular-dynamics simulations of the peptide in solution at 298 and 340 K have been analyzed. In both simulations reversible folding to the hairpin conformation is observed. Interestingly, there is a significant conformational overlap between the unfolded state of the peptide at each of the temperatures. As already observed in previous studies of peptide folding, the unfolded state is composed of a (relatively) small number of predominant conformers and in this case lacks any type of secondary-structure element. The trajectories provide an excellent ground for the interpretation of the NMR-derived data in terms of ensemble averages and distributions as opposed to single-conformation interpretations. From this perspective, a relative population of the hairpin conformation of 20% to 30% would suffice to explain the NMR-derived data. Surprisingly, however, the ensemble of structures from the simulation at 340 K reproduces more accurately the NMR-derived data than the ensemble from the simulation at 298 K, a question that needs further investigation.     

Molecular Dynamics Simulations of a β-Hairpin Fragment of Protein G by Means of Atom-Bond Electronegativity Equalization Method Fused into Molecular Mechanics (ABEEMσπ/MM)

There are some controversial opinions about the origin of folding β‐hairpin stability in aqueous solution. In this study, the structural and dynamic behavior of a 16‐residue β‐hairpin from B1 domain of protein G has been investigated at 280, 300, 350 and 450 K using molecular dynamics (MD) simulations by means of Atom‐Bond Electronegativity Equalization Method Fused into Molecular Mechanics i.e., ABEEMδπ/MM and the explicit ABEEM‐7P water solvent model. In addition, a 300 K simulation of one mutant having the aromatic residues substituted with alanines has been performed. The hydrophobic surface area, hydrophilic surface area and some structural properties have been used to measure the role of the hydrophobic interactions. It is found that the aromatic residues substituted with alanines have shown an evident destabilization of the structure and unfolding started after 1.5 ns. It is also found that the number of the main chain hydrogen bonds have different distributions through three different simulations. All above demonstrate that the hydrophobic interactions and the main chain hydrogen bonds play an important role in the stability of the folding structure of β‐hairpin in solution. Furthermore, through the structural analyses of the β‐hairpin structures from four temperature simulations and the comparison with other MD simulations of β‐hairpin peptides, the new ABEEMδπ force field can reproduce the structural data in good agreement with the experimental data.