Novel eGZ-motif formed by regularly extruded guanine bases in a left-handed Z-DNA helix as a major motif behind CGG trinucleotide repeats.

The expansion of d(CGG) trinucleotide repeats (TRs) lies behind several important neurodegenerative diseases. Atypical DNA secondary structures have been shown to trigger TR expansion: their characterization is important for a molecular understanding of TR disease. CD spectroscopy experiments in the last decade have unequivocally demonstrated that CGG runs adopt a left-handed Z-DNA conformation, whose features remain uncertain because it entails accommodating GG mismatches. In order to find this missing motif, we have carried out molecular dynamics (MD) simulations to explore all the possible Z-DNA helices that potentially form after the transition from B- to Z-DNA. Such helices combine either CpG or GpC Watson-Crick steps in Z-DNA form with GG-mismatch conformations set as either intrahelical or extrahelical; and participating in BZ or ZZ junctions or in alternately extruded conformations. Characterization of the stability and structural features (especially overall left-handedness, higher-temperature and steered MD simulations) identified two novel Z-DNA helices: the most stable one displays alternately extruded Gs, and is followed by a helix with symmetrically extruded ZZ junctions. The G-extrusion favors a seamless stacking of the Watson-Crick base pairs; extruded Gs favor syn conformations and display hydrogen-bonding and stacking interactions. Such conformations could have the potential to hijack the MMR complex, thus triggering further expansion.

Conformationally Restricted Glycopeptide Backbone Inhibits Gas-Phase H/D Scrambling between Glycan and Peptide Moieties.

Protein glycosylation is a common post-translational modification on extracellular proteins. The conformational dynamics of several glycoproteins have been characterized by hydrogen/deuterium exchange mass spectrometry (HDX-MS). However, it is, in most cases, not possible to extract information about glycan conformation and dynamics due to the general difficulty of separating the deuterium content of the glycan from that of the peptide (in particular, for O-linked glycans). Here, we investigate whether the fragmentation of protonated glycopeptides by collision-induced dissociation (CID) can be used to determine the solution-specific deuterium content of the glycan. Central to this concept is that glycopeptides can undergo a facile loss of glycans upon CID, thereby allowing for the determination of their masses. However, an essential prerequisite is that hydrogen and deuterium (H/D) scrambling can be kept in check. Therefore, we have measured the degree of scrambling upon glycosidic bond cleavage in glycopeptides that differ in the conformational flexibility of their backbone and glycosylation pattern. Our results show that complete scrambling precedes the glycosidic bond cleavage in normal glycopeptides derived from a glycoprotein; i.e., all labile hydrogens have undergone positional randomization prior to loss of the glycan. In contrast, the glycosidic bond cleavage occurs without any scrambling in the glycopeptide antibiotic vancomycin, reflecting that the glycan cannot interact with the peptide moiety due to a conformationally restricted backbone as revealed by molecular dynamics simulations. Scrambling is also inhibited, albeit to a lesser degree, in the conformationally restricted glycopeptides ristocetin and its pseudoaglycone, demonstrating that scrambling depends on an intricate interplay between the flexibility and proximity of the glycan and the peptide backbone.

Atypical structures of GAA/TTC trinucleotide repeats underlying Friedreich's ataxia: DNA triplexes and RNA/DNA hybrids.

Expansion of the GAA/TTC repeats in the first intron of the FXN gene causes Friedreich's ataxia. Non-canonical structures are linked to this expansion. DNA triplexes and R-loops are believed to arrest transcription, which results in frataxin deficiency and eventual neurodegeneration. We present a systematic in silico characterization of the possible DNA triplexes that could be assembled with GAA and TTC strands; the two hybrid duplexes [r(GAA):d(TTC) and d(GAA):r(UUC)] in an R-loop; and three hybrid triplexes that could form during bidirectional transcription when the non-template DNA strand bonds with the hybrid duplex (collapsed R-loops, where the two DNA strands remain antiparallel). For both Y·R:Y and R·R:Y DNA triplexes, the parallel third strand orientation is more stable; both parallel and antiparallel protonated d(GA+A)·d(GAA):d(TTC) triplexes are stable. Apparent contradictions in the literature about the R·R:Y triplex stability is probably due to lack of molecular resolution, since shifting the third strand by a single nucleotide alters the stability ranking. In the collapsed R-loops, antiparallel d(TTC+)·d(GAA):r(UUC) is unstable, while parallel d(GAA)·r(GAA):d(TTC) and d(GA+A)·r(GAA):d(TTC) are stable. In addition to providing new structural perspectives for specific therapeutic aims, our results contribute to a systematic structural basis for the emerging field of quantitative R-loop biology.

Dynamics of strand slippage in DNA hairpins formed by CAG repeats: roles of sequence parity and trinucleotide interrupts.

DNA trinucleotide repeats (TRs) can exhibit dynamic expansions by integer numbers of trinucleotides that lead to neurodegenerative disorders. Strand slipped hairpins during DNA replication, repair and/or recombination may contribute to TR expansion. Here, we combine single-molecule FRET experiments and molecular dynamics studies to elucidate slipping dynamics and conformations of (CAG)n TR hairpins. We directly resolve slipping by predominantly two CAG units. The slipping kinetics depends on the even/odd repeat parity. The populated states suggest greater stability for 5'-AGCA-3' tetraloops, compared with alternative 5'-CAG-3' triloops. To accommodate the tetraloop, even(odd)-numbered repeats have an even(odd) number of hanging bases in the hairpin stem. In particular, a paired-end tetraloop (no hanging TR) is stable in (CAG)n = even, but such situation cannot occur in (CAG)n = odd, where the hairpin is "frustrated'' and slips back and forth between states with one TR hanging at the 5' or 3' end. Trinucleotide interrupts in the repeating CAG pattern associated with altered disease phenotypes select for specific conformers with favorable loop sequences. Molecular dynamics provide atomic-level insight into the loop configurations. Reducing strand slipping in TR hairpins by sequence interruptions at the loop suggests disease-associated variations impact expansion mechanisms at the level of slipped hairpins.

E-motif formed by extrahelical cytosine bases in DNA homoduplexes of trinucleotide and hexanucleotide repeats.

Atypical DNA secondary structures play an important role in expandable trinucleotide repeat (TR) and hexanucleotide repeat (HR) diseases. The cytosine mismatches in C-rich homoduplexes and hairpin stems are weakly bonded; experiments show that for certain sequences these may flip out of the helix core, forming an unusual structure termed an 'e-motif'. We have performed molecular dynamics simulations of C-rich TR and HR DNA homoduplexes in order to characterize the conformations, stability and dynamics of formation of the e-motif, where the mismatched cytosines symmetrically flip out in the minor groove, pointing their base moieties towards the 5'-direction in each strand. TRs have two non-equivalent reading frames, (GCC)n and (CCG)n; while HRs have three: (CCCGGC)n, (CGGCCC)n, (CCCCGG)n. We define three types of pseudo basepair steps related to the mismatches and show that the e-motif is only stable in (GCC)n and (CCCGGC)n homoduplexes due to the favorable stacking of pseudo GpC steps (whose nature depends on whether TRs or HRs are involved) and the formation of hydrogen bonds between the mismatched cytosine at position i and the cytosine (TRs) or guanine (HRs) at position i - 2 along the same strand. We also characterize the extended e-motif, where all mismatched cytosines are extruded, their extra-helical stacking additionally stabilizing the homoduplexes.

Structure and Dynamics of DNA and RNA Double Helices of CAG and GAC Trinucleotide Repeats.

CAG trinucleotide repeats are known to cause 10 late-onset progressive neurodegenerative disorders as the repeats expand beyond a threshold, whereas GAC repeats are associated with skeletal dysplasias and expand from the normal five to a maximum of seven repeats. The TR secondary structure is believed to play a role in CAG expansions. We have carried out free energy and molecular dynamics studies to determine the preferred conformations of the A-A noncanonical pairs in (CAG)n and (GAC)n trinucleotide repeats (n = 1, 4) and the consequent changes in the overall structure of the RNA and DNA duplexes. We find that the global free energy minimum corresponds to A-A pairs stacked inside the core of the helix with anti-anti conformations in RNA and (high-anti)-(high-anti) conformations in DNA. The next minimum corresponds to anti-syn conformations, whereas syn-syn conformations are higher in energy. Transition rates of the A-A conformations are higher for RNA than DNA. Mechanisms for these various transitions are identified. Additional structural and dynamical aspects of the helical conformations are explored, with a focus on contrasting CAG and GAC duplexes. The neutralizing ion distribution around the noncanonical pairs is described.

Picosecond infrared laser-induced all-atom nonequilibrium molecular dynamics simulation of dissociation of viruses.

Since the discovery of the plant pathogen tobacco mosaic virus as the first viral entity in the late 1800s, viruses traditionally have been mainly thought of as pathogens for disease-resistances. However, viruses have recently been exploited as nanoplatforms with applications in biomedicine and materials science. To this aim, a large majority of current methods and tools have been developed to improve the physical stability of viral particles, which may be critical to the extreme physical or chemical conditions that viruses may encounter during purification, fabrication processes, storage and use. However, considerably fewer studies are devoted to developing efficient methods to degrade or recycle such enhanced stability biomaterials. With this in mind, we carry out all-atom nonequilibrium molecular dynamics simulation, inspired by the recently developed mid-infrared free-electron laser pulse technology, to dissociate viruses. Adopting the poliovirus as a representative example, we find that the primary step in the dissociation process is due to the strong resonance between the amide I vibrational modes of the virus and the tuned laser frequencies. This process is determined by a balance between the formation and dissociation of the protein shell, reflecting the highly plasticity of the virus. Furthermore, our method should provide a feasible approach to simulate viruses, which is otherwise too expensive for conventional equilibrium all-atom simulations of such very large systems. Our work shows a proof of concept which may open a new, efficient way to cleave or to recycle virus-based materials, provide an extremely valuable tool for elucidating mechanical aspects of viruses, and may well play an important role in future fighting against virus-related diseases.

Comparative melting and healing of B-DNA and Z-DNA by an infrared laser pulse.

We explore the use of a fast laser melting simulation approach combined with atomistic molecular dynamics simulations in order to determine the melting and healing responses of B-DNA and Z-DNA dodecamers with the same d(5'-CGCGCGCGCGCG-3')2 sequence. The frequency of the laser pulse is specifically tuned to disrupt Watson-Crick hydrogen bonds, thus inducing melting of the DNA duplexes. Subsequently, the structures relax and partially refold, depending on the field strength. In addition to the inherent interest of the nonequilibrium melting process, we propose that fast melting by an infrared laser pulse could be used as a technique for a fast comparison of relative stabilities of same-sequence oligonucleotides with different secondary structures with full atomistic detail of the structures and solvent. This could be particularly useful for nonstandard secondary structures involving non-canonical base pairs, mismatches, etc.

Ion distributions around left- and right-handed DNA and RNA duplexes: a comparative study.

The ion atmosphere around nucleic acids is an integral part of their solvated structure. However, detailed aspects of the ionic distribution are difficult to probe experimentally, and comparative studies for different structures of the same sequence are almost non-existent. Here, we have used large-scale molecular dynamics simulations to perform a comparative study of the ion distribution around (5'-CGCGCGCGCGCG-3')2 dodecamers in solution in B-DNA, A-RNA, Z-DNA and Z-RNA forms. The CG sequence is very sensitive to ionic strength and it allows the comparison with the rare but important left-handed forms. The ions investigated include Na(+), K(+) and Mg(2 +), with various concentrations of their chloride salts. Our results quantitatively describe the characteristics of the ionic distributions for different structures at varying ionic strengths, tracing these differences to nucleic acid structure and ion type. Several binding pockets with rather long ion residence times are described, both for the monovalent ions and for the hexahydrated Mg[(H2O)6](2+) ion. The conformations of these binding pockets include direct binding through desolvated ion bridges in the GpC steps in B-DNA and A-RNA; direct binding to backbone oxygens; binding of Mg[(H2O)6](2+) to distant phosphates, resulting in acute bending of A-RNA; tight 'ion traps' in Z-RNA between C-O2 and the C-O2' atoms in GpC steps; and others.

Picosecond melting of peptide nanotubes using an infrared laser: a nonequilibrium simulation study.

Self-assembled functional peptide biomaterials are emerging with a wide range of envisioned applications in the field of nanotechnology. Currently, methods and tools have been developed to control and manipulate as well as to explore new properties of self-assembled structures. However, considerably fewer studies are being devoted to developing efficient methods to degrade or recycle such extremely stable biomaterials. With this in mind, here we suggest a theoretical framework, inspired by the recent developed mid-infrared free-electron laser pulse technology, to dissociate peptide nanotubes. Adopting a diphenylalanine channel as a prototypical example, we find that the primary step in the dissociation process occurs due to the strong resonance between the carboxylate bond vibrations of the diphenylalanine peptides and the tuned laser frequencies. The effects of laser irradiation are determined by a balance between tube formation and dissociation. Our work shows a proof of concept and should provide a motivation for future experimental developments with the final aim to open a new and efficient way to cleave or to recycle bio-inspired materials.

Picosecond dissociation of amyloid fibrils with infrared laser: A nonequilibrium simulation study.

Recently, mid-infrared free-electron laser technology has been developed to dissociate amyloid fibrils. Here, we present a theoretical framework for this type of experiment based on laser-induced nonequilibrium all-atom molecular dynamics simulations. We show that the fibril is destroyed due to the strong resonance between its amide I vibrational modes and the laser field. The effects of laser irradiation are determined by a balance between fibril formation and dissociation. While the overall rearrangements of the fibril finish over short time scales, the interaction between the peptides and the solvent continues over much longer times indicating that the waters play an important role in the dissociation process. Our results thus provide new insights into amyloid fibril dissociation by laser techniques and open up new venues to investigate the complex phenomena associated with amyloidogenesis.

Reaction path ensemble of the B-Z-DNA transition: a comprehensive atomistic study.

Since its discovery in 1979, left-handed Z-DNA has evolved from an in vitro curiosity to a challenging DNA structure with crucial roles in gene expression, regulation and recombination. A fundamental question that has puzzled researchers for decades is how the transition from B-DNA, the prevalent right-handed form of DNA, to Z-DNA is accomplished. Due to the complexity of the B-Z-DNA transition, experimental and computational studies have resulted in several different, apparently contradictory models. Here, we use molecular dynamics simulations coupled with state-of-the-art enhanced sampling techniques operating through non-conventional reaction coordinates, to investigate the B-Z-DNA transition at the atomic level. Our results show a complex free energy landscape, where several phenomena such as over-stretching, unpeeling, base pair extrusion and base pair flipping are observed resulting in interconversions between different DNA conformations such as B-DNA, Z-DNA and S-DNA. In particular, different minimum free energy paths allow for the coexistence of different mechanisms (such as zipper and stretch-collapse mechanisms) that previously had been proposed as independent, disconnected models. We find that the B-Z-DNA transition--in absence of other molecular partners--can encompass more than one mechanism of comparable free energy, and is therefore better described in terms of a reaction path ensemble.

Investigating rare events with nonequilibrium work measurements. II. Transition and reaction rates.

We present a formalism for investigating transition pathways and transition probabilities for rare events in biomolecular systems. The formalism is based on combining Transition Path Theory with the results of nonequilibrium work relations, and shows that the equilibrium and nonequilibrium transition rates are in fact related. Aside from its fundamental importance, this allows for the calculation of relative equilibrium reaction rates with driven nonequilibrium simulations such as Steered Molecular Dynamics. The workings of the formalism are illustrated with a few typical numerical examples.

Investigating rare events with nonequilibrium work measurements. I. Nonequilibrium transition path probabilities.

We have developed a formalism for investigating transition pathways and transition probabilities for rare events in biomolecular systems. In this paper, we set the theoretical framework for employing nonequilibrium work relations to estimate the relative reaction rates associated with different classes of transition pathways. Particularly, we derive an extension of Crook's transient fluctuation theorem, which relates the relative transition rates of driven systems in the forward and reverse directions, and allows for the calculation of these relative rates using work measurements (e.g., in Steered Molecular Dynamics). The formalism presented here can be combined with Transition Path Theory to relate the equilibrium and driven transition rates. The usefulness of this framework is illustrated by means of a Gaussian model and a driven proline dimer.

Are long-range structural correlations behind the aggregration phenomena of polyglutamine diseases?

We have characterized the conformational ensembles of polyglutamine Qn peptides of various lengths n (ranging from 6 to 40), both with and without the presence of a C-terminal polyproline hexapeptide. For this, we used state-of-the-art molecular dynamics simulations combined with a novel statistical analysis to characterize the various properties of the backbone dihedral angles and secondary structural motifs of the glutamine residues. For Q40 (i.e., just above the pathological length ≃36 for Huntington's disease), the equilibrium conformations of the monomer consist primarily of disordered, compact structures with non-negligible α-helical and turn content. We also observed a relatively small population of extended structures suitable for forming aggregates including ß- and α-strands, and ß- and α-hairpins. Most importantly, for Q40 we find that there exists a long-range correlation (ranging for at least 20 residues) among the backbone dihedral angles of the Q residues. For polyglutamine peptides below the pathological length, the population of the extended strands and hairpins is considerably smaller, and the correlations are short-range (at most 5 residues apart). Adding a C-terminal hexaproline to Q40 suppresses both the population of these rare motifs and the long-range correlation of the dihedral angles. We argue that the long-range correlation of the polyglutamine homopeptide, along with the presence of these rare motifs, could be responsible for its aggregation phenomena.

Structure and Dynamics of DNA and RNA Double Helices Formed by d(CTG), d(GTC), r(CUG), and r(GUC) Trinucleotide Repeats and Associated DNA-RNA Hybrids.

Myotonic dystrophy type 1 is the most frequent form of muscular dystrophy in adults caused by an abnormal expansion of the CTG trinucleotide. Both the expanded DNA and the expanded CUG RNA transcript can fold into hairpins. Co-transcriptional formation of stable RNA·DNA hybrids can also enhance the instability of repeat tracts. We performed molecular dynamics simulations of homoduplexes associated with the disease, d(CTG)n and r(CUG)n, and their corresponding r(CAG)n:d(CTG)n and r(CUG)n:d(CAG)n hybrids that can form under bidirectional transcription and of non-pathological d(GTC)n and d(GUC)n homoduplexes. We characterized their conformations, stability, and dynamics and found that the U·U and T·T mismatches are dynamic, favoring anti-anti conformations inside the helical core, followed by anti-syn and syn-syn conformations. For DNA, the secondary minima in the non-expanding d(GTC)n helices are deeper, wider, and longer-lived than those in d(CTG)n, which constitutes another biophysical factor further differentiating the expanding and non-expanding sequences. The hybrid helices are closer to A-RNA, with the A-T and A-U pairs forming two stable Watson-Crick hydrogen bonds. The neutralizing ion distribution around the non-canonical pairs is also described.

Frustration Between Preferred States of Complementary Trinucleotide Repeat DNA Hairpins Anticorrelates with Expansion Disease Propensity.

DNA trinucleotide repeat (TRs) expansion beyond a threshold often results in human neurodegenerative diseases. The mechanisms causing expansions remain unknown, although the tendency of TR ssDNA to self-associate into hairpins that slip along their length is widely presumed related. Here we apply single molecule FRET (smFRET) experiments and molecular dynamics simulations to determine conformational stabilities and slipping dynamics for CAG, CTG, GAC and GTC hairpins. Tetraloops are favored in CAG (89%), CTG (89%) and GTC (69%) while GAC favors triloops. We also determined that TTG interrupts near the loop in the CTG hairpin stabilize the hairpin against slipping. The different loop stabilities have implications for intermediate structures that may form when TR-containing duplex DNA opens. Opposing hairpins in the (CAG) âˆ™ (CTG) duplex would have matched stability whereas opposing hairpins in a (GAC) âˆ™ (GTC) duplex would have unmatched stability, introducing frustration in the (GAC) âˆ™ (GTC) opposing hairpins that could encourage their resolution to duplex DNA more rapidly than in (CAG) âˆ™ (CTG) structures. Given that the CAG and CTG TR can undergo large, disease-related expansion whereas the GAC and GTC sequences do not, these stability differences can inform and constrain models of expansion mechanisms of TR regions.

Unpolarized laser method for infrared spectrum calculation of amide I CO bonds in proteins using molecular dynamics simulation.

The investigation of the strong infrared (IR)-active amide I modes of peptides and proteins has received considerable attention because a wealth of detailed information on hydrogen bonding, dipole-dipole interactions, and the conformations of the peptide backbone can be derived from the amide I bands. The interpretation of experimental spectra typically requires substantial theoretical support, such as direct ab-initio molecular dynamics simulation or mixed quantum-classical description. However, considering the difficulties associated with these theoretical methods and their applications are limited in small peptides, it is highly desirable to develop a simple yet efficient approach for simulating the amide I modes of any large proteins in solution. In this work, we proposed a comprehensive computational method that extends the well-established molecular dynamics (MD) simulation method to include an unpolarized IR laser for exciting the CO bonds of proteins. We showed the amide I frequency corresponding to the frequency of the laser pulse which resonated with the CO bond vibration. At this frequency, the protein energy and the CO bond length fluctuation were maximized. Overall, the amide I bands of various single proteins and amyloids agreed well with experimental data. The method has been implemented into the AMBER simulation package, making it widely available to the scientific community. Additionally, the application of the method to simulate the transient amide I bands of amyloid fibrils during the IR laser-induced disassembly process was discussed in details.

Conformations and free energy landscapes of polyproline peptides.

The structure of the proline amino acid allows folded polyproline peptides to exist as both left- (PPII) and right-handed (PPI) helices. We have characterized the free energy landscapes of hexamer, nanomer, and tridecamer polyproline peptides in gas phase and implicit water as well as explicit hexane and 1-propanol for the nanomer. To enhance the sampling provided by regular molecular dynamics, we used the recently developed adaptively biased molecular dynamics method, which describes Landau free energy maps in terms of relevant collective variables. These maps, as a function of the collective variables of handedness, radius of gyration, and three others based on the peptide torsion angle omega, were used to determine the relative stability of the different structures, along with an estimate of the transition pathways connecting the different minima. Results show the existence of several metastable isomers and therefore provide a complementary view to experimental conclusions based on photo-induced electron transfer experiments with regard to the existence of stable heterogeneous subpopulations in PPII polyproline.

RNA as a Major-Groove Ligand: RNA-RNA and RNA-DNA Triplexes Formed by GAA and UUC or TTC Sequences.

Friedreich's ataxia is associated with noncanonical nucleic acid structures that emerge when GAA:TTC repeats in the first intron of the FXN gene expand beyond a critical number of repeats. Specifically, the noncanonical repeats are associated with both triplexes and R-loops. Here, we present an in silico investigation of all possible triplexes that form by attaching a third RNA strand to an RNA:RNA or DNA:DNA duplex, complementing previous DNA-based triplex studies. For both new triplexes results are similar. For a pyridimine UUC+ third strand, the parallel orientation is stable while its antiparallel counterpart is unstable. For a neutral GAA third strand, the parallel conformation is stable. A protonated GA+A third strand is stable in both parallel and antiparallel orientations. We have also investigated Na+ and Mg2+ ion distributions around the triplexes. The presence of Mg2+ ions helps stabilize neutral, antiparallel GAA triplexes. These results (along with previous DNA-based studies) allow for the emergence of a complete picture of the stability and structural characteristics of triplexes based on the GAA and TTC/UUC sequences, thereby contributing to the field of trinucleotide repeats and the associated unusual structures that trigger expansion.