Conformational fluctuations and phases in fused in sarcoma (FUS) low-complexity domain.

The well-known phenomenon of phase separation in synthetic polymers and proteins has become a major topic in biophysics because it has been invoked as a mechanism of compartment formation in cells, without the need for membranes. Most of the coacervates (or condensates) are composed of Intrinsically Disordered Proteins (IDPs) or regions that are structureless, often in interaction with RNA and DNA. One of the more intriguing IDPs is the 526-residue RNA-binding protein, Fused in Sarcoma (FUS), whose monomer conformations and condensates exhibit unusual behavior that are sensitive to solution conditions. By focussing principally on the N-terminus low-complexity domain (FUS-LC comprising residues 1-214) and other truncations, we rationalize the findings of solid-state NMR experiments, which show that FUS-LC adopts a non-polymorphic fibril structure (core-1) involving residues 39-95, flanked by fuzzy coats on both the N- and C-terminal ends. An alternate structure (core-2), whose free energy is comparable to core-1, emerges only in the truncated construct (residues 110-214). Both core-1 and core-2 fibrils are stabilized by a Tyrosine ladder as well as hydrophilic interactions. The morphologies (gels, fibrils, and glass-like) adopted by FUS seem to vary greatly, depending on the experimental conditions. The effect of phosphorylation is site-specific. Simulations show that phosphorylation of residues within the fibril has a greater destabilization effect than residues that are outside the fibril region, which accords well with experiments. Many of the peculiarities associated with FUS may also be shared by other IDPs, such as TDP43 and hnRNPA2. We outline a number of problems for which there is no clear molecular explanation.

Differences in the free energies between the excited states of Aß40 and Aß42 monomers encode their aggregation propensities.

The early events in the aggregation of the intrinsically disordered peptide, amyloid-ß (Aß), involve transitions from the disordered free energy ground state to assembly-competent states. Are the fingerprints of order found in the amyloid fibrils encoded in the conformations that the monomers access at equilibrium? If so, could the enhanced aggregation rate of Aß42 compared to Aß40 be rationalized from the sparsely populated high free energy states of the monomers? Here, we answer these questions in the affirmative using coarse-grained simulations of the self-organized polymer-intrinsically disordered protein (SOP-IDP) model of Aß40 and Aß42. Although both the peptides have practically identical ensemble-averaged properties, characteristic of random coils (RCs), the conformational ensembles of the two monomers exhibit sequence-specific heterogeneity. Hierarchical clustering of conformations reveals that both the peptides populate high free energy aggregation-prone ([Formula: see text]) states, which resemble the monomers in the fibril structure. The free energy gap between the ground (RC) and the [Formula: see text] states of Aß42 peptide is smaller than that for Aß40. By relating the populations of excited states of the two peptides to the fibril formation time scales using an empirical formula, we explain nearly quantitatively the faster aggregation rate of Aß42 relative to Aß40. The [Formula: see text] concept accounts for fibril polymorphs, leading to the prediction that the less stable [Formula: see text] state of Aß42, encoding for the U-bend fibril, should form earlier than the structure with the S-bend topology, which is in accord with Ostwald's rule rationalizing crystal polymorph formation.

Lipase sensing by naphthalene diimide based fluorescent organic nanoparticles: a solvent induced manifestation of self-assembly.

The precise control of supramolecular self-assembly is gaining utmost interest for the demanding applications of manifested nano-architecture across the scientific domain. This study delineates the morphological transformation of naphthalene diimide (NDI) derived amphiphiles with varying water content in dimethyl sulfoxide (DMSO) and the selective sensing of lipase using its aggregation-induced emission (AIE) properties. To this end, NDI-based, benzyl alcohol protected alkyl chain (C1, C5, and C10) linked amphiphilic molecules (NDI-1,2,3) were synthesized. Among the synthesized amphiphiles, benzyl ester linked C5 tailored naphthalene diimide (NDI-2) exhibited AIE with an emission maximum at 490 nm in a DMSO-water binary solvent system from fw = 30% and above water content. The fibrous morphology of NDI-2 at fw = 30% got gradually transformed to spherical aggregated particles along with steady increment in the emission intensity upon increasing the amount of water in DMSO. At fw = 99% water in DMSO, complete transformation to fluorescent organic nanoparticles (FONPs) was observed. Microscopic and spectroscopic techniques demonstrated the solvent driven morphological transformation and the AIE property of NDI-2. Moreover, this AIE of NDI-2 FONPs was employed in the selective turn-off sensing of lipase against many other enzymes including esterase, through hydrolysis of a benzyl ester linkage with a limit of detection 10.0 ± 0.8 µg L-1. The NDI-2 FONP also exhibited its lipase sensing efficiency in vitro using a human serum sample.

A multifunnel energy landscape encodes the competing α-helix and ß-hairpin conformations for a designed peptide.

Depending on the amino acid sequence, as well as the local environment, some peptides have the capability to fold into multiple secondary structures. Conformational switching between such structures is a key element of protein folding and aggregation. Specifically, understanding the molecular mechanism underlying the transition from an α-helix to a ß-hairpin is critical because it is thought to be a harbinger of amyloid assembly. In this study, we explore the energy landscape for an 18-residue peptide (DP5), designed by Araki and Tamura to exhibit equal propensities for the α-helical and ß-hairpin forms. We find that the degeneracy is encoded in the multifunnel nature of the underlying free energy landscape. In agreement with experiment, we also observe that mutation of tyrosine at position 12 to serine shifts the equilibrium in favor of the α-helix conformation, by altering the landscape topography. The transition from the α-helix to the ß-hairpin is a complex stepwise process, and occurs via collapsed coil-like intermediates. Our findings suggest that even a single mutation can tune the emergent features of the landscape, providing an efficient route to protein design. Interestingly, the transition pathways for the conformational switch seem to be minimally perturbed upon mutation, suggesting that there could be universal microscopic features that are conserved among different switch-competent protein sequences.

Dynamics of an adenine-adenine RNA conformational switch from discrete path sampling.

The study of "rare event" dynamics can be challenging despite continuing advances in computer hardware. A wide variety of methods based on the master equation approach have been developed to tackle such problems, where the focus is on Markovian dynamics between appropriately defined states. In this contribution, we employ the discrete path sampling approach to characterize pathways and rates for an adenine-adenine RNA conformational switch. The underlying free energy landscape supports competing structures separated by relatively high barriers, with the two principal funnels leading to the major and minor conformations identified by NMR experiments. The interconversion time scale is predicted to be a few hundred seconds, consistent with the experimental lower bound estimates. We find that conformational switching occurs via stacked intermediates, through a sliding mechanism, in agreement with a previous simulation study. By retaining full dimensionality and avoiding low-dimensional projections, the mechanism can be described at an atomistic level of detail.

Charge fluctuation effects on the shape of flexible polyampholytes with applications to intrinsically disordered proteins.

Random polyampholytes (PAs) contain positively and negatively charged monomers that are distributed randomly along the polymer chain. The interaction between charges is assumed to be given by the Debye-Huckel potential. We show that the size of the PA is determined by an interplay between electrostatic interactions, giving rise to the polyelectrolyte effect due to net charge per monomer (σ) and an effective attractive PA interaction due to charge fluctuations, δσ. The interplay between these terms gives rise to non-monotonic dependence of the radius of gyration, R g , on the inverse Debye length, κ, when PA effects are important ( δ σ σ > 1 ). In the opposite limit, R g decreases monotonically with increasing κ. Simulations of PA chains, using a charged bead-spring model, further corroborate our theoretical predictions. The simulations unambiguously show that conformational heterogeneity manifests itself among sequences that have identical PA parameters. A clear implication is that the phases of PA sequences, and by inference intrinsically disordered proteins (IDPs), cannot be determined using only the bare PA parameters (σ and δσ). The theory is used to calculate the changes in R g on N, the number of residues for a set of IDPs. For a certain class of IDPs, with N between 24 and 441, the size grows as R g ∼ N 0.6, which agrees with data from small angle X-ray scattering experiments.

Multifunctional energy landscape for a DNA G-quadruplex: An evolved molecular switch.

We explore the energy landscape for a four-fold telomere repeat, obtaining interconversion pathways between six experimentally characterised G-quadruplex topologies. The results reveal a multi-funnel system, with a variety of intermediate configurations and misfolded states. This organisation is identified with the intrinsically multi-functional nature of the system, suggesting a new paradigm for the classification of such biomolecules and clarifying issues regarding apparently conflicting experimental results.

Probing helical transitions in a DNA duplex.

The complex conformational change from B-DNA to Z-DNA requires inversion of helix-handedness. Multiple degrees of freedom are intricately coupled during this transition, and formulating an appropriate reaction coordinate that captures the underlying complexity would be problematic. In this contribution, we adopt an alternative approach, based on the potential energy landscape perspective, to construct a kinetic transition network. Microscopic insight into the B → Z transition is provided in terms of geometrically defined discrete paths consisting of local minima and the transition states that connect them. We find that the inversion of handedness can occur via two competing mechanisms, either involving stretched intermediates, or a B-Z junction, in agreement with previous predictions. The organisation of the free energy landscape further suggests that this process is likely to be slow under physiological conditions. Our results represent a key step towards decoding the more intriguing features of the B → Z transition, such as the role of ionic strength and negative supercoiling in reshaping the landscape.

Energy landscapes, folding mechanisms, and kinetics of RNA tetraloop hairpins.

RNA hairpins play a pivotal role in a diverse range of cellular functions, and are integral components of ribozymes, mRNA, and riboswitches. However, the mechanistic and kinetic details of RNA hairpin folding, which are key determinants of most of its biological functions, are poorly understood. In this work, we use the discrete path sampling (DPS) approach to explore the energy landscapes of two RNA tetraloop hairpins, and provide insights into their folding mechanisms and kinetics in atomistic detail. Our results show that the potential energy landscapes have a distinct funnel-like bias toward the folded hairpin state, consistent with efficient structure-seeking properties. Mechanistic and kinetic information is analyzed in terms of kinetic transition networks. We find microsecond folding times, consistent with temperature jump experiments, for hairpin folding initiated from relatively compact unfolded states. This process is essentially driven by an initial collapse, followed by rapid zippering of the helix stem in the final phase. Much lower folding rates are predicted when the folding is initiated from extended chains, which undergo longer excursions on the energy landscape before nucleation events can occur. Our work therefore explains recent experiments and coarse-grained simulations, where the folding kinetics exhibit precisely this dependency on the initial conditions.

Brewing COFFEE: A Sequence-Specific Coarse-Grained Energy Function for Simulations of DNA-Protein Complexes.

DNA-protein interactions are pervasive in a number of biophysical processes ranging from transcription and gene expression to chromosome folding. To describe the structural and dynamic properties underlying these processes accurately, it is important to create transferable computational models. Toward this end, we introduce Coarse-grained Force Field for Energy Estimation, COFFEE, a robust framework for simulating DNA-protein complexes. To brew COFFEE, we integrated the energy function in the self-organized polymer model with side-chains for proteins and the three interaction site model for DNA in a modular fashion, without recalibrating any of the parameters in the original force-fields. A unique feature of COFFEE is that it describes sequence-specific DNA-protein interactions using a statistical potential (SP) derived from a data set of high-resolution crystal structures. The only parameter in COFFEE is the strength (λDNAPRO) of the DNA-protein contact potential. For an optimal choice of λDNAPRO, the crystallographic B-factors for DNA-protein complexes with varying sizes and topologies are quantitatively reproduced. Without any further readjustments to the force-field parameters, COFFEE predicts scattering profiles that are in quantitative agreement with small-angle X-ray scattering experiments, as well as chemical shifts that are consistent with NMR. We also show that COFFEE accurately describes the salt-induced unraveling of nucleosomes. Strikingly, our nucleosome simulations explain the destabilization effect of ARG to LYS mutations, which do not alter the balance of electrostatic interactions but affect chemical interactions in subtle ways. The range of applications attests to the transferability of COFFEE, and we anticipate that it would be a promising framework for simulating DNA-protein complexes at the molecular length-scale.

Competition between Stacking and Divalent Cation-Mediated Electrostatic Interactions Determines the Conformations of Short DNA Sequences.

Interplay between divalent cations (Mg2+ and Ca2+) and single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), as well as stacking interactions, is important in nucleosome stability and phase separation in nucleic acids. Quantitative techniques accounting for ion-DNA interactions are needed to obtain insights into these and related problems. Toward this end, we created a sequence-dependent computational TIS-ION model that explicitly accounts for monovalent and divalent ions. Simulations of the rigid 24 base-pair (bp) dsDNA and flexible ssDNA sequences, dT30 and dA30, with varying amounts of the divalent cations show that the calculated excess number of ions around the dsDNA and ssDNA agree quantitatively with ion-counting experiments. Using an ensemble of all-atom structures generated from coarse-grained simulations, we calculated the small-angle X-ray scattering profiles, which are in excellent agreement with experiments. Although ion-counting experiments mask the differences between Mg2+ and Ca2+, we find that Mg2+ binds to the minor grooves and phosphate groups, whereas Ca2+ binds specifically to the minor groove. Both Mg2+ and Ca2+ exhibit a tendency to bind to the minor groove of DNA as opposed to the major groove. The dA30 conformations are dominated by stacking interactions, resulting in structures with considerable helical order. The near cancellation of the favorable stacking and unfavorable electrostatic interactions leads to dT30 populating an ensemble of heterogeneous conformations. The successful applications of the TIS-ION model are poised to confront many problems in DNA biophysics.

Coarse-grained simulations of DNA overstretching.

We use a recently developed coarse-grained model to simulate the overstretching of duplex DNA. Overstretching at 23 °C occurs at 74 pN in the model, about 6-7 pN higher than the experimental value at equivalent salt conditions. Furthermore, the model reproduces the temperature dependence of the overstretching force well. The mechanism of overstretching is always force-induced melting by unpeeling from the free ends. That we never see S-DNA (overstretched duplex DNA), even though there is clear experimental evidence for this mode of overstretching under certain conditions, suggests that S-DNA is not simply an unstacked but hydrogen-bonded duplex, but instead probably has a more exotic structure.

Brewing COFFEE: A sequence-specific coarse-grained energy function for simulations of DNA-protein complexes.

DNA-protein interactions are pervasive in a number of biophysical processes ranging from transcription, gene expression, to chromosome folding. To describe the structural and dynamic properties underlying these processes accurately, it is important to create transferable computational models. Toward this end, we introduce Coarse grained force field for energy estimation, COFFEE, a robust framework for simulating DNA-protein complexes. To brew COFFEE, we integrated the energy function in the Self-Organized Polymer model with Side Chains for proteins and the Three Interaction Site model for DNA in a modular fashion, without re-calibrating any of the parameters in the original force-fields. A unique feature of COFFEE is that it describes sequence-specific DNA-protein interactions using a statistical potential (SP) derived from a dataset of high-resolution crystal structures. The only parameter in COFFEE is the strength (λDNAPRO) of the DNA-protein contact potential. For an optimal choice of λDNAPRO, the crystallographic B-factors for DNA-protein complexes, with varying sizes and topologies, are quantitatively reproduced. Without any further readjustments to the force-field parameters, COFFEE predicts the scattering profiles that are in quantitative agreement with SAXS experiments as well as chemical shifts that are consistent with NMR. We also show that COFFEE accurately describes the salt-induced unraveling of nucleosomes. Strikingly, our nucleosome simulations explain the destabilization effect of ARG to LYS mutations, which does not alter the balance of electrostatic interactions, but affects chemical interactions in subtle ways. The range of applications attests to the transferability of COFFEE, and we anticipate that it would be a promising framework for simulating DNA-protein complexes at the molecular length-scale.

Energy landscapes of Aß monomers are sculpted in accordance with Ostwald's rule of stages.

The transition from a disordered to an assembly-competent monomeric state (N*) in amyloidogenic sequences is a crucial event in the aggregation cascade. Using a well-calibrated model for intrinsically disordered proteins (IDPs), we show that the N* states, which bear considerable resemblance to the polymorphic fibril structures found in experiments, not only appear as excitations in the free energy landscapes of Aß40 and Aß42, but also initiate the aggregation cascade. For Aß42, the transitions to the different N* states are in accord with Ostwald's rule of stages, with the least stable structures forming ahead of thermodynamically favored ones. The Aß40 and Aß42 monomer landscapes exhibit different extents of local frustration, which we show have profound implications in dictating subsequent self-assembly. Using kinetic transition networks, we illustrate that the most favored dimerization routes proceed via N* states. We argue that Ostwald's rule also holds for the aggregation of fused in sarcoma and polyglutamine proteins.

Conformational Fluctuations and Phases in Fused in Sarcoma (FUS) Low-Complexity Domain.

The well known phenomenon of phase separation in synthetic polymers and proteins has become a major topic in biophysics because it has been invoked as a mechanism of compartment formation in cells, without the need for membranes. Most of the coacervates (or condensates) are composed of Intrinsically Disordered Proteins (IDPs) or regions that are structureless, often in interaction with RNA and DNA. One of the more intriguing IDPs is the 526-residue RNA binding protein, Fused In Sarcoma (FUS), whose monomer conformations and condensates exhibit unusual behavior that are sensitive to solution conditions. By focussing principally on the N-terminus low complexity domain (FUS-LC comprising residues 1-214) and other truncations, we rationalize the findings of solid state NMR experiments, which show that FUS-LC adopts a non-polymorphic fibril (core-1) involving residues 39-95, flanked by fuzzy coats on both the N- and C- terminal ends. An alternate structure (core-2), whose free energy is comparable to core-1, emerges only in the truncated construct (residues 110-214). Both core-1 and core-2 fibrils are stabilized by a Tyrosine ladder as well as hydrophilic interactions. The morphologies (gels, fibrils, and glass-like behavior) adopted by FUS seem to vary greatly, depending on the experimental conditions. The effect of phosphorylation is site specific and affects the stability of the fibril depending on the sites that are phosphorylated. Many of the peculiarities associated with FUS may also be shared by other IDPs, such as TDP43 and hnRNPA2. We outline a number of problems for which there is no clear molecular understanding.

Energy Landscapes for Base-Flipping in a Model DNA Duplex.

We explore the process of base-flipping for four central bases, adenine, guanine, cytosine, and thymine, in a deoxyribonucleic acid (DNA) duplex using the energy landscape perspective. NMR imino-proton exchange and fluorescence correlation spectroscopy studies have been used in previous experiments to obtain lifetimes for bases in paired and extrahelical states. However, the difference of almost 4 orders of magnitude in the base-flipping rates obtained by the two methods implies that they are exploring different pathways and possibly different open states. Our results support the previous suggestion that minor groove opening may be favored by distortions in the DNA backbone and reveal links between sequence effects and the direction of opening, i.e., whether the base flips toward the major or the minor groove side. In particular, base flipping along the minor groove pathway was found to align toward the 5' side of the backbone. We find that bases align toward the 3' side of the backbone when flipping along the major groove pathway. However, in some cases for cytosine and thymine, the base flipping along the major groove pathway also aligns toward the 5' side. The sequence effect may be caused by the polar interactions between the flipping-base and its neighboring bases on either of the strands. For guanine flipping toward the minor groove side, we find that the equilibrium constant for opening is large compared to flipping via the major groove. We find that the estimated rates of base opening, and hence the lifetimes of the closed state, obtained for thymine flipping through small and large angles along the major groove differ by 6 orders of magnitude, whereas for thymine flipping through small angles along the minor groove and large angles along the major groove, the rates differ by 3 orders of magnitude.

Side-Chain Polarity Modulates the Intrinsic Conformational Landscape of Model Dipeptides.

The intrinsic conformational preferences of small peptides may provide additional insight into the thermodynamics and kinetics of protein folding. In this study, we explore the underlying energy landscapes of two model peptides, namely, Ac-Ala-NH2 and Ac-Ser-NH2, using geometry-optimization-based tools developed within the context of energy landscape theory. We analyze not only how side-chain polarity influences the structural preferences of the dipeptides, but also other emergent properties of the landscape, including heat capacity profiles, and kinetics of conformational rearrangements. The contrasting topographies of the free energy landscape agree with recent results from Fourier transform microwave spectroscopy experiments, where Ac-Ala-NH2 was found to exist as a mixture of two conformers, while Ac-Ser-NH2 remained structurally locked, despite exhibiting an apparently rich conformational landscape.

Sequence Determines the Switch in the Fibril Forming Regions in the Low-Complexity FUS Protein and Its Variants.

Residues spanning distinct regions of the low-complexity domain of the RNA-binding protein, Fused in Sarcoma (FUS-LC), form fibril structures with different core morphologies. Solid-state NMR experiments show that the 214-residue FUS-LC forms a fibril with an S-bend (core-1, residues 39-95), while the rest of the protein is disordered. In contrast, the fibrils of the C-terminal variant (FUS-LC-C; residues 111-214) have a U-bend topology (core-2, residues 112-150). Absence of the U-bend in FUS-LC implies that the two fibril cores do not coexist. Computer simulations show that these perplexing findings could be understood in terms of the population of sparsely populated fibril-like excited states in the monomer. The propensity to form core-1 is higher compared to core-2. We predict that core-2 forms only in truncated variants that do not contain the core-1 sequence. At the monomer level, sequence-dependent enthalpic effects determine the relative stabilities of the core-1 and core-2 topologies.

Energy Landscapes and Hybridization Pathways for DNA Hexamer Duplexes.

Strand hybridization is not only a fundamental molecular mechanism underlying the biological functions of nucleic acids but is also a key step in the design of efficient nanodevices. Despite recent efforts, the microscopic rules governing the hybridization mechanisms remain largely unknown. In this study, we exploit the energy landscape framework to assess how sequence-specificity modulates the hybridization mechanisms in DNA. We find that GG-tracts hybridize much more rapidly compared to GC-tracts, via either zippering or slithering pathways. For the hybridization of GG-tracts, both zippering and slithering mechanisms appear to be kinetically relevant. In contrast, for the GC-tracts, the zippering mechanism is dominant. Our work reveals that even for the relatively small systems considered, the energy landscapes feature multiple metastable states and kinetic traps, which is at odds with the conventional "all-or-nothing" model of DNA hybridization formulated on the basis of thermodynamic arguments alone. Interestingly, entropic effects are found to play an important role in determining the thermal stability of competing conformational ensembles and in determining the preferred hybridization pathways.

Morphological transformation of self-assemblies by tuning hydrophobic segment of small amphiphiles.

HYPOTHESIS: With increasing surge in the development of supramolecular self-assemblies, it is crucial to predict the influence of amphiphilic segment in dictating the morphology of self-aggregates. This article reports the design and synthesis of low molecular weight amphiphiles with varying hydrophobicity both in the spacer unit and at the terminal moiety. EXPERIMENTS: Hydrophobicity at the spacer moiety was modulated by altering alkyl chain length and by inclusion of aromatic ring and the same was changed at hydrophobic terminal using pyrene, naphthalene, n-hexadecane having 2-aminopyridine as polar head. Microscopy and spectroscopy were used to investigate the morphologies of self-aggregated amphiphiles. FINDINGS: Variation of hydrophobicity of the spacer moiety either by changing the alkyl chain length (C0, C2, C6, C11 and phenyl ring) having pyrene as terminal hydrophobic unit led to the formation of only spherical vesicles in respective solvent system. Morphological transformation of self-aggregates from vesicle to fused-vesicle to gel was observed in DMSO-water upon alteration in the hydrophobic end of amphiphile from pyrene to naphthyl to C16 alkyl chain having C6 alkyl chain as spacer. Hence, the hydrophobicity at the terminal of the amphiphile has the predominant role in tuning the morphology of self-aggregates through modulation in the hydrophobic-lipophilic balance (HLB) of amphiphiles.