publications

2024

1. 

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

   Balaka Mondal, Debayan Chakraborty, Naoto Hori, Hung T. Nguyen, and D. Thirumalai

   J. Chem. Theory Comput., 2024

   Abstract bioRxiv HTML

   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.

2. 

   Salt-Dependent Self-Association of Trinucleotide Repeat RNA Sequences

   Hiranmay Maity, Hung T. Nguyen, Naoto Hori, and D. Thirumalai

   J. Phys. Chem. Lett., 2024

   Abstract bioRxiv HTML

   Repeat RNA sequences self-associate to form condensates. Simulations of a coarse-grained single-interaction site model for (CAG)n (n = 30 and 31) show that the salt-dependent free energy gap, ΔGS, between the ground (perfect hairpin) and the excited state (slipped hairpin (SH) with one CAG overhang) of the monomer for (n even) is the primary factor that determines the rates and yield of self-assembly. For odd n, the free energy (GS) of the ground state, which is an SH, is used to predict the self-association kinetics. As the monovalent salt concentration, CS, increases, ΔGS and GS increase, which decreases the rates of dimer formation. In contrast, ΔGS for shuffled sequences, with the same length and sequence composition as (CAG)31, is larger, which suppresses their propensities to aggregate. Although demonstrated explicitly for (CAG) polymers, the finding of inverse correlation between the free energy gap and RNA aggregation is general.

2023

1. 

   Sizes, Conformational fluctuations, and SAXS profiles for Intrinsically Disordered Proteins

   Mauro L. Mugnai, Debayan Chakraborty, Abhinaw Kumar, Hung T. Nguyen, Wade Zeno, Jeanne C. Stachowiak, John E. Straub, and D. Thirumalai

   bioRxiv, 2023

   Abstract bioRxiv Code

   The preponderance of Intrinsically Disordered Proteins (IDPs) in the eukaryotic proteome, and their ability to interact with each other, proteins, RNA, and DNA for functional purposes have made it important to quantitatively characterize their biophysical properties. Towards this end, we developed the transferable Self-Organized Polymer (SOP-IDP) model in order to calculate the properties of a number of IDPs. The calculated and measured radius of gyration values (Rgs) are in excellent agreement, with a correlation coefficient of 0.96. For AP180 and Epsin, the predicted and values obtained using Fluorescence Correlation Spectroscopy for the hydrodynamic radii (Rhs) are also in quantitative agreement. Strikingly, the calculated SAXS profiles for thirty six IDPs also nearly match the experiments. The dependence of Rg, the mean end-to-end distance (Re), and Rh obey the Flory scaling law, Rα ≈ ααN0.59 (α = g, e, and h), suggesting that globally IDPs behave as polymers in a good solvent. The values of αg, αe, and αh are 0.21 nm, 0.53 nm, and 0.16 nm, respectively. Surprisingly, finite size corrections to scaling, expected on theoretical grounds, for all the three quantities are negligible. Sequence dependencies, masked in ensemble properties, emerge through a fine structure analyses of the conformational ensembles using a hierarchical clustering method. Typically, the ensemble of conformations partition into three distinct clusters, with differing extent of population and structural properties. The subpopulations could dictate phase separation tendencies and association with ligands. Without any adjustments to the three parameters in the SOP-IDP model, we obtained excellent agreement with paramagnetic relaxation enhancement (PRE) measurements for α-synuclein. The transferable SOP-IDP model sets the stage for a number of promising applications, including the study of phase separation in IDPs and interactions with nucleic acids.

2. 

   Odd-even disparity in the population of slipped hairpins in RNA repeat sequences with implications for phase separation

   Hiranmay Maity, Hung T. Nguyen, Naoto Hori, and D. Thirumalai

   Proc. Nat. Acad. Sci., 2023

   Abstract bioRxiv HTML

   Low-complexity nucleotide repeat sequences, which are implicated in several neurological disorders, undergo liquid–liquid phase separation (LLPS) provided the number of repeat units, n, exceeds a critical value. Here, we establish a link between the folding landscapes of the monomers of trinucleotide repeats and their propensity to self-associate. Simulations using a coarse-grained Self-Organized Polymer (SOP) model for (CAG)n repeats in monovalent salt solutions reproduce experimentally measured melting temperatures, which are available only for small n. By extending the simulations to large n, we show that the free-energy gap, ΔGS, between the ground state (GS) and slipped hairpin (SH) states is a predictor of aggregation propensity. The GS for even n is a perfect hairpin (PH), whereas it is a SH when n is odd. The value of ΔGS (zero for odd n) is larger for even n than for odd n. As a result, the rate of dimer formation is slower in (CAG)30 relative to (CAG)31, thus linking ΔGS to RNA–RNA association. The yield of the dimer decreases dramatically, compared to the wild type, in mutant sequences in which the population of the SH decreases substantially. Association between RNA chains is preceded by a transition to the SH even if the GS is a PH. The finding that the excitation spectrum—which depends on the exact sequence, n, and ionic conditions—is a predictor of self-association should also hold for other RNAs (mRNA for example) that undergo LLPS.

2022

1. 

   Condensates in RNA repeat sequences are heterogeneously organized and exhibit reptation dynamics

   Hung T. Nguyen, Naoto Hori, and D. Thirumalai

   Nat. Chem., 2022

   Abstract bioRxiv HTML Code

   Although it is known that RNA undergoes liquid–liquid phase separation, the interplay between the molecular driving forces and the emergent features of the condensates, such as their morphologies and dynamic properties, is not well understood. We introduce a coarse-grained model to simulate phase separation of trinucleotide repeat RNAs, which are implicated in neurological disorders. After establishing that the simulations reproduce key experimental findings, we show that once recruited inside the liquid droplets, the monomers transition from hairpin-like structures to extended states. Interactions between the monomers in the condensates result in the formation of an intricate and dense intermolecular network, which severely restrains the fluctuations and mobilities of the RNAs inside large droplets. In the largest densely packed high-viscosity droplets, the mobility of RNA chains is best characterized by reptation, reminiscent of the dynamics in polymer melts. Our work provides a microscopic framework for understanding liquid–liquid phase separation in RNA, which is not easily discernible in current experiments.

2020

1. 

   Charge Density of Cation Determines Inner versus Outer Shell Coordination to Phosphate in RNA

   Hung T. Nguyen, and D. Thirumalai

   J. Phys. Chem. B, 2020

   Abstract bioRxiv HTML

   Divalent cations are often required to fold RNA, which is a highly charged polyanion. Condensation of ions, such as Mg2+ or Ca2+, in the vicinity of RNA renormalizes the effective charges on the phosphate groups, thus minimizing the intra RNA electrostatic repulsion. The prevailing view is that divalent ions bind diffusively in a nonspecific manner. In sharp contrast, we arrive at the exact opposite conclusion using a theory for the interaction of ions with the phosphate groups using RISM theory in conjunction with simulations based on an accurate three-interaction-site RNA model. The divalent ions bind in a nucleotide-specific manner using either the inner (partially dehydrated) or outer (fully hydrated) shell coordination. The high charge density Mg2+ ion has a preference to bind to the outer shell, whereas the opposite is the case for Ca2+. Surprisingly, we find that bridging interactions, involving ions that are coordinated to two or more phosphate groups, play a crucial role in maintaining the integrity of the folded state. Their importance could become increasingly prominent as the size of the RNA increases. Because the modes of interaction of divalent ions with DNA are likely to be similar, we propose that specific inner and outer shell coordination could play a role in DNA condensation, and perhaps genome organization as well.

2019

1. 

   Theory and simulations for RNA folding in mixtures of monovalent and divalent cations

   Hung T. Nguyen, Naoto Hori, and D. Thirumalai

   Proc. Nat. Acad. Sci., 2019

   Abstract bioRxiv HTML Code

   RNA molecules cannot fold in the absence of counterions. Experiments are typically performed in the presence of monovalent and divalent cations. How to treat the impact of a solution containing a mixture of both ion types on RNA folding has remained a challenging problem for decades. By exploiting the large concentration difference between divalent and monovalent ions used in experiments, we develop a theory based on the reference interaction site model (RISM), which allows us to treat divalent cations explicitly while keeping the implicit screening effect due to monovalent ions. Our theory captures both the inner shell and outer shell coordination of divalent cations to phosphate groups, which we demonstrate is crucial for an accurate calculation of RNA folding thermodynamics. The RISM theory for ion–phosphate interactions when combined with simulations based on a transferable coarse-grained model allows us to predict accurately the folding of several RNA molecules in a mixture containing monovalent and divalent ions. The calculated folding free energies and ion-preferential coefficients for RNA molecules (pseudoknots, a fragment of the rRNA, and the aptamer domain of the adenine riboswitch) are in excellent agreement with experiments over a wide range of monovalent and divalent ion concentrations. Because the theory is general, it can be readily used to investigate ion and sequence effects on DNA properties.

2016

1. 

   Extracting water and ion distributions from solution x-ray scattering experiments

   Hung T. Nguyen, Suzette A. Pabit, Lois Pollack, and David A. Case

   J. Chem. Phys., 2016

   Abstract HTML

   Small-angle X-ray scattering measurements can provide valuable information about the solvent environment around biomolecules, but it can be difficult to extract solvent-specific information from observed intensity profiles. Intensities are proportional to the square of scattering amplitudes, which are complex quantities. Amplitudes in the forward direction are real, and the contribution from a solute of known structure (and from the waters it excludes) can be estimated from theory; hence, the amplitude arising from the solvent environment can be computed by difference. We have found that this “square root subtraction scheme” can be extended to non-zero q values, out to 0.1 Å−1 for the systems considered here, since the phases arising from the solute and from the water environment are nearly identical in this angle range. This allows us to extract aspects of the water and ion distributions (beyond their total numbers), by combining experimental data for the complete system with calculations for the solutes. We use this approach to test molecular dynamics and integral-equation (3D-RISM (three-dimensional reference interaction site model)) models for solvent structure around myoglobin, lysozyme, and a 25 base-pair duplex DNA. Comparisons can be made both in Fourier space and in terms of the distribution of interatomic distances in real space. Generally, computed solvent distributions arising from the MD simulations fit experimental data better than those from 3D-RISM, even though the total small-angle X-ray scattering patterns are very similar; this illustrates the potential power of this sort of analysis to guide the development of computational models.

2014

1. 

   Accurate small and wide angle x-ray scattering profiles from atomic models of proteins and nucleic acids

   Hung T. Nguyen, Suzette A. Pabit, Steve P. Meisburger, Lois Pollack, and David A. Case

   J. Chem. Phys., 2014

   Abstract HTML Code

   A new method is introduced to compute X-ray solution scattering profiles from atomic models of macromolecules. The three-dimensional version of the Reference Interaction Site Model (RISM) from liquid-state statistical mechanics is employed to compute the solvent distribution around the solute, including both water and ions. X-ray scattering profiles are computed from this distribution together with the solute geometry. We describe an efficient procedure for performing this calculation employing a Lebedev grid for the angular averaging. The intensity profiles (which involve no adjustable parameters) match experiment and molecular dynamics simulations up to wide angle for two proteins (lysozyme and myoglobin) in water, as well as the small-angle profiles for a dozen biomolecules taken from the BioIsis.net database. The RISM model is especially well-suited for studies of nucleic acids in salt solution. Use of fiber-diffraction models for the structure of duplex DNA in solution yields close agreement with the observed scattering profiles in both the small and wide angle scattering (SAXS and WAXS) regimes. In addition, computed profiles of anomalous SAXS signals (for Rb+ and Sr2+) emphasize the ionic contribution to scattering and are in reasonable agreement with experiment. In cases where an absolute calibration of the experimental data at q = 0 is available, one can extract a count of the excess number of waters and ions; computed values depend on the closure that is assumed in the solution of the Ornstein–Zernike equations, with results from the Kovalenko–Hirata closure being closest to experiment for the cases studied here.

2010

1. 

   Mechanical Unfolding of Acylphosphatase Studied by Single-Molecule Force Spectroscopy and MD Simulations

   Gali Arad-Haase, Silvia G. Chuartzman, Shlomi Dagan, Reinat Nevo, Maksim Kouza, Binh Khanh Mai, Hung T. Nguyen, Mai Suan Li, and 1 more author

   Biophys. J., 2010

   Abstract HTML

   Single-molecule manipulation methods provide a powerful means to study protein transitions. Here we combined single-molecule force spectroscopy and steered molecular-dynamics simulations to study the mechanical properties and unfolding behavior of the small enzyme acylphosphatase (AcP). We find that mechanical unfolding of AcP occurs at relatively low forces in an all-or-none fashion and is decelerated in the presence of a ligand, as observed in solution measurements. The prominent energy barrier for the transition is separated from the native state by a distance that is unusually long for α/β proteins. Unfolding is initiated at the C-terminal strand (βT) that lies at one edge of the β-sheet of AcP, followed by unraveling of the strand located at the other. The central strand of the sheet and the two helices in the protein unfold last. Ligand binding counteracts unfolding by stabilizing contacts between an arginine residue (Arg-23) and the catalytic loop, as well as with βT of AcP, which renders the force-bearing units of the protein resistant to force. This stabilizing effect may also account for the decelerated unfolding of ligand-bound AcP in the absence of force.

2009

1. Top-hits for H1N1pdm identified by virtual screening using ensemble-based docking

   Hung T. Nguyen, Ly Le, and Thanh N. Truong

   PLoS currents, 2009