Steve P. Meisburger

Ionic strength-dependent persistence lengths of single-stranded RNA and DNA

Dynamic RNA molecules carry out essential processes in the cell including translation and splicing. Base-pair interactions stabilize RNA into relatively rigid structures, while flexible non-base-paired regions allow RNA to undergo conformational changes required for function. To advance our understanding of RNA folding and dynamics it is critical to know the flexibility of these un-base-paired regions and how it depends on counterions. Yet, information about nucleic acid polymer properties is mainly derived from studies of ssDNA. Here we measure the persistence lengths (lp) of ssRNA. We observe valence and ionic strength-dependent differences in lp in a direct comparison between 40-mers of deoxythymidylate (dT40) and uridylate (rU40) measured using the powerful combination of SAXS and smFRET. We also show that nucleic acid flexibility is influenced by local environment (an adjoining double helix). Our results illustrate the complex interplay between conformation and ion environment that modulates nucleic acid function in vivo.

Both helix topology and counterion distribution contribute to the more effective charge screening in dsRNA compared with dsDNA

The recent discovery of the RNA interference mechanism emphasizes the biological importance of short, isolated, double-stranded (ds) RNA helices and calls for a complete understanding of the biophysical properties of dsRNA. However, most previous studies of the electrostatics of nucleic acid duplexes have focused on DNA. Here, we present a comparative investigation of electrostatic effects in RNA and DNA. Using resonant (anomalous) and non-resonant small-angle X-ray scattering, we characterized the charge screening efficiency and counterion distribution around short (25 bp) dsDNA and RNA molecules of comparable sequence. Consistent with theoretical predictions, we find counterion mediated screening to be more efficient for dsRNA than dsDNA. Furthermore, the topology of the RNA A-form helix alters the spatial distribution of counterions relative to B-form DNA. The experimental results reported here agree well with ion-size-corrected non-linear Poisson–Boltzmann calculations. We propose that differences in electrostatic properties aid in selective recognition of different types of short nucleic acid helices by target binding partners.

RNA and Its Ionic Cloud: Solution Scattering Experiments and Atomically Detailed Simulations

RNA molecules play critical roles in many cellular processes. Traditionally viewed as genetic messengers, RNA molecules were recently discovered to have diverse functions related to gene regulation and expression. RNA also has great potential as a therapeutic and a tool for further investigation of gene regulation. Metal ions are an integral part of RNA structure and should be considered in any experimental or theoretical study of RNA. Here, we report a multidisciplinary approach that combines anomalous small-angle x-ray scattering and molecular-dynamics (MD) simulations with explicit solvent and ions around RNA. From experiment and simulation results, we find excellent agreement in the number and distribution of excess monovalent and divalent ions around a short RNA duplex. Although similar agreement can be obtained from a continuum description of the solvent and mobile ions (by solving the Poisson-Boltzmann equation and accounting for finite ion size), the use of MD is easily extended to flexible RNA systems with thermal fluctuations. Therefore, we also model a short RNA pseudoknot and find good agreement between the MD results and the experimentally derived solution structures. Surprisingly, both deviate from crystal structure predictions. These favorable comparisons of experiment and simulations encourage work on RNA in all-atom dynamic models.

Determining the Locations of Ions and Water around DNA from X-Ray Scattering Measurements

Nucleic acids carry a negative charge, attracting salt ions and water. Interactions with these components of the solvent drive DNA to condense, RNA to fold, and proteins to bind. To understand these biological processes, knowledge of solvent structure around the nucleic acids is critical. Yet, because they are often disordered, ions and water evade detection by x-ray crystallography and other high-resolution methods. Small-angle x-ray scattering (SAXS) is uniquely sensitive to the spatial correlations between solutes and the surrounding solvent. Thus, SAXS provides an experimental constraint to guide or test emerging solvation theories. However, the interpretation of SAXS profiles is nontrivial because of the difficulty in separating the scattering signals of each component: the macromolecule, ions, and hydration water. Here, we demonstrate methods for robustly deconvoluting these signals, facilitating a more straightforward comparison with theory. Using SAXS data collected on an absolute intensity scale for short DNA duplexes in solution with Na(+), K(+), Rb(+), or Cs(+) counterions, we mathematically decompose the scattering profiles into components (DNA, water, and ions) and validate the decomposition using anomalous scattering measurements. In addition, we generate a library of physically motivated ion atmosphere models and rank them by agreement with the scattering data. The best-fit models have relatively compact ion atmospheres when compared to predictions from the mean-field Poisson-Boltzmann theory of electrostatics. Thus, the x-ray scattering methods presented here provide a valuable measurement of the global structure of the ion atmosphere that can be used to test electrostatics theories that go beyond the mean-field approximation.

Revealing transient structures of nucleosomes as DNA unwinds

The modulation of DNA accessibility by nucleosomes is a fundamental mechanism of gene regulation in eukaryotes. The nucleosome core particle (NCP) consists of 147 bp of DNA wrapped around a symmetric octamer of histone proteins. The dynamics of DNA packaging and unpackaging from the NCP affect all DNA-based chemistries, but depend on many factors, including DNA positioning sequence, histone variants and modifications. Although the structure of the intact NCP has been studied by crystallography at atomic resolution, little is known about the structures of the partially unwrapped, transient intermediates relevant to nucleosome dynamics in processes such as transcription, DNA replication and repair. We apply a new experimental approach combining contrast variation with time-resolved small angle X-ray scattering (TR-SAXS) to determine transient structures of protein and DNA constituents of NCPs during salt-induced disassembly. We measure the structures of unwrapping DNA and monitor protein dissociation from Xenopus laevis histones reconstituted with two model NCP positioning constructs: the Widom 601 sequence and the sea urchin 5S ribosomal gene. Both constructs reveal asymmetric release of DNA from disrupted histone cores, but display different patterns of protein dissociation. These kinetic intermediates may be biologically important substrates for gene regulation.

Domain Movements upon Activation of Phenylalanine Hydroxylase Characterized by Crystallography and Chromatography-Coupled Small-Angle X-ray Scattering.

Mammalian phenylalanine hydroxylase (PheH) is an allosteric enzyme that catalyzes the first step in the catabolism of the amino acid phenylalanine. Following allosteric activation by high phenylalanine levels, the enzyme catalyzes the pterin-dependent conversion of phenylalanine to tyrosine. Inability to control elevated phenylalanine levels in the blood leads to increased risk of mental disabilities commonly associated with the inherited metabolic disorder, phenylketonuria. Although extensively studied, structural changes associated with allosteric activation in mammalian PheH have been elusive. Here, we examine the complex allosteric mechanisms of rat PheH using X-ray crystallography, isothermal titration calorimetry (ITC), and small-angle X-ray scattering (SAXS). We describe crystal structures of the preactivated state of the PheH tetramer depicting the regulatory domains docked against the catalytic domains and preventing substrate binding. Using SAXS, we further describe the domain movements involved in allosteric activation of PheH in solution and present the first demonstration of chromatography-coupled SAXS with Evolving Factor Analysis (EFA), a powerful method for separating scattering components in a model-independent way. Together, these results support a model for allostery in PheH in which phenylalanine stabilizes the dimerization of the regulatory domains and exposes the active site for substrate binding and other structural changes needed for activity.

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

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 Sr(2+)) 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.

Polyelectrolyte properties of single stranded DNA measured using SAXS and single-molecule FRET: Beyond the wormlike chain model

Nucleic acids are highly charged polyelectrolytes that interact strongly with salt ions. Rigid, base-paired regions are successfully described with wormlike chain models, but nonbase-paired single stranded regions have fundamentally different polymer properties because of their greater flexibility. Recently, attention has turned to single stranded nucleic acids due to the growing recognition of their biological importance, as well as the availability of sophisticated experimental techniques sensitive to the conformation of individual molecules. We investigate polyelectrolyte properties of poly(dT), an important and widely studied model system for flexible single stranded nucleic acids, in physiologically important mixed mono- and divalent salt. We report measurements of the form factor and interparticle interactions using SAXS, end-to-end distances using smFRET, and number of excess ions using ASAXS. We present a coarse-grained model that accounts for flexibility, excluded volume, and electrostatic interactions in these systems. Predictions of the model are validated against experiment. We also discuss the state of all-atom, explicit solvent molecular dynamics simulations of poly(dT), the next step in understanding the complexities of ion interactions with these highly charged and flexible polymers.

X-ray Scattering Studies of Protein Structural Dynamics

X-ray scattering is uniquely suited to the study of disordered systems and thus has the potential to provide insight into dynamic processes where diffraction methods fail. In particular, while X-ray crystallography has been a staple of structural biology for more than half a century and will continue to remain so, a major limitation of this technique has been the lack of dynamic information. Solution X-ray scattering has become an invaluable tool in structural and mechanistic studies of biological macromolecules where large conformational changes are involved. Such systems include allosteric enzymes that play key roles in directing metabolic fluxes of biochemical pathways, as well as large, assembly-line type enzymes that synthesize secondary metabolites with pharmaceutical applications. Furthermore, crystallography has the potential to provide information on protein dynamics via the diffuse scattering patterns that are overlaid with Bragg diffraction. Historically, these patterns have been very difficult to interpret, but recent advances in X-ray detection have led to a renewed interest in diffuse scattering analysis as a way to probe correlated motions. Here, we will review X-ray scattering theory and highlight recent advances in scattering-based investigations of protein solutions and crystals, with a particular focus on complex enzymes.

Asymmetric unwrapping of nucleosomal DNA propagates asymmetric opening and dissociation of the histone core.

Significance Nucleosomes are fundamental protein–DNA structures through which eukaryotes package and organize DNA inside the nucleus. Nucleosomes are disassembled to gain access to the critical information stored in DNA. Here, we describe a new experimental approach that characterizes the kinetics of nucleosome disassembly and the synergy between DNA conformation and protein components. Using NaCl to disrupt electrostatic interactions, we identify kinetic pathways and transient intermediates that reveal how DNA unwrapping and protein dissociation are linked in this macromolecular complex. These dynamic structures may provide new insight into the regulation of DNA access during transcription, replication, and repair. The nucleosome core particle (NCP) is the basic structural unit for genome packaging in eukaryotic cells and consists of DNA wound around a core of eight histone proteins. DNA access is modulated through dynamic processes of NCP disassembly. Partly disassembled structures, such as the hexasome (containing six histones) and the tetrasome (four histones), are important for transcription regulation in vivo. However, the pathways for their formation have been difficult to characterize. We combine time-resolved (TR) small-angle X-ray scattering and TR-FRET to correlate changes in the DNA conformations with composition of the histone core during salt-induced disassembly of canonical NCPs. We find that H2A–H2B histone dimers are released sequentially, with the first dimer being released after the DNA has formed an asymmetrically unwrapped, teardrop-shape DNA structure. This finding suggests that the octasome-to-hexasome transition is guided by the asymmetric unwrapping of the DNA. The link between DNA structure and histone composition suggests a potential mechanism for the action of proteins that alter nucleosome configurations such as histone chaperones and chromatin remodeling complexes.