Conformational Dynamics of the Hepatitis C Virus 3'X RNA.

The 3' end of the hepatitis C virus genome is terminated by a highly conserved, 98-nucleotide sequence called 3'X. This untranslated structural element is thought to regulate several essential RNA-dependent processes associated with infection. 3'X has two proposed conformations comprised of either three- or two stem-loop structures that result from different base pairing interactions within the first 55 nucleotides. Here, we used single-molecule FRET spectroscopy to monitor the conformational status of fluorescently labeled constructs that isolate this region of the RNA (3'X55). We observed that 3'X55 can adopt both proposed conformations and the relative abundance of them can be modulated by either solution conditions or nucleotide deletions. Furthermore, interconversion between the two conformations is slow and takes place over the course of several hours. The simultaneous existence of two slowly interconverting conformations may help prime individual copies of the viral genome for either viral protein or RNA synthesis, thereby minimizing conflicts between these two competing processes.

Single-molecule-binding studies of antivirals targeting the hepatitis C virus core protein.

IMPORTANCE: The hepatitis C virus is associated with nearly 300,000 deaths annually. At the core of the virus is an RNA-protein complex called the nucleocapsid, which consists of the viral genome and many copies of the core protein. Because the assembly of the nucleocapsid is a critical step in viral replication, a considerable amount of effort has been devoted to identifying antiviral therapeutics that can bind to the core protein and disrupt assembly. Although several candidates have been identified, little is known about how they interact with the core protein or how those interactions alter the structure and thus the function of this viral protein. Our work biochemically characterizes several of these binding interactions, highlighting both similarities and differences as well as strengths and weaknesses. These insights bolster the notion that this viral protein is a viable target for novel therapeutics and will help to guide future developments of these candidate antivirals.

Mechanism of Ligand Binding to Theophylline RNA Aptamer.

Studying RNA-ligand interactions and quantifying their binding thermodynamics and kinetics are of particular relevance in the field of drug discovery. Here, we combined biochemical binding assays and accelerated molecular simulations to investigate ligand binding and dissociation in RNA using the theophylline-binding RNA as a model system. All-atom simulations using a Ligand Gaussian accelerated Molecular Dynamics method (LiGaMD) have captured repetitive binding and dissociation of theophylline and caffeine to RNA. Theophylline's binding free energy and kinetic rate constants align with our experimental data, while caffeine's binding affinity is over 10,000 times weaker, and its kinetics could not be determined. LiGaMD simulations allowed us to identify distinct low-energy conformations and multiple ligand binding pathways to RNA. Simulations revealed a "conformational selection" mechanism for ligand binding to the flexible RNA aptamer, which provides important mechanistic insights into ligand binding to the theophylline-binding model. Our findings suggest that compound docking using a structural ensemble of representative RNA conformations would be necessary for structure-based drug design of flexible RNA.

Recognition of single-stranded nucleic acids by small-molecule splicing modulators.

Risdiplam is the first approved small-molecule splicing modulator for the treatment of spinal muscular atrophy (SMA). Previous studies demonstrated that risdiplam analogues have two separate binding sites in exon 7 of the SMN2 pre-mRNA: (i) the 5'-splice site and (ii) an upstream purine (GA)-rich binding site. Importantly, the sequence of this GA-rich binding site significantly enhanced the potency of risdiplam analogues. In this report, we unambiguously determined that a known risdiplam analogue, SMN-C2, binds to single-stranded GA-rich RNA in a sequence-specific manner. The minimum required binding sequence for SMN-C2 was identified as GAAGGAAGG. We performed all-atom simulations using a robust Gaussian accelerated molecular dynamics (GaMD) method, which captured spontaneous binding of a risdiplam analogue to the target nucleic acids. We uncovered, for the first time, a ligand-binding pocket formed by two sequential GAAG loop-like structures. The simulation findings were highly consistent with experimental data obtained from saturation transfer difference (STD) NMR and structure-affinity-relationship studies of the risdiplam analogues. Together, these studies illuminate us to understand the molecular basis of single-stranded purine-rich RNA recognition by small-molecule splicing modulators with an unprecedented binding mode.

The SARS-CoV-2 Conserved Macrodomain Is a Mono-ADP-Ribosylhydrolase.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other SARS-related CoVs encode 3 tandem macrodomains within nonstructural protein 3 (nsp3). The first macrodomain, Mac1, is conserved throughout CoVs and binds to and hydrolyzes mono-ADP-ribose (MAR) from target proteins. Mac1 likely counters host-mediated antiviral ADP-ribosylation, a posttranslational modification that is part of the host response to viral infections. Mac1 is essential for pathogenesis in multiple animal models of CoV infection, implicating it as a virulence factor and potential therapeutic target. Here, we report the crystal structure of SARS-CoV-2 Mac1 in complex with ADP-ribose. SARS-CoV-2, SARS-CoV, and Middle East respiratory syndrome coronavirus (MERS-CoV) Mac1 domains exhibit similar structural folds, and all 3 proteins bound to ADP-ribose with affinities in the low micromolar range. Importantly, using ADP-ribose-detecting binding reagents in both a gel-based assay and novel enzyme-linked immunosorbent assays (ELISAs), we demonstrated de-MARylating activity for all 3 CoV Mac1 proteins, with the SARS-CoV-2 Mac1 protein leading to a more rapid loss of substrate than the others. In addition, none of these enzymes could hydrolyze poly-ADP-ribose. We conclude that the SARS-CoV-2 and other CoV Mac1 proteins are MAR-hydrolases with similar functions, indicating that compounds targeting CoV Mac1 proteins may have broad anti-CoV activity.IMPORTANCE SARS-CoV-2 has recently emerged into the human population and has led to a worldwide pandemic of COVID-19 that has caused more than 1.2 million deaths worldwide. With no currently approved treatments, novel therapeutic strategies are desperately needed. All coronaviruses encode a highly conserved macrodomain (Mac1) that binds to and removes ADP-ribose adducts from proteins in a dynamic posttranslational process that is increasingly being recognized as an important factor that regulates viral infection. The macrodomain is essential for CoV pathogenesis and may be a novel therapeutic target. Thus, understanding its biochemistry and enzyme activity are critical first steps for these efforts. Here, we report the crystal structure of SARS-CoV-2 Mac1 in complex with ADP-ribose and describe its ADP-ribose binding and hydrolysis activities in direct comparison to those of SARS-CoV and MERS-CoV Mac1 proteins. These results are an important first step for the design and testing of potential therapies targeting this unique protein domain.

Real-time compaction of nanoconfined DNA by an intrinsically disordered macromolecular counterion.

We demonstrate how a recently developed nanofluidic device can be used to study protein-induced compaction of genome-length DNA freely suspended in solution. The protein we use in this study is the hepatitis C virus core protein (HCVcp), which is a positively charged, intrinsically disordered protein. Using nanofluidic devices in combination with fluorescence microscopy, we observe that protein-induced compaction preferentially begins at the ends of linear DNA. This observation would be difficult to make with many other single-molecule techniques, which generally require the DNA ends to be anchored to a substrate. We also demonstrate that this protein-induced compaction is reversible and can be dynamically modulated by exposing the confined DNA molecules to solutions containing either HCVcp (to promote compaction) or Proteinase K (to disassemble the compact nucleo-protein complex). Although the natural binding partner for HCVcp is genomic viral RNA, the general biophysical principles governing protein-induced compaction of DNA are likely relevant for a broad range of nucleic acid-binding proteins and their targets.

Biophysical Insights from Temperature-Dependent Single-Molecule Förster Resonance Energy Transfer.

Single-molecule fluorescence microscopy techniques can be used in combination with micrometer length-scale temperature control and Förster resonance energy transfer (FRET) in order to gain detailed information about fundamental biophysical phenomena. In particular, this combination of techniques has helped foster the development of remarkable quantitative tools for studying both time- and temperature-dependent structural kinetics of biopolymers. Over the past decade, multiple research efforts have successfully incorporated precise spatial and temporal control of temperature into single-molecule FRET (smFRET)-based experiments, which have uncovered critical thermodynamic information on a wide range of biological systems such as conformational dynamics of nucleic acids. This review provides an overview of various temperature-dependent smFRET approaches from our laboratory and others, highlighting efforts in which such methods have been successfully applied to studies of single-molecule nucleic acid folding.

Molecular-crowding effects on single-molecule RNA folding/unfolding thermodynamics and kinetics.

The effects of "molecular crowding" on elementary biochemical processes due to high solute concentrations are poorly understood and yet clearly essential to the folding of nucleic acids and proteins into correct, native structures. The present work presents, to our knowledge, first results on the single-molecule kinetics of solute molecular crowding, specifically focusing on GAAA tetraloop-receptor folding to isolate a single RNA tertiary interaction using time-correlated single-photon counting and confocal single-molecule FRET microscopy. The impact of crowding by high-molecular-weight polyethylene glycol on the RNA folding thermodynamics is dramatic, with up to ΔΔG° ∼ -2.5 kcal/mol changes in free energy and thus >60-fold increase in the folding equilibrium constant (Keq) for excluded volume fractions of 15%. Most importantly, time-correlated single-molecule methods permit crowding effects on the kinetics of RNA folding/unfolding to be explored for the first time (to our knowledge), which reveal that this large jump in Keq is dominated by a 35-fold increase in tetraloop-receptor folding rate, with only a modest decrease in the corresponding unfolding rate. This is further explored with temperature-dependent single-molecule RNA folding measurements, which identify that crowding effects are dominated by entropic rather than enthalpic contributions to the overall free energy change. Finally, a simple "hard-sphere" treatment of the solute excluded volume is invoked to model the observed kinetic trends, and which predict ΔΔG° ∼ -5 kcal/mol free-energy stabilization at excluded volume fractions of 30%.

Single-Molecule FRET Reveals Three Conformations for the TLS Domain of Brome Mosaic Virus Genome.

Metabolite-dependent conformational switching in RNA riboswitches is now widely accepted as a critical regulatory mechanism for gene expression in bacterial systems. More recently, similar gene regulation mechanisms have been found to be important for viral systems as well. One of the most abundant and best-studied systems is the tRNA-like structure (TLS) domain, which has been found to occur in many plant viruses spread across numerous genera. In this work, folding dynamics for the TLS domain of Brome Mosaic Virus have been investigated using single-molecule fluorescence resonance energy transfer techniques. In particular, burst fluorescence methods are exploited to observe metal-ion ([M(n+)])-induced folding in freely diffusing RNA constructs resembling the minimal TLS element of brome mosaic virus RNA3. The results of these experiments reveal a complex equilibrium of at least three distinct populations. A stepwise, or consecutive, thermodynamic model for TLS folding is developed, which is in good agreement with the [M(n+)]-dependent evolution of conformational populations and existing structural information in the literature. Specifically, this folding pathway explains the metal-ion dependent formation of a functional TLS domain from unfolded RNAs via two consecutive steps: 1) hybridization of a long-range stem interaction, followed by 2) formation of a 3'-terminal pseudoknot. These two conformational transitions are well described by stepwise dissociation constants for [Mg(2+)] (K1 = 328 ± 30 µM and K2 = 1092 ± 183 µM) and [Na(+)] (K1 = 74 ± 6 mM and K2 = 243 ± 52 mM)-induced folding. The proposed thermodynamic model is further supported by inhibition studies of the long-range stem interaction using a complementary DNA oligomer, which effectively shifts the dynamic equilibrium toward the unfolded conformation. Implications of this multistep conformational folding mechanism are discussed with regard to regulation of virus replication.

Entropic origin of Mg2+-facilitated RNA folding.

Mg(2+) is essential for the proper folding and function of RNA, though the effect of Mg(2+) concentration on the free energy, enthalpy, and entropy landscapes of RNA folding is unknown. This work exploits temperature-controlled single-molecule FRET methods to address the thermodynamics of RNA folding pathways by probing the intramolecular docking/undocking kinetics of the ubiquitous GAAA tetraloop-receptor tertiary interaction as a function of [Mg(2+)]. These measurements yield the barrier and standard state enthalpies, entropies, and free energies for an RNA tertiary transition, in particular, revealing the thermodynamic origin of [Mg(2+)]-facilitated folding. Surprisingly, these studies reveal that increasing [Mg(2+)] promotes tetraloop-receptor interaction by reducing the entropic barrier (-TΔS(++)(dock)) and the overall entropic penalty (-TΔS(+) (dock)) for docking, with essentially negligible effects on both the activation enthalpy (ΔH(++)(dock)) and overall exothermicity (ΔH(+)(dock)). These observations contrast with the conventional notion that increasing [Mg(2+)] facilitates folding by minimizing electrostatic repulsion of opposing RNA helices, which would incorrectly predict a decrease in ΔH(++)(dock)) and ΔH(+)(dock)) with [Mg(2+)]. Instead we propose that higher [Mg(2+)] can aid RNA folding by decreasing the entropic penalty of counterion uptake and by reducing disorder of the unfolded conformational ensemble.

Pulsed IR heating studies of single-molecule DNA duplex dissociation kinetics and thermodynamics.

Single-molecule fluorescence spectroscopy is a powerful technique that makes it possible to observe the conformational dynamics associated with biomolecular processes. The addition of precise temperature control to these experiments can yield valuable thermodynamic information about equilibrium and kinetic rate constants. To accomplish this, we have developed a microscopy technique based on infrared laser overtone/combination band absorption to heat small (≈10(-11) liter) volumes of water. Detailed experimental characterization of this technique reveals three major advantages over conventional stage heating methods: 1), a larger range of steady-state temperatures (20-100°C); 2), substantially superior spatial (≤20 µm) control; and 3), substantially superior temporal (≈1 ms) control. The flexibility and breadth of this spatial and temporally resolved laser-heating approach is demonstrated in single-molecule fluorescence assays designed to probe the dissociation of a 21 bp DNA duplex. These studies are used to support a kinetic model based on nucleic acid end fraying that describes dissociation for both short (<10 bp) and long (>10 bp) DNA duplexes. These measurements have been extended to explore temperature-dependent kinetics for the 21 bp construct, which permit determination of single-molecule activation enthalpies and entropies for DNA duplex dissociation.

Single-molecule conformational dynamics of a biologically functional hydroxocobalamin riboswitch.

Riboswitches represent a family of highly structured regulatory elements found primarily in the leader sequences of bacterial mRNAs. They function as molecular switches capable of altering gene expression; commonly, this occurs via a conformational change in a regulatory element of a riboswitch that results from ligand binding in the aptamer domain. Numerous studies have investigated the ligand binding process, but little is known about the structural changes in the regulatory element. A mechanistic description of both processes is essential for deeply understanding how riboswitches modulate gene expression. This task is greatly facilitated by studying all aspects of riboswitch structure/dynamics/function in the same model system. To this end, single-molecule fluorescence resonance energy transfer (smFRET) techniques have been used to directly observe the conformational dynamics of a hydroxocobalamin (HyCbl) binding riboswitch (env8HyCbl) with a known crystallographic structure.1 The single-molecule RNA construct studied in this work is unique in that it contains all of the structural elements both necessary and sufficient for regulation of gene expression in a biological context. The results of this investigation reveal that the undocking rate constant associated with the disruption of a long-range kissing-loop (KL) interaction is substantially decreased when the ligand is bound to the RNA, resulting in a preferential stabilization of the docked conformation. Notably, the formation of this tertiary KL interaction directly sequesters the Shine-Dalgarno sequence (i.e., the ribosome binding site) via base-pairing, thus preventing translation initiation. These results reveal that the conformational dynamics of this regulatory switch are quantitatively described by a four-state kinetic model, whereby ligand binding promotes formation of the KL interaction. The results of complementary cell-based gene expression experiments conducted in Escherichia coli are highly correlated with the smFRET results, suggesting that KL formation is directly responsible for regulating gene expression.

Single-molecule kinetics reveal cation-promoted DNA duplex formation through ordering of single-stranded helices.

In this work, the kinetics of short, fully complementary oligonucleotides are investigated at the single-molecule level. Constructs 6-9 bp in length exhibit single exponential kinetics over 2 orders of magnitude time for both forward (kon, association) and reverse (koff, dissociation) processes. Bimolecular rate constants for association are weakly sensitive to the number of basepairs in the duplex, with a 2.5-fold increase between 9 bp (k'on = 2.1(1) × 10(6) M(-1) s(-1)) and 6 bp (k'on = 5.0(1) × 10(6) M(-1) s(-1)) sequences. In sharp contrast, however, dissociation rate constants prove to be exponentially sensitive to sequence length, varying by nearly 600-fold over the same 9 bp (koff = 0.024 s(-1)) to 6 bp (koff = 14 s(-1)) range. The 8 bp sequence is explored in more detail, and the NaCl dependence of kon and koff is measured. Interestingly, kon increases by >40-fold (kon = 0.10(1) s(-1) to 4.0(4) s(-1) between [NaCl] = 25 mM and 1 M), whereas in contrast, koff decreases by fourfold (0.72(3) s(-1) to 0.17(7) s(-1)) over the same range of conditions. Thus, the equilibrium constant (Keq) increases by ≈160, largely due to changes in the association rate, kon. Finally, temperature-dependent measurements reveal that increased [NaCl] reduces the overall exothermicity (ΔΔH° > 0) of duplex formation, albeit by an amount smaller than the reduction in entropic penalty (-TΔΔS° < 0). This reduced entropic cost is attributed to a cation-facilitated preordering of the two single-stranded species, which lowers the association free-energy barrier and in turn accelerates the rate of duplex formation.

Thermodynamic origins of monovalent facilitated RNA folding.

Cations have long been associated with formation of native RNA structure and are commonly thought to stabilize the formation of tertiary contacts by favorably interacting with the electrostatic potential of the RNA, giving rise to an "ion atmosphere". A significant amount of information regarding the thermodynamics of structural transitions in the presence of an ion atmosphere has accumulated and suggests stabilization is dominated by entropic terms. This work provides an analysis of how RNA-cation interactions affect the entropy and enthalpy associated with an RNA tertiary transition. Specifically, temperature-dependent single-molecule fluorescence resonance energy transfer studies have been exploited to determine the free energy (ΔG°), enthalpy (ΔH°), and entropy (ΔS°) of folding for an isolated tetraloop-receptor tertiary interaction as a function of Na(+) concentration. Somewhat unexpectedly, increasing the Na(+) concentration changes the folding enthalpy from a strongly exothermic process [e.g., ΔH° = -26(2) kcal/mol at 180 mM] to a weakly exothermic process [e.g., ΔH° = -4(1) kcal/mol at 630 mM]. As a direct corollary, it is the strong increase in folding entropy [Δ(ΔS°) > 0] that compensates for this loss of exothermicity for the achievement of more favorable folding [Δ(ΔG°) < 0] at higher Na(+) concentrations. In conjunction with corresponding measurements of the thermodynamics of the transition state barrier, these data provide a detailed description of the folding pathway associated with the GAAA tetraloop-receptor interaction as a function of Na(+) concentration. The results support a potentially universal mechanism for monovalent facilitated RNA folding, whereby an increasing monovalent concentration stabilizes tertiary structure by reducing the entropic penalty for folding.

Apolar chemical environments compact unfolded RNAs and can promote folding.

It is well documented that the structure, and thus function, of nucleic acids depends on the chemical environment surrounding them, which often includes potential proteinaceous binding partners. The nonpolar amino acid side chains of these proteins will invariably alter the polarity of the local chemical environment around the nucleic acid. However, we are only beginning to understand how environmental polarity generally influences the structural and energetic properties of RNA folding. Here, we use a series of aqueous-organic cosolvent mixtures to systematically modulate the solvent polarity around two different RNA folding constructs that can form either secondary or tertiary structural elements. Using single-molecule Förster resonance energy transfer spectroscopy to simultaneously monitor the structural and energetic properties of these RNAs, we show that the unfolded conformations of both model RNAs become more compact in apolar environments characterized by dielectric constants less than that of pure water. In the case of tertiary structure formation, this compaction also gives rise to more energetically favorable folding. We propose that these physical changes arise from an enhanced accumulation of counterions in the low dielectric environment surrounding the unfolded RNA.

Integrating single-molecule FRET and biomolecular simulations to study diverse interactions between nucleic acids and proteins.

The conformations of biological macromolecules are intimately related to their cellular functions. Conveniently, the well-characterized dipole-dipole distance-dependence of Förster resonance energy transfer (FRET) makes it possible to measure and monitor the nanoscale spatial dimensions of these conformations using fluorescence spectroscopy. For this reason, FRET is often used in conjunction with single-molecule detection to study a wide range of conformationally dynamic biochemical processes. Written for those not yet familiar with the subject, this review aims to introduce biochemists to the methodology associated with single-molecule FRET, with a particular emphasis on how it can be combined with biomolecular simulations to study diverse interactions between nucleic acids and proteins. In the first section, we highlight several conceptual and practical considerations related to this integrative approach. In the second section, we review a few recent research efforts wherein various combinations of single-molecule FRET and biomolecular simulations were used to study the structural and dynamic properties of biochemical systems involving different types of nucleic acids (e.g., DNA and RNA) and proteins (e.g., folded and disordered).

Binding without folding - the biomolecular function of disordered polyelectrolyte complexes.

Recent evidence shows that oppositely charged intrinsically disordered proteins (IDPs) can form high-affinity complexes that involve neither the formation of secondary or tertiary structure nor site-specific interactions between individual residues. Similar electrostatically dominated interactions have also been identified for positively charged IDPs binding to nucleic acids. These highly disordered polyelectrolyte complexes constitute an extreme case within the spectrum of biomolecular interactions involving disorder. Such interactions are likely to be widespread, since sequence analysis predicts proteins with highly charged disordered regions to be surprisingly numerous. Here, we summarize the insights that have emerged from the highly disordered polyelectrolyte complexes identified so far, and we highlight recent developments and future challenges in (i) establishing models for the underlying highly dynamic structural ensembles, (ii) understanding the novel binding mechanisms associated with them, and (iii) identifying the functional consequences.

The SARS-CoV-2 conserved macrodomain is a mono-ADP-ribosylhydrolase.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other SARS-like-CoVs encode 3 tandem macrodomains within non-structural protein 3 (nsp3). The first macrodomain, Mac1, is conserved throughout CoVs, and binds to and hydrolyzes mono-ADP-ribose (MAR) from target proteins. Mac1 likely counters host-mediated anti-viral ADP-ribosylation, a posttranslational modification that is part of the host response to viral infections. Mac1 is essential for pathogenesis in multiple animal models of CoV infection, implicating it as a virulence factor and potential therapeutic target. Here we report the crystal structure of SARS-CoV-2 Mac1 in complex with ADP-ribose. SARS-CoV-2, SARS-CoV and MERS-CoV Mac1 exhibit similar structural folds and all 3 proteins bound to ADP-ribose with low µM affinities. Importantly, using ADP-ribose detecting binding reagents in both a gel-based assay and novel ELISA assays, we demonstrated de-MARylating activity for all 3 CoV Mac1 proteins, with the SARS-CoV-2 Mac1 protein leading to a more rapid loss of substrate compared to the others. In addition, none of these enzymes could hydrolyze poly-ADP-ribose. We conclude that the SARS-CoV-2 and other CoV Mac1 proteins are MAR-hydrolases with similar functions, indicating that compounds targeting CoV Mac1 proteins may have broad anti-CoV activity. IMPORTANCE: SARS-CoV-2 has recently emerged into the human population and has led to a worldwide pandemic of COVID-19 that has caused greater than 900 thousand deaths worldwide. With, no currently approved treatments, novel therapeutic strategies are desperately needed. All coronaviruses encode for a highly conserved macrodomain (Mac1) that binds to and removes ADP-ribose adducts from proteins in a dynamic post-translational process increasingly recognized as an important factor that regulates viral infection. The macrodomain is essential for CoV pathogenesis and may be a novel therapeutic target. Thus, understanding its biochemistry and enzyme activity are critical first steps for these efforts. Here we report the crystal structure of SARS-CoV-2 Mac1 in complex with ADP-ribose, and describe its ADP-ribose binding and hydrolysis activities in direct comparison to SARS-CoV and MERS-CoV Mac1 proteins. These results are an important first step for the design and testing of potential therapies targeting this unique protein domain.

Disordered RNA chaperones can enhance nucleic acid folding via local charge screening.

RNA chaperones are proteins that aid in the folding of nucleic acids, but remarkably, many of these proteins are intrinsically disordered. How can these proteins function without a well-defined three-dimensional structure? Here, we address this question by studying the hepatitis C virus core protein, a chaperone that promotes viral genome dimerization. Using single-molecule fluorescence spectroscopy, we find that this positively charged disordered protein facilitates the formation of compact nucleic acid conformations by acting as a flexible macromolecular counterion that locally screens repulsive electrostatic interactions with an efficiency equivalent to molar salt concentrations. The resulting compaction can bias unfolded nucleic acids towards folding, resulting in faster folding kinetics. This potentially widespread mechanism is supported by molecular simulations that rationalize the experimental findings by describing the chaperone as an unstructured polyelectrolyte.

Conformational Plasticity of Hepatitis C Virus Core Protein Enables RNA-Induced Formation of Nucleocapsid-like Particles.

Many of the unanswered questions associated with hepatitis C virus assembly are related to the core protein (HCVcp), which forms an oligomeric nucleocapsid encompassing the viral genome. The structural properties of HCVcp have been difficult to quantify, at least in part because it is an intrinsically disordered protein. We have used single-molecule Förster Resonance Energy Transfer techniques to study the conformational dimensions and dynamics of the HCVcp nucleocapsid domain (HCVncd) at various stages during the RNA-induced formation of nucleocapsid-like particles. Our results indicate that HCVncd is a typical intrinsically disordered protein. When it forms small ribonucleoprotein complexes with various RNA hairpins from the 3' end of the HCV genome, it compacts but remains intrinsically disordered and conformationally dynamic. Above a critical RNA concentration, these ribonucleoprotein complexes rapidly and cooperatively assemble into large nucleocapsid-like particles, wherein the individual HCVncd subunits become substantially more extended.