Folding kinetics for the conformational switch between alternative RNA structures.

RNA molecules with novel functions have revived interest in the accurate prediction of RNA three-dimensional (3D) structure and folding dynamics. However, existing methods are inefficient in automated 3D structure prediction. Here, we report a robust computational approach for rapid folding of RNA molecules. We develop a simplified RNA model for discrete molecular dynamics (DMD) simulations, incorporating base-pairing and base-stacking interactions. We demonstrate correct folding of 150 structurally diverse RNA sequences. The majority of DMD-predicted 3D structures have <4 A deviations from experimental structures. The secondary structures corresponding to the predicted 3D structures consist of 94% native base-pair interactions. Folding thermodynamics and kinetics of tRNA(Phe), pseudoknots, and mRNA fragments in DMD simulations are in agreement with previous experimental findings. Folding of RNA molecules features transient, non-native conformations, suggesting non-hierarchical RNA folding. Our method allows rapid conformational sampling of RNA folding, with computational time increasing linearly with RNA length. We envision this approach as a promising tool for RNA structural and functional analyses.

In proteins, empirical correlations have shown that changes in heat capacity (DeltaC(P)) scale linearly with the hydrophobic surface area buried upon folding. The influence of DeltaC(P) on RNA folding has been widely overlooked and is poorly understood. In addition to considerations of solvent reorganization, electrostatic effects might contribute to DeltaC(P)s of folding in polyanionic species such as RNAs. Here, we employ a perturbation method based on electrostatic theory to probe the hot and cold denaturation behavior of the hammerhead ribozyme. This treatment avoids much of the error associated with imposing two-state folding models on non-two-state systems. Ribozyme stability is perturbed across a matrix of solvent conditions by varying the concentration of NaCl and methanol co-solvent. Temperature-dependent unfolding is then monitored by circular dichroism spectroscopy. The resulting array of unfolding transitions can be used to calculate a DeltaC(P) of folding that accurately predicts the observed cold denaturation temperature. We confirm the accuracy of the calculated DeltaC(P) by using isothermal titration calorimetry, and also demonstrate a methanol-dependence of the DeltaC(P). We weigh the strengths and limitations of this method for determining DeltaC(P) values. Finally, we discuss the data in light of the physical origins of the DeltaC(P)s for RNA folding and consider their impact on biological function.

The increasing significance of RNAs in transcriptional or post-transcriptional gene regulation processes has generated considerable interest towards the prediction of RNA folding and its sensitivity to environmental factors. We use Boltzmann-weighted sampling to generate RNA secondary structures, which are used to characterize the energy landscape, via the distributions of energies and base-pair distances. Depending upon the length of an RNA, the number of sequences investigated, and the sample size of generated structures --- generating and analyzing sufficient samples can be computationally challenging. We introduce and develop a lightweight and extensible runtime environment that is effective across a range of RNA sizes and other parameters, as well as over a range of infrastructure -- from traditional HPC grids to clouds, without requiring any changes at the application or user level. The Adaptive Distributed Application Management System (ADAMS) is built upon an extensbile and interoperable pilot-job and supports the concurrent execution of a broad range of task sizes across a range of infrastructure. We use ADAMS to investigate the folding energy landscape for two RNA systems of different sizes: a set of S-adenosyl methionine (SAM) binding RNA sequences known as SAM-I riboswitches and the S gene of the Bovine Corona Virus (BCoV) RNA genome that comprises 4092 nucleotides. Results of the energy and base-pair distance distributions suggest different energy landscapes, implying different folding dynamics. With obtained results, we demonstrated the possibility of utilizing this protocol to explore microscopic origins for reported sequence-dependent variation of binding affinity and gene expression in the two RNA systems.

pH-Dependent Free Energy Landscape, Conformational Selection, and Thermodynamics of Protein Folding

Toward protein fingerprinting with a solid-state nanopore.

Several small-molecule ligands specifically bind and stabilize G-quadruplex (G4) nucleic acid structures, which are considered to be promising therapeutic targets. G4s are polymorphic structures of varying stability, and their formation is dynamic. Here, we investigate the mechanisms of ligand binding to dynamically populated human telomere G4 DNA by using the bisquinolinium based ligand Phen-DC3 and a combination of single-molecule FRET microscopy, ensemble FRET and CD spectroscopies. Different cations are used to tune G4 polymorphism and folding dynamics. We find that ligand binding occurs to pre-folded G4 structures and that Phen-DC3 also induces G4 formation in unfolded single strands. Following ligand binding to dynamically populated G4s, the DNA undergoes pronounced conformational redistributions that do not involve direct ligand-induced G4 conformational interconversion. On the contrary, the redistribution is driven by ligand-induced G4 folding and trapping of dynamically populated short-lived conformation states. Thus, ligand-induced stabilization does not necessarily require the initial presence of stably folded G4s.

Substrate recognition by Clp chaperones is dependent on interactions with motifs composed of specific peptide sequences. We studied the binding of short motif-bearing peptides to ClpA, the chaperone component of the ATP-dependent ClpAP protease of Escherichia coli in the presence of ATPgammaS and Mg2+ at pH 7.5. Binding was measured by isothermal titration calorimetry (ITC) using the peptide, AANDENYALAA, which corresponds to the SsrA degradation motif found at the C terminus of abnormal nascent polypeptides in vivo. One SsrA peptide was bound per hexamer of ClpA with an association constant (K(A)) of 5 x 10(6) m(-1). Binding was also assayed by changes in fluorescence of an N-terminal dansylated SsrA peptide, which bound with the same stoichiometry of one per ClpA hexamer (K(A) approximately 1 x 10(7) m(-1)). Similar results were obtained when ATP was substituted for ATPgammaS at 6 degrees C. Two additional peptides, derived from the phage P1 RepA protein and the E. coli HemA protein, which bear different substrate motifs, were competitive inhibitors of SsrA binding and bound to ClpA hexamers with K(A)' > 3 x 10(7) m(-1). DNS-SsrA bound with only slightly reduced affinity to deletion mutants of ClpA missing either the N-terminal domain or the C-terminal nucleotide-binding domain, indicating that the binding site for SsrA lies within the N-terminal nucleotide-binding domain. Because only one protein at a time can be unfolded and translocated by ClpA hexamers, restricting the number of peptides initially bound should avoid nonproductive binding of substrates and aggregation of partially processed proteins.

RNA secondary structure is important in a wide variety of biological processes, but relatively little is known about the pathways and kinetics of RNA folding. When the IS10 transposase (tnp) gene is transcribed from a promoter outside the element, little increase in tnp expression is observed. This protection from outside transcription (pot) occurs at the translational level, presumably resulting from mRNA secondary structure proposed to sequester the tnp ribosome-binding site. Here, we confirm the pot RNA structure and show that it blocks 30S ribosomal subunit binding in vitro. Point mutations that abolish protection in vivo map to the pot structure. Surprisingly, these pot mutations do not severely alter the pot secondary structure or increase 30S subunit binding in vitro, except in one case. Using an oligonucleotide hybridization assay, we show that most of the pot mutations slow the kinetics of pot structure formation, with little or no effect on the inhibitory function of the final structure. Moreover, a suppressor mutation reverses this effect. We propose a pathway for pot mRNA folding that is consistent with the mutations and implicates the formation of important kinetic intermediates. The significance of these observations for the RNA folding problem in general is discussed.

We have analysed the regulatory behaviour of deletion mutants lacking different segments of the leader region of the tryptophan operon of Serratia marcescens. Our results support the model in which a particular RNA structure, the terminator, is recognized during transcription as a transcription termination signal, and an alternative RNA structure, the anti-terminator, prevents formation of the terminator. It appears that the role of translation, ribosome stalling and shifts between alternative RNA secondary structures, is simply to regulate formation of the terminator.

I was fortunate to practice science during the last half of the previous century, when many basic biological and biochemical concepts could be experimentally addressed for the first time. My introduction to research involved isolating and identifying intermediates in the niacin biosynthetic pathway. These studies were followed by investigations focused on determining the properties of genes and enzymes essential to metabolism and examining how they were alterable by mutation. The most challenging problem I initially attacked was establishing the colinear relationship between gene and protein. Subsequent research emphasized identification and characterization of regulatory mechanisms that microorganisms use to control gene expression. An elaborate regulatory strategy, transcription attenuation, was discovered that is often based on selection between alternative RNA structures. Throughout my career I enjoyed the excitement of solving basic scientific problems. Most rewarding, however, was the feeling that I was helping young scientists experience the pleasure of performing creative research.

Protein folding is an inherently complex process involving coordination of the intricate networks of weak interactions that stabilize native three-dimensional structures. In the conventional paradigm, simple protein structures are assumed to fold in an all-or-none process that is inaccessible to experiment. Existing experimental methods therefore probe folding mechanisms indirectly. A widely used approach interprets changes in protein stability and/or folding kinetics, induced by engineered mutations, in terms of the structure of the native protein. In addition to limitations in connecting energetics with structure, mutational methods have significant experimental uncertainties and are unable to map complex networks of interactions. In contrast, analytical theory predicts small barriers to folding and the possibility of downhill folding. These theoretical predictions have been confirmed experimentally in recent years, including the observation of global downhill folding. However, a key remaining question is whether downhill folding can indeed lead to the high-resolution analysis of protein folding processes. Here we show, with the use of nuclear magnetic resonance (NMR), that the downhill protein BBL from Escherichia coli unfolds atom by atom starting from a defined three-dimensional structure. Thermal unfolding data on 158 backbone and side-chain protons out of a total of 204 provide a detailed view of the structural events during folding. This view confirms the statistical nature of folding, and exposes the interplay between hydrogen bonding, hydrophobic forces, backbone conformation and side-chain entropy. From the data we also obtain a map of the interaction network in this protein, which reveals the source of folding cooperativity. Our approach can be extended to other proteins with marginal barriers (less than 3RT), providing a new tool for the study of protein folding.

Strigolactones (SLs) are a class of plant hormones that regulate numerous processes of growth and development. SL perception and signal activation involves interaction between F-box E3 ubiquitin ligase D3/MAX2 and DWARF14 (D14) α/β-hydrolase in a SL-dependent manner and targeting of D53/SMXL6/7/8 transcriptional repressors (SMXLs) for proteasome-mediated degradation. D3/MAX2 has been shown to exist in multiple conformational states in which the C-terminal helix (CTH) undergoes a closed-to-open dynamics and regulates D14 binding and SL perception. Despite the multiple modes of D3-D14 interactions found in vitro, the residues that regulate the conformational switch of D3/MAX2 CTH in targeting D53/SMXLs and the subsequent effect on SL signalling remain unclear. Here we elucidate the functional dynamics of ASK1-D3/MAX2 in SL signalling by leveraging conformational switch mutants in vitro and in plants. We report the crystal structure of a dislodged CTH of the ASK1-D3 mutant and demonstrate that disruptions in CTH plasticity via either CRISPR-Cas9 genome editing or expression of point mutation mutants result in impairment of SL signalling. We show that the conformational switch in ASK1-D3/MAX2 CTH directly regulates ubiquitin-mediated protein degradation. A dislodged conformation involved in D53/SMXLs SL-dependent recruitment and ubiquitination and an engaged conformation are required for the release of polyubiquitinated D53/SMXLs and subsequently D14 for proteasomal degradation. Finally, we uncovered an organic acid metabolite that can directly trigger the D3/MAX2 CTH conformational switch. Our findings unravel a new regulatory function of a SKP1-CUL1-F-box ubiquitin ligase in plant signalling.

How ions affect RNA folding thermodynamics and kinetics is an important but a vexing problem that remains unsolved. Experiments have shown that the free-energy change, Δ G( c), of RNA upon folding varies with the salt concentration ( c) as, Δ G( c) = k c ln c + const, where the coefficient k c is proportional to the difference in the ion preferential coefficient, ΔΓ. We performed simulations of a coarse-grained model, by modeling electrostatic interactions implicitly and with explicit representation of ions, to elucidate the molecular underpinnings of the relationship between Δ G and ΔΓ. The simulations quantitatively reproduce the heat capacity for a pseudoknot, thus validating the model. We show that Δ G( c), calculated directly from ΔΓ, varies linearly with ln c ( c < 0.2 M), for a hairpin and the pseudoknot, demonstrating a molecular link between the two quantities. Explicit ion simulations also show the linear dependence of Δ G( c) on ln c at all c with k c = 2 kB T, except that Δ G( c) values are shifted by ∼2 kcal/mol higher than experiments. The discrepancy is due to an underestimation of Γ for both the folded and unfolded states while giving accurate values for ΔΓ. The predictions for the salt dependence of ΔΓ are amenable to test using single-molecule pulling experiments. The framework provided here can be used to obtain accurate thermodynamics for other RNA molecules as well.

Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins constitute a microbial, adaptive immune system countering invading nucleic acids. Cas2 is a universal Cas protein found in all types of CRISPR-Cas systems, and its role is implicated in new spacer acquisition into CRISPR loci. In subtype I-C CRISPR-Cas systems, Cas2 proteins are metal-dependent double-stranded DNA (dsDNA) nucleases, and a pH-dependent conformational transition has been proposed as a prerequisite for catalytic action. Here, we report the crystal structure of Xanthomonas albilineans Cas2 (XaCas2) and provide experimental evidence of a pH-dependent conformational change during functional activation. XaCas2 crystallized at an acidic pH represented a catalytically inactive conformational state in which two Asp8 residues were too far apart to coordinate a single catalytic metal ion. Consistently, XaCas2 exhibited dsDNA nuclease activity only under neutral and basic conditions. Despite the overall structural similarity of the two protomers, significant conformational heterogeneity was evident in the putative hinge regions, suggesting that XaCas2 engages in hinge-bending conformational switching. The presence of a Trp residue in the hinge region enabled the investigation of hinge dynamics by fluorescence spectroscopy. The pH dependence of the fluorescence intensity overlapped precisely with that of nuclease activity. Mutational analyses further suggested that conformational activation proceeded via a rigid-body hinge-bending motion as both D8E and hinge mutations significantly reduced nuclease activity. Together, our results reveal strong correlations between the conformational states, catalytic activity, and hinge dynamics of XaCas2, and provide structural and dynamic insights into the conformational activation of the nuclease function of Cas2.

Folding Kinetics of Large RNAs