Michael T. Woodside

Direct Observation of Hierarchical Folding in Single Riboswitch Aptamers

Riboswitches regulate genes through structural changes in ligand-binding RNA aptamers. With the use of an optical-trapping assay based on in situ transcription by a molecule of RNA polymerase, single nascent RNAs containing pbuE adenine riboswitch aptamers were unfolded and refolded. Multiple folding states were characterized by means of both force-extension curves and folding trajectories under constant force by measuring the molecular contour length, kinetics, and energetics with and without adenine. Distinct folding steps correlated with the formation of key secondary or tertiary structures and with ligand binding. Adenine-induced stabilization of the weakest helix in the aptamer, the mechanical switch underlying regulatory action, was observed directly. These results provide an integrated view of hierarchical folding in an aptamer, demonstrating how complex folding can be resolved into constituent parts, and supply further insights into tertiary structure formation.

Direct measurement of the full, sequence-dependent folding landscape of a nucleic acid

Nucleic acid hairpins provide a powerful model system for understanding macromolecular folding, with free-energy landscapes that can be readily manipulated by changing the hairpin sequence. The full shapes of energy landscapes for the reversible folding of DNA hairpins under controlled loads exerted by an optical force clamp were obtained by deconvolution from high-resolution, single-molecule trajectories. The locations and heights of the energy barriers for hairpin folding could be tuned by adjusting the number and location of G:C base pairs, and the presence and position of folding intermediates were controlled by introducing single-nucleotide mismatches.

Reconstructing Folding Energy Landscapes by Single-Molecule Force Spectroscopy

Folding may be described conceptually in terms of trajectories over a landscape of free energies corresponding to different molecular configurations. In practice, energy landscapes can be difficult to measure. Single-molecule force spectroscopy (SMFS), whereby structural changes are monitored in molecules subjected to controlled forces, has emerged as a powerful tool for probing energy landscapes. We summarize methods for reconstructing landscapes from force spectroscopy measurements under both equilibrium and nonequilibrium conditions. Other complementary, but technically less demanding, methods provide a model-dependent characterization of key features of the landscape. Once reconstructed, energy landscapes can be used to study critical folding parameters, such as the characteristic transition times required for structural changes and the effective diffusion coefficient setting the timescale for motions over the landscape. We also discuss issues that complicate measurement and interpretation, including the possibility of multiple states or pathways and the effects of projecting multiple dimensions onto a single coordinate.

Direct observation of multiple misfolding pathways in a single prion protein molecule

Protein misfolding is a ubiquitous phenomenon associated with a wide range of diseases. Single-molecule approaches offer a powerful tool for deciphering the mechanisms of misfolding by measuring the conformational fluctuations of a protein with high sensitivity. We applied single-molecule force spectroscopy to observe directly the misfolding of the prion protein PrP, a protein notable for having an infectious misfolded state that is able to propagate by recruiting natively folded PrP. By measuring folding trajectories of single PrP molecules held under tension in a high-resolution optical trap, we found that the native folding pathway involves only two states, without evidence for partially folded intermediates that have been proposed to mediate misfolding. Instead, frequent but fleeting transitions were observed into off-pathway intermediates. Three different misfolding pathways were detected, all starting from the unfolded state. Remarkably, the misfolding rate was even higher than the rate for native folding. A mutant PrP with higher aggregation propensity showed increased occupancy of some of the misfolded states, suggesting these states may act as intermediates during aggregation. These measurements of individual misfolding trajectories demonstrate the power of single-molecule approaches for characterizing misfolding directly by mapping out nonnative folding pathways.

Energy landscape analysis of native folding of the prion protein yields the diffusion constant, transition path time, and rates.

Protein folding is described conceptually in terms of diffusion over a configurational free-energy landscape, typically reduced to a one-dimensional profile along a reaction coordinate. In principle, kinetic properties can be predicted directly from the landscape profile using Kramers theory for diffusive barrier crossing, including the folding rates and the transition time for crossing the barrier. Landscape theory has been widely applied to interpret the time scales for protein conformational dynamics, but protein folding rates and transition times have not been calculated directly from experimentally measured free-energy profiles. We characterized the energy landscape for native folding of the prion protein using force spectroscopy, measuring the change in extension of a single protein molecule at high resolution as it unfolded/refolded under tension. Key parameters describing the landscape profile were first recovered from the distributions of unfolding and refolding forces, allowing the diffusion constant for barrier crossing and the transition path time across the barrier to be calculated. The full landscape profile was then reconstructed from force-extension curves, revealing a double-well potential with an extended, partially unfolded transition state. The barrier height and position were consistent with the previous results. Finally, Kramers theory was used to predict the folding rates from the landscape profile, recovering the values observed experimentally both under tension and at zero force in ensemble experiments. These results demonstrate how advances in single-molecule theory and experiment are harnessing the power of landscape formalisms to describe quantitatively the mechanics of folding.

Single-molecule force spectroscopy of the add adenine riboswitch relates folding to regulatory mechanism.

Riboswitches regulate gene expression via ligand binding to an aptamer domain which induces conformational changes in a regulatory expression platform. By unfolding and refolding single add adenine riboswitch molecules in an optical trap, an integrated picture of the folding was developed and related to the regulatory mechanism. Force-extension curves (FECs) and constant-force folding trajectories measured on the aptamer alone revealed multiple partially-folded states, including several misfolded states not on the native folding pathway. All states were correlated to key structural components and interactions within hierarchical folding pathways. FECs of the full-length riboswitch revealed that the thermodynamically stable conformation switches upon ligand binding from a structure repressing translation to one permitting it. Along with rapid equilibration of the two structures in the absence of adenine, these results support a thermodynamically-controlled regulatory mechanism, in contrast with the kinetic control of the closely-related pbuE adenine riboswitch. Comparison of the folding of these riboswitches revealed many similarities arising from shared structural features but also essential differences related to their different regulatory mechanisms.

Folding and unfolding single RNA molecules under tension.

Single-molecule force spectroscopy constitutes a powerful method for probing RNA folding: It allows the kinetic, energetic, and structural properties of intermediate and transition states to be determined quantitatively, yielding new insights into folding pathways and energy landscapes. Recent advances in experimental and theoretical methods, including fluctuation theorems, kinetic theories, novel force clamps, and ultrastable instruments, have opened new avenues for study. These tools have been used to probe folding in simple model systems, for example, RNA and DNA hairpins. Knowledge gained from such systems is helping to build our understanding of more complex RNA structures composed of multiple elements, as well as how nucleic acids interact with proteins involved in key cellular activities, such as transcription and translation.

Direct observation of transition paths during the folding of proteins and nucleic acids.

How biomolecules fold In order to fold, biomolecules must search a conformational energy landscape to find low-energy states. There are peaks in the landscape where the molecules must occupy unstable conformations for a short time. Neupane et al. used optical tweezers to observe these transition paths directly for single nucleic acid and protein molecules (see the Perspective by Wolynes). They measured a distribution of times taken to cross the transition path and found that the shape of the distribution agrees well with theory that assumes one-dimensional diffusion over the landscape. Science, this issue p. 239; see also p. 150 Optical tweezers reveal how a protein and a DNA hairpin cross the barrier between the folded and unfolded states. [Also see Perspective by Wolynes] Transition paths, the fleeting trajectories through the transition states that dominate the dynamics of biomolecular folding reactions, encapsulate the critical information about how structure forms. Owing to their brief duration, however, they have not previously been observed directly. We measured transition paths for both nucleic acid and protein folding, using optical tweezers to observe the microscopic diffusive motion of single molecules traversing energy barriers. The average transit times and the shapes of the transit-time distributions agreed well with theoretical expectations for motion over the one-dimensional energy landscapes reconstructed for the same molecules, validating the physical theory of folding reactions. These measurements provide a first look at the critical microscopic events that occur during folding, opening exciting new avenues for investigating folding phenomena.

Scanned potential microscopy of edge and bulk currents in the quantum Hall regime

Using an atomic force microscope as a local voltmeter, we measure the Hall voltage profile in a two-dimensional electron gas in the quantum Hall (QH) regime. We observe a linear profile in the bulk of the sample in the transition regions between QH plateaus and a distinctly nonlinear profile on the plateaus. In addition, localized voltage drops are observed at the sample edges in the transition regions. We interpret these results in terms of theories of edge and bulk current in the QH regime. {copyright} {ital 1999} {ital The American Physical Society}

Programmed −1 frameshifting efficiency correlates with RNA pseudoknot conformational plasticity, not resistance to mechanical unfolding

Programmed −1 frameshifting, whereby the reading frame of a ribosome on messenger RNA is shifted in order to generate an alternate gene product, is often triggered by a pseudoknot structure in the mRNA in combination with an upstream slippery sequence. The efficiency of frameshifting varies widely for different sites, but the factors that determine frameshifting efficiency are not yet fully understood. Previous work has suggested that frameshifting efficiency is related to the resistance of the pseudoknot against mechanical unfolding. We tested this hypothesis by studying the mechanical properties of a panel of pseudoknots with frameshifting efficiencies ranging from 2% to 30%: four pseudoknots from retroviruses, two from luteoviruses, one from a coronavirus, and a nonframeshifting bacteriophage pseudoknot. Using optical tweezers to apply tension across the RNA, we measured the distribution of forces required to unfold each pseudoknot. We found that neither the average unfolding force, nor the unfolding kinetics, nor the parameters describing the energy landscape for mechanical unfolding of the pseudoknot (energy barrier height and distance to the transition state) could be correlated to frameshifting efficiency. These results indicate that the resistance of pseudoknots to mechanical unfolding is not a primary determinant of frameshifting efficiency. However, increased frameshifting efficiency was correlated with an increased tendency to form alternate, incompletely folded structures, suggesting a more complex picture of the role of the pseudoknot involving the conformational dynamics.