A two-dimensional mutate-and-map strategy for non-coding RNA structure

Wipapat Kladwang, Christopher C. VanLang, Pablo Cordero, and Rhiju Das


Non-coding RNAs fold into precise base-pairing patterns to carry out critical roles in genetic regulation and protein synthesis, but determining RNA structure remains difficult. Here, we show that coupling systematic mutagenesis with high-throughput chemical mapping enables accurate base-pair inference of domains from ribosomal RNA, ribozymes and riboswitches. For a six-RNA benchmark that has challenged previous chemical/computational methods, this ‘mutate-and-map’ strategy gives secondary structures that are in agreement with crystallography (helix error rates, 2%), including a blind test on a double-glycine riboswitch. Through modelling of partially ordered states, the method enables the first test of an interdomain helix-swap hypothesis for ligand-binding cooperativity in a glycine riboswitch. Finally, the data report on tertiary contacts within non-coding RNAs, and coupling to the Rosetta/FARFAR algorithm gives nucleotide-resolution three-dimensional models (helix root-mean-squared deviation, 5.7 Å) of an adenine riboswitch. These results establish a promising two-dimensional chemical strategy for inferring the secondary and tertiary structures that underlie non-coding RNA behaviour.


DOI


Journal: Nature Chemistry

Metastability of Native Proteins and the Phenomenon of Amyloid Formation

Andrew J. Baldwin, Tuomas P. J. Knowles, Gian Gaetano Tartaglia, Anthony W. Fitzpatrick, Glyn L. Devlin, Sarah Lucy Shammas, Christopher A. Waudby, Maria F. Mossuto†, Sarah Meehan, Sally L. Gras, John Christodoulou, Spencer J. Anthony-Cahill, Paul D. Barker, Michele Vendruscolo, and Christopher M. Dobson

An experimental determination of the thermodynamic stabilities of a series of amyloid fibrils reveals that this structural form is likely to be the most stable one that protein molecules can adopt even under physiological conditions. This result challenges the conventional assumption that functional forms of proteins correspond to the global minima in their free energy surfaces and suggests that living systems are conformationally as well as chemically metastable.

DOI

Journal: Journal of the American Chemical Society 

Mechanics on Myocardium Deficient in the N2B Region of Titin: The Cardiac-Unique Spring Element Improves Efficiency of the Cardiac Cycle

Joshua Nedrud, Siegfried Labeit, Michael Gotthardt, Henk Granzier


Titin (also known as connectin) is an intrasarcomeric muscle protein that functions as a molecular spring and generates passive tension upon muscle stretch. The N2B element is a cardiac-specific spring element within titin's extensible region. Our goal was to study the contribution of the N2B element to the mechanical properties of titin, particularly its hypothesized role in limiting energy loss during repeated stretch (diastole)-shortening (systole) cycles of the heart. We studied energy loss by measuring hysteresis from the area between the stretch and release passive force-sarcomere length curves and used both wild-type (WT) mice and N2B knockout (KO) mice in which the N2B element has been deleted. A range of protocols was used, including those that mimic physiological loading conditions. KO mice showed significant increases in hysteresis. Most prominently, in tissue that had been preconditioned with a physiological stretch-release protocol, hysteresis increased significantly from 320 ± 46 pJ/mm2/sarcomere in WT to 650 ± 94 pJ/mm2/sarcomere in N2B KO myocardium. These results are supported by experiments in which oxidative stress was used to mechanically inactivate portions of the N2B-Us of WT titin through cysteine cross-linking. Studies on muscle from which the thin filaments had been extracted (using the actin severing protein gelsolin) showed that the difference in hysteresis between WT and KO tissue cannot be explained by filament sliding-based viscosity. Instead the results suggest that hysteresis arises from within titin and most likely involves unfolding of immunoglobulin-like domains. These studies support that the mechanical function of the N2B element of titin includes reducing hysteresis and increasing the efficiency of the heart.


DOI


Journal: Biophysical Journal

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

1. Krishna Neupane, 
2. Hao Yu, 
3. Daniel A. N. Foster, 
4. Feng Wang, and 
5. Michael T. Woodside

1. 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.

1. DOI

1. Journal: Nucleic Acids Research

Kinetic Partitioning Mechanism Governs the Folding of the Third FnIII Domain of Tenascin-C: Evidence at the Single-Molecule Level

Qing Peng, Jie Fang, Meijia Wang, and Hongbin Li


Statistical mechanics and molecular dynamics simulations proposed that the folding of proteins can follow multiple parallel pathways on a rugged energy landscape from unfolded state en route to their folded native states. Kinetic partitioning mechanism is one of the possible mechanisms underlying such complex folding dynamics. Here, we use single-molecule atomic force microscopy technique to directly probe the multiplicity of the folding pathways of the third fibronectin type III domain from theextracellular matrix protein tenascin-C (TNfn3). By stretching individual (TNfn3)8 molecules, we forced TNfn3 domains to undergo mechanical unfolding and refolding cycles, allowing us to directly observe the folding pathways of TNfn3. We found that, after being mechanically unraveled and then relaxed to zero force, TNfn3 follows multiple parallel pathways to fold into their native states. The majority of TNfn3 fold into the native state in a simple two-state fashion, while a small percentage of TNfn3 were found to be trapped into kinetically stable folding intermediate states with well-defined three-dimensional structures. Furthermore, the folding of TNfn3 was also influenced by its neighboring TNfn3 domains. Complex misfolded states of TNfn3 were observed, possibly due to the formation of domain-swapped dimeric structures. Our studies revealed the ruggedness of the folding energy landscape of TNfn3 and provided direct experimental evidence that the folding dynamics of TNfn3 are governed by the kinetic partitioning mechanism. Our results demonstrated the unique capability of single-molecule AFM to probe the folding dynamics of proteins at the single-molecule level.


DOI


Journal: Biophysical Journal

Structural characterization of a misfolded intermediate populated during the folding process of a PDZ domain

• Stefano Gianni,
• Ylva Ivarsson,
• Alfonso De Simone,
• Carlo Travaglini-Allocatelli,
• Maurizio Brunori, and 
• Michele Vendruscolo

• Incorrectly folded states transiently populated during the protein folding process are potentially prone to aggregation and have been implicated in a range of misfolding disorders that include Alzheimer's and Parkinson's diseases. Despite their importance, however, the structures of these states and the mechanism of their formation have largely escaped detailed characterization because of their short-lived nature. Here we present the structures of all the major states involved in the folding process of a PDZ domain, which include an off-pathway misfolded intermediate. By using a combination of kinetic, protein engineering, biophysical and computational techniques, we show that the misfolded intermediate is characterized by an alternative packing of the N-terminal β-hairpin onto an otherwise native-like scaffold. Our results suggest a mechanism of formation of incorrectly folded transient compact states by which misfolded structural elements are assembled together with more extended native-like regions.

• DOI

• Journal: 
Nature Structural and Molecular Biology

Conformations of the Huntingtin N-term in aqueous solution from atomistic simulations

Giulia Rossetti, Pilar Cossio, Alessandro Laio, and Paolo Carloni


The first 17 amino acids of Huntingtin protein (N17) play a crucial role in the protein’s aggregation. Here we predict its free energy landscape in aqueous solution by using bias exchange metadynamics. All our findings are consistent with experimental data. N17 populates four main kinetic basins, which interconvert on the microsecond time-scale. The most populated basin (about 75%) is a random coil, with an extended flat exposed hydrophobic surface. This might create a hydrophobic seed promoting Huntingtin aggregation. The other main populated basins contain helical conformations, which could facilitate N17 binding on its cellular targets.


DOI


Journal: Febs Letters

Toward A Mechanism of Prion Misfolding and Structural Models of PrPSc: Current Knowledge and Future Directions

Will C. Guest, Steven S. Plotkin, and Neil R. Cashman


Despite extensive investigation, many features of prion protein misfolding remain enigmatic. Physicochemical variables known to influence misfolding are reviewed to help elucidate the mechanism of prionogenesis and identify salient features of PrP(Sc), the misfolded conformer of the prion protein. Prospective work on refinement of candidate PrP(Sc) models based on thermodynamic considerations will help to complete atomic-scale structural details missing from experimental studies and may explain the basis for the templating activity of PrP(Sc) in disease.


DOI


Journal: Journal Of Toxicology And Environmental Health-Part A-Current Issues 

Ergodic and nonergodic processes coexist in the plasma membrane as observed by single-molecule tracking

Aubrey V. Weigel, Blair Simon, Michael M. Tamkun, and Diego Krapf

Diffusion in the plasma membrane of living cells is often found to display anomalous dynamics. However, the mechanism underlying this diffusion pattern remains highly controversial. Here, we study the physical mechanism underlying Kv2.1 potassium channel anomalous dynamics using single-molecule tracking. Our analysis includes both time series of individual trajectories and ensemble averages. We show that an ergodic and a nonergodic process coexist in the plasma membrane. The ergodic process resembles a fractal structure with its origin in macromolecular crowding in the cell membrane. The nonergodic process is found to be regulated by transient binding to the actin cytoskeleton and can be accurately modeled by a continuous-time random walk. When the cell is treated with drugs that inhibit actin polymerization, the diffusion pattern of Kv2.1 channels recovers ergodicity. However, the fractal structure that induces anomalous diffusion remains unaltered. These results have direct implications on the regulation of membrane receptor trafficking and signaling.

DOI

Journal: Proceedings of the National Academy of Sciences

Random-Coil:α-Helix Equilibria as a Reporter for the LewisX–LewisX Interaction

1. Timothy M. Altamore, 
2. Christian Fernández-García, 
3. Andrew H. Gordon, 
4. Tina Hübscher, 
5. Dr. Netnapa Promsawan, 
6. Maxim G. Ryadnov, 
7.  Andrew J. Doig, 
8. Derek N. Woolfson, 
9. Timothy Gallagher

1. Probing weak interactions: A peptide random-coil:α-helix equilibrium has been used to identify a weak carbohydrate–carbohydrate interaction (CCI). Glucose and lactose destabilized the helical conformer while Lewis^X trisaccharide led to increased helicity. Though small, the trend observed indicates that this peptide reporter can detect a single CCI in isolation.

1. DOI

1. Journal: Angewandte Chemie

Locating the Barrier for Folding of Single Molecules under an External Force

Olga K. Dudko, Thomas G. W. Graham, and Robert B. Best


Single-molecule pulling experiments on the folding of biomolecules are usually interpreted with one-dimensional models in which the dynamics occurs on the “pulling coordinate.” Paradoxically, the free-energy profile along this coordinate may lack a refolding barrier, yet a barrier is known to exist for folding; thus, it has been argued that pulling experiments do not probe folding. Here, we show that transitions monitored in pulling experiments probe the true folding barrier but that the barrier may be hidden in the projection onto the pulling coordinate. However, one-dimensional theory using the pulling coordinate still yields physically meaningful energy landscape parameters.


DOI


Journal: Physical Review Letters

GB1 Is Not a Two-State Folder: Identification and Characterization of an On-Pathway Intermediate

Angela Morrone, Rajanish Giri, Rudesh D. Toofanny, Carlo Travaglini-Allocatelli, Maurizio Brunori, Valerie Daggett, and Stefano Gianni

The folding pathway of the small α/β protein GB1 has been extensively studied during the past two decades using both theoretical and experimental approaches. These studies provided a consensus view that the protein folds in a two-state manner. Here, we reassessed the folding of GB1, both by experiments and simulations, and detected the presence of an on-pathway intermediate. This intermediate has eluded earlier experimental characterization and is distinct from the collapsed state previously identified using ultrarapid mixing. Failure to identify the presence of an intermediate affects some of the conclusions that have been drawn for GB1, a popular model for protein folding studies.


DOI


Journal: Biophysical Journal

Probing ribosomal protein–RNA interactions with an external force

Pierre Mangeol, Thierry Bizebard, Claude Chiaruttini, Marc Dreyfus, Mathias Springer, and  Ulrich Bockelmann


Ribosomal (r-) RNA adopts a well-defined structure within the ribosome, but the role of r-proteins in stabilizing this structure is poorly understood. To address this issue, we use optical tweezers to unfold RNA fragments in the presence or absence of r-proteins. Here, we focus on Escherichia coli r-protein L20, whose globular C-terminal domain (L20C) recognizes an irregular stem in domain II of 23S rRNA. L20C also binds its own mRNA and represses its translation; binding occurs at two different sites—i.e., a pseudoknot and an irregular stem. We find that L20C makes rRNA and mRNA fragments encompassing its binding sites more resistant to mechanical unfolding. The regions of increased resistance correspond within two base pairs to the binding sites identified by conventional methods. While stabilizing specific RNA structures, L20C does not accelerate their formation from alternate conformations—i.e., it acts as a clamp but not as a chaperone. In the ribosome, L20C contacts only one side of its target stem but interacts with both strands, explaining its clamping effect. Other r-proteins bind rRNA similarly, suggesting that several rRNA structures are stabilized by “one-side” clamping.


DOI


Journal: Proceedings of the National Academy of Sciences 

Thermodynamic efficiency and mechanochemical coupling of F1-ATPase

1. Shoichi Toyabe, 
2. Takahiro Watanabe-Nakayama, 
3. Tetsuaki Okamoto,
4. Seishi Kudo, and 
5. Eiro Muneyuki

1. F₁-ATPase is a nanosized biological energy transducer working as part of F_oF₁-ATP synthase. Its rotary machinery transduces energy between chemical free energy and mechanical work and plays a central role in the cellular energy transduction by synthesizing most ATP in virtually all organisms. However, information about its energetics is limited compared to that of the reaction scheme. Actually, fundamental questions such as how efficiently F₁-ATPase transduces free energy remain unanswered. Here, we demonstrated reversible rotations of isolated F₁-ATPase in discrete 120° steps by precisely controlling both the external torque and the chemical potential of ATP hydrolysis as a model system of F_oF₁-ATP synthase. We found that the maximum work performed by F₁-ATPase per 120° step is nearly equal to the thermodynamical maximum work that can be extracted from a single ATP hydrolysis under a broad range of conditions. Our results suggested a 100% free-energy transduction efficiency and a tight mechanochemical coupling of F₁-ATPase.

1. DOI

1. Journal: 
Proceedings of the National Academy of Sciences

Dynamics of protein folding and cofactor binding monitored by single-molecule force spectroscopy

Yi Cao and Hongbin Li


Many proteins in living cells require cofactors to carry out their biological functions. To reach their functional states, these proteins need to fold into their unique three-dimensional structures in the presence of their cofactors. Two processes, folding of the protein and binding of cofactors, intermingle with each other, making the direct elucidation of the folding mechanism of proteins in the presence of cofactors challenging. Here we use single-molecule atomic force microscopy to directly monitor the folding and cofactor binding dynamics of an engineered metal-binding protein G6-53 at the single-molecule level. Using the mechanical stability of different conformers of G6-53 as sensitive probes, we directly identified different G6-53 conformers (unfolded, apo- and Ni²⁺-bound) populated along the folding pathway of G6-53 in the presence of its cofactor Ni²⁺. By carrying out single-molecule atomic force microscopy refolding experiments, we monitored kinetic evolution processes of these different conformers. Our results suggested that the majority of G6-53 folds through a binding-after-folding mechanism, whereas a small fraction follows a binding-before-folding pathway. Our study opens an avenue to utilizing force spectroscopy techniques to probe the folding dynamics of proteins in the presence of cofactors at the single-molecule level, and we anticipated that this method can be used to study a wide variety of proteins requiring cofactors for their function.


DOI


Journal: Biophysical Journal