Nanoengineering a single-molecule mechanical switch using DNA self-assembly

Ken Halvorsen, Diane Schaak, and Wesley P Wong

The ability to manipulate and observe single biological molecules has led to both fundamental scientific discoveries and new methods in nanoscale engineering. A common challenge in many single-molecule experiments is reliably linking molecules to surfaces, and identifying their interactions. We have met this challenge by nanoengineering a novel DNA-based linker that behaves as a force-activated switch, providing a molecular signature that can eliminate errant data arising from non-specific and multiple interactions. By integrating a receptor and ligand into a single piece of DNA using DNA self-assembly, a single tether can be positively identified by force–extension behavior, and receptor–ligand unbinding easily identified by a sudden increase in tether length. Additionally, under proper conditions the exact same pair of molecules can be repeatedly bound and unbound. Our approach is simple, versatile and modular, and can be easily implemented using standard commercial reagents and laboratory equipment. In addition to improving the reliability and accuracy of force measurements, this single-molecule mechanical switch paves the way for high-throughput serial measurements, single-molecule on-rate studies, and investigations of population heterogeneity.

DOI

Jounal: Nanotechnology

A Size Barrier Limits Protein Diffusion at the Cell Surface to Generate Lipid-Rich Myelin-Membrane Sheets

Shweta Aggarwal, Larisa Yurlova, Nicolas Snaidero, Christina Reetz, Steffen Frey, Johannes Zimmermann, Gesa Pähler, Andreas Janshoff, Jens Friedrichs, Daniel J. Müller, Cornelia Goebel, and Mikael Simons


The insulating layers of myelin membrane wrapped around axons by oligodendrocytes are essential for the rapid conduction of nerve impulses in the central nervous system. To fulfill this function as an electrical insulator, myelin requires a unique lipid and protein composition. Here we show that oligodendrocytes employ a barrier that functions as a physical filter to generate the lipid-rich myelin-membrane sheets. Myelin basic protein (MBP) forms this molecular sieve and restricts the diffusion of proteins with large cytoplasmic domains into myelin. The barrier is generated from MBP molecules that line the entire sheet and is, thus, intimately intertwined with the biogenesis of the polarized cell surface. This system might have evolved in oligodendrocytes in order to generate an anisotropic membrane organization that facilitates the assembly of highly insulating lipid-rich membranes.


DOI


Journal: Development Cell

Texture-Induced Modulations of Friction Force: The Fingerprint Effect

E. Wandersman, R. Candelier, G. Debrégeas, and A. Prevost


Modulations of the friction force in dry solid friction are usually attributed to macroscopic stick-slip instabilities. Here we show that a distinct, quasistatic mechanism can also lead to nearly periodic force oscillations during sliding contact between an elastomer patterned with parallel grooves, and abraded glass slides. The dominant oscillation frequency is set by the ratio between the sliding velocity and the grooves period. A model is derived which quantitatively captures the dependence of the force modulations amplitude with the normal load, the grooves period, and the slides roughness characteristics. The model’s main ingredient is the nonlinearity of the friction law. Since such nonlinearity is ubiquitous for soft solids, this “fingerprint effect” should be relevant to a large class of frictional configurations and have important consequences in human digital touch.


DOI


Journal: Physical Review Letters

Poly(zwitterionic)protein conjugates offer increased stability without sacrificing binding affinity or bioactivity

Andrew J. Keefe, and Shaoyi Jiang


Treatment with therapeutic proteins is an attractive approach to targeting a number of challenging diseases. Unfortunately, the native proteins themselves are often unstable in physiological conditions, reducing bioavailability and therefore increasing the dose that is required. Conjugation with poly(ethylene glycol) (PEG) is often used to increase stability, but this has a detrimental effect on bioactivity. Here, we introduce conjugation with zwitterionic polymers such as poly(carboxybetaine). We show that poly(carboxybetaine) conjugation improves stability in a manner similar to PEGylation, but that the new conjugates retain or even improve the binding affinity as a result of enhanced protein–substrate hydrophobic interactions. This chemistry opens a new avenue for the development of protein therapeutics by avoiding the need to compromise between stability and affinity.


DOI


Journal: Nature Chemistry

Actin filaments function as a tension sensor by tension-dependent binding of cofilin to the filament

1. Kimihide Hayakawa, 
2. Hitoshi Tatsumi, and 
3. Masahiro Sokabe

1. Intracellular and extracellular mechanical forces affect the structure and dynamics of the actin cytoskeleton. However, the underlying molecular and biophysical mechanisms, including how mechanical forces are sensed, are largely unknown. Actin-depolymerizing factor/cofilin proteins are actin-modulating proteins that are ubiquitously distributed in eukaryotes, and they are the most likely candidate as proteins to drive stress fiber disassembly in response to changes in tension in the fiber. In this study, we propose a novel hypothesis that tension in an actin filament prevents the filament from being severed by cofilin. To test this, we placed single actin filaments under tension using optical tweezers. When a fiber was tensed, it was severed after the application of cofilin with a significantly larger delay in comparison with control filaments suspended in solution. The binding rate of cofilin to an actin bundle decreased when the bundle was tensed. These results suggest that tension in an actin filament reduces the cofilin binding, resulting in a decrease in its effective severing activity.

   DOI

   
   

   Journal: The Journal of Cell Biology

Beta structure motifs of islet amyloid polypeptides identified through surface-mediated assemblies

Xiao-Bo Mao, 

Chen-Xuan Wang, 

Xing-Kui Wu, 

Xiao-Jing Ma, 

Lei Liu, 

Lan Zhang, 

Lin Niu, 

Yuan-Yuan Guo, 

Deng-Hua Li, 

Yan-Lian Yang, and 

Chen Wang

We report here the identification of the key sites for the beta structure motifs of the islet amyloid polypeptide (IAPP) analogs by using scanning tunneling microscopy (STM). Duplex folding structures in human IAPP_8–37 (hIAPP_8–37) assembly were observed featuring a hairpin structure. The multiplicity in rIAPP assembly structures indicates the polydispersity of the rat IAPP_8–37 (rIAPP_8–37) beta-like motifs. The bimodal length distribution of beta structure motifs for rIAPP_8–37 R18H indicates the multiple beta segments linked by turns. The IAPP_8–37 analogs share common structure motifs of IAPP_8–17 and IAPP_26–37 with the most probable key sites at positions around Ser₁₉/Ser₂₀ and Gly₂₄. These observations reveal the similar amyloid formation tendency in the C and N terminus segments because of the sequence similarity, while the differences in specific amino acids at each key site manifest the effect of sequence variations. The results could be beneficial for studying structural polymorphism of amyloidal peptides with multiple beta structure motifs.

DOI

Journal: Proceedings of the National Academy of Sciences

A nanomechanical interface to rapid single-molecule interactions

• Mingdong Dong, and 
• Ozgur Sahin

• Single-molecule techniques provide opportunities for molecularly precise imaging, manipulation, assembly and biophysical studies. Owing to the kinetics of bond rupture processes, rapid single-molecule measurements can reveal novel bond rupture mechanisms, probe single-molecule events with short lifetimes and enhance the interaction forces supplied by single molecules. Rapid measurements will also increase throughput necessary for technological use of single-molecule techniques. Here we report a nanomechanical sensor that allows single-molecule force spectroscopy on the previously unexplored microsecond timescale. We probed bond lifetimes around 5 μs and observed significant enhancements in molecular interaction forces. Our loading-rate-dependent measurements provide experimental evidence for an additional energy barrier in the biotin–streptavidin complex. We also demonstrate quantitative mapping of rapid single-molecule interactions with high spatial resolution. This nanomechanical interface may allow studies of molecular processes with short lifetimes and development of novel biological imaging, single-molecule manipulation and assembly technologies.

DOI

Journal: Nature Communications

A Dynamic Knockout Reveals That Conformational Fluctuations Influence the Chemical Step of Enzyme Catalysis

Gira Bhabha, Jeeyeon Lee, Damian C. Ekiert, Jongsik Gam, Ian A. Wilson, H. Jane Dyson, Stephen J. Benkovic, and Peter E. Wright 


Conformational dynamics play a key role in enzyme catalysis. Although protein motions have clear implications for ligand flux, a role for dynamics in the chemical step of enzyme catalysis has not been clearly established. We generated a mutant of Escherichia coli dihydrofolate reductase that abrogates millisecond-time-scale fluctuations in the enzyme active site without perturbing its structural and electrostatic preorganization. This dynamic knockout severely impairs hydride transfer. Thus, we have found a link between conformational fluctuations on the millisecond time scale and the chemical step of an enzymatic reaction, with broad implications for our understanding of enzyme mechanisms and for design of novel protein catalysts.

DOI

Journal: Science

Catalysis by dihydrofolate reductase and other enzymes arises from electrostatic preorganization, not conformational motions

Andrew J. Adamczyk, Jie Cao, Shina C. L. Kamerlin, and Arieh Warshel

The proposal that enzymatic catalysis is due to conformational fluctuations has been previously promoted by means of indirect considerations. However, recent works have focused on cases where the relevant motions have components toward distinct conformational regions, whose population could be manipulated by mutations. In particular, a recent work has claimed to provide direct experimental evidence for a dynamical contribution to catalysis in dihydrofolate reductase, where blocking a relevant conformational coordinate was related to the suppression of the motion toward the occluded conformation. The present work utilizes computer simulations to elucidate the true molecular basis for the experimentally observed effect. We start by reproducing the trend in the measured change in catalysis upon mutations (which was assumed to arise as a result of a “dynamical knockout” caused by the mutations). This analysis is performed by calculating the change in the corresponding activation barriers without the need to invoke dynamical effects. We then generate the catalytic landscape of the enzyme and demonstrate that motions in the conformational space do not help drive catalysis. We also discuss the role of flexibility and conformational dynamics in catalysis, once again demonstrating that their role is negligible and that the largest contribution to catalysis arises from electrostatic preorganization. Finally, we point out that the changes in the reaction potential surface modify the reorganization free energy (which includes entropic effects), and such changes in the surface also alter the corresponding motion. However, this motion is never the reason for catalysis, but rather simply a reflection of the shape of the reaction potential surface.

DOI

Journal: Proceedings of the National Academy of Sciences

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