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.

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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.


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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.


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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.


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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.

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   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.

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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.

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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.

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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.

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Journal: Proceedings of the National Academy of Sciences