Izhar D. Medalsy and Daniel J. Müller
Knowing the dynamic mechanical response of tissue, cells, membranes, proteins, nucleic acids, and carbohydrates to external perturbations is important to understand various biological and biotechnological problems. Atomic force microscopy (AFM)-based approaches are the most frequently used nanotechnologies to determine the mechanical properties of biological samples that range in size from microscopic to (sub)nanoscopic. However, the dynamic nature of biomechanical properties has barely been addressed by AFM imaging. In this work, we characterizethe viscoelastic properties of the native light-driven proton pump bacteriorhodopsin of the purple membrane of Halobacterium salinarum. Using force–distance curve (F–D)-based AFM we imaged purple membranes while force probing their mechanical response over a wide range of loading rates (from 0.5 to 100 μN/s). Our results show that the mechanical stiffness of protein and membrane increases with the loading rate up to a factor of 10 (from 0.3 to 3.2 N/m). In addition, the electrostatic repulsion between AFM tip and sample can alter the mechanical stiffness measured by AFM up to 60% (from 0.8 to 1.3 N/m).These findings indicate that the mechanical response of membranes and proteins and probably of other biomolecular systems should be determined at different loading rates to fully understand their properties.
DOI
Journal: ACS Nano
C. David Williams, Mary K. Salcedo, Thomas C. Irving, Michael Regnier and Thomas L. Daniel
Classic interpretations of the striated muscle length–tension curve focus on how force varies with overlap of thin (actin) and thick (myosin) filaments. New models of sarcomere geometry and experiments with skinned synchronous insect flight muscle suggest that changes in the radial distance between the actin and myosin filaments, the filament lattice spacing, are responsible for between 20% and 50% of the change in force seen between sarcomere lengths of 1.4 and 3.4 µm. Thus, lattice spacing is a significant force regulator, increasing the slope of muscle's force–length dependence.
DOI
Journal: Proceedings of the Royal Society B
Bian Liu, Ronald J. Baskin, and Stephen C. Kowalczykowski
Single-molecule studies can overcome the complications of asynchrony and ensemble-averaging in bulk-phase measurements, provide mechanistic insights into molecular activities, and reveal interesting variations between individual molecules. The application of these techniques to the RecBCD helicase of Escherichia coli has resolved some long-standing discrepancies, and has provided otherwise unattainable mechanistic insights into its enzymatic behaviour. Enigmatically, the DNA unwinding rates of individual enzyme molecules are seen to vary considerably, but the origin of this heterogeneity remains unknown. Here we investigate the physical basis for this behaviour. Although any individual RecBCD molecule unwound DNA at a constant rate for an average of approximately 30,000 steps, we discover that transiently halting a single enzyme–DNA complex by depleting Mg2+-ATP could change the subsequent rates of DNA unwinding by that enzyme after reintroduction to ligand. The proportion of molecules that changed rate increased exponentially with the duration of the interruption, with a half-life of approximately 1 second, suggesting that a conformational change occurred during the time that the molecule was arrested. The velocity after pausing an individual molecule was any velocity found in the starting distribution of the ensemble. We suggest that substrate binding stabilizes the enzyme in one of many equilibrium conformational sub-states that determine the rate-limiting translocation behaviour of each RecBCD molecule. Each stabilized sub-state can persist for the duration (approximately 1 minute) of processive unwinding of a DNA molecule, comprising tens of thousands of catalytic steps, each of which is much faster than the time needed for the conformational change required to alter kinetic behaviour. This ligand-dependent stabilization of rate-defining conformational sub-states results in seemingly static molecule-to-molecule variation in RecBCD helicase activity, but in fact reflects one microstate from the equilibrium ensemble that a single molecule manifests during an individual processive translocation event.
DOI
Journal: Nature
Elizabeth Natkanski, Wing-Yiu Lee, Bhakti Mistry, Antonio Casal, Justin E. Molloy, Pavel Tolar
The generation of high-affinity antibodies depends on the ability of B cells to extract antigens from the surfaces of antigen-presenting cells. B cells that express high-affinity B cell receptors (BCRs) acquire more antigen and obtain better T cell help. However, the mechanisms by which B cells extract antigen remain unclear. Using fluid and flexible membrane substrates to mimic antigen-presenting cells, we showed that B cells acquire antigen by dynamic myosin IIa–mediated contractions that pull out and invaginate the presenting membranes. The forces generated by myosin IIa contractions ruptured most individual BCR-antigen bonds and promoted internalization of only high-affinity, multivalent BCR microclusters. Thus, B cell contractility contributes to affinity discrimination by mechanically testing the strength of antigen binding.
DOI
Journal: Science
Neil T. Roach, Madhusudhan Venkadesan, Michael J. Rainbow, and Daniel E. Lieberman
Some primates, including chimpanzees, throw objects occasionally, but only humans regularly throw projectiles with high speed and accuracy. Darwin noted that the unique throwing abilities of humans, which were made possible when bipedalism emancipated the arms, enabled foragers to hunt effectively using projectiles. However, there has been little consideration of the evolution of throwing in the years since Darwin made his observations, in part because of a lack of evidence of when, how and why hominins evolved the ability to generate high-speed throws. Here we use experimental studies of humans throwing projectiles to show that our throwing capabilities largely result from several derived anatomical features that enable elastic energy storage and release at the shoulder. These features first appear together approximately 2 million years ago in the species Homo erectus. Taking into consideration archaeological evidence suggesting that hunting activity intensified around this time, we conclude that selection for throwing as a means to hunt probably had an important role in the evolution of the genus Homo.
DOI
Journal: Nature
Dong-Hua Chen, Damian Madan, Jeremy Weaver, Zong Lin, Gunnar F. Schröder, Wah Chiu, Hays S. Rye
The GroEL/ES chaperonin system is required for the assisted folding of many proteins. How these substrate proteins are encapsulated within the GroEL-GroES cavity is poorly understood. Using symmetry-free, single-particle cryo-electron microscopy, we have characterized a chemically modified mutant of GroEL (EL43Py) that is trapped at a normally transient stage of substrate protein encapsulation. We show that the symmetric pattern of the GroEL subunits is broken as the GroEL cis-ring apical domains reorient to accommodate the simultaneous binding of GroES and an incompletely folded substrate protein (RuBisCO). The collapsed RuBisCO folding intermediate binds to the lower segment of two apical domains, as well as to the normally unstructured GroEL C-terminal tails. A comparative structural analysis suggests that the allosteric transitions leading to substrate protein release and folding involve concerted shifts of GroES and the GroEL apical domains and C-terminal tails.
DOI
Journal: Cell
Knowing the dynamic mechanical response of tissue, cells, membranes, proteins, nucleic acids, and carbohydrates to external perturbations is important to understand various biological and biotechnological problems. Atomic force microscopy (AFM)-based approaches are the most frequently used nanotechnologies to determine the mechanical properties of biological samples that range in size from microscopic to (sub)nanoscopic. However, the dynamic nature of biomechanical properties has barely been addressed by AFM imaging. In this work, we characterizethe viscoelastic properties of the native light-driven proton pump bacteriorhodopsin of the purple membrane of Halobacterium salinarum. Using force–distance curve (F–D)-based AFM we imaged purple membranes while force probing their mechanical response over a wide range of loading rates (from 0.5 to 100 μN/s). Our results show that the mechanical stiffness of protein and membrane increases with the loading rate up to a factor of 10 (from 0.3 to 3.2 N/m). In addition, the electrostatic repulsion between AFM tip and sample can alter the mechanical stiffness measured by AFM up to 60% (from 0.8 to 1.3 N/m).These findings indicate that the mechanical response of membranes and proteins and probably of other biomolecular systems should be determined at different loading rates to fully understand their properties.
