Cooperativity in Thermal and Force-Induced Protein Unfolding: Integration of Crack Propagation and Network Elasticity Models

Amit Srivastava and Rony Granek

We investigate force-induced and temperature-induced unfolding of proteins using the combination of a Gaussian network model and a crack propagation model based on “bond”-breaking independent events. We assume the existence of threshold values for the mean strain and strain fluctuations that dictate bond rupture. Surprisingly, we find that this stepwise process usually leads to a few cooperative, first-order-like, transitions in which several bonds break simultaneously, reminiscent of the “avalanches” seen in disordered networks.

DOI

Journal: Physical Review Letters

Multistep protein unfolding during nanopore translocation

David Rodriguez-Larrea, and Hagan Bayley

Cells are divided into compartments and separated from the environment by lipid bilayer membranes. Essential molecules are transported back and forth across the membranes. We have investigated how folded proteins use narrow transmembrane pores to move between compartments. During this process, the proteins must unfold. To examine co-translocational unfolding of individual molecules, we tagged protein substrates with oligonucleotides to enable potential-driven unidirectional movement through a model protein nanopore, a process that differs fundamentally from extension during force spectroscopy measurements. Our findings support a four-step translocation mechanism for model thioredoxin substrates. First, the DNA tag is captured by the pore. Second, the oligonucleotide is pulled through the pore, causing local unfolding of the C terminus of the thioredoxin adjacent to the pore entrance. Third, the remainder of the protein unfolds spontaneously. Finally, the unfolded polypeptide diffuses through the pore into the recipient compartment. The unfolding pathway elucidated here differs from those revealed by denaturation experiments in solution, for which two-state mechanisms have been proposed.

DOI

Journal: Nature Nanotechnology

Mechanical Activation of a Multimeric Adhesive Protein Through Domain Conformational Change

Sithara S. Wijeratne, Eric Botello, Hui-Chun Yeh, Zhou Zhou, Angela L. Bergeron, Eric W. Frey, Jay M. Patel, Leticia Nolasco, Nancy A. Turner, Joel L. Moake, Jing-fei Dong, and Ching-Hwa Kiang

The mechanical force-induced activation of the adhesive protein von Willebrand factor (VWF), which experiences high hydrodynamic forces, is essential in initiating platelet adhesion. The importance of the mechanical force-induced functional change is manifested in the multimeric VWF’s crucial role in blood coagulation, when high fluid shear stress activates plasma VWF (PVWF) multimers to bind platelets. Here, we showed that a pathological level of high shear stress exposure of PVWF multimers results in domain conformational changes, and the subsequent shifts in the unfolding force allow us to use force as a marker to track the dynamic states of the multimeric VWF. We found that shear-activated PVWF multimers are more resistant to mechanical unfolding than nonsheared PVWF multimers, as indicated in the higher peak unfolding force. These results provide insight into the mechanism of shear-induced activation of PVWF multimers.

DOI

Journal: Physical Review Letters

A transgenic mouse for in vivo detection of endogenous labeled mRNA

Timothée Lionnet, Kevin Czaplinski, Xavier Darzacq, Yaron Shav-Tal, Amber L Wells, Jeffrey A Chao, Hye Yoon Park, Valeria de Turris, Melissa Lopez-Jones, and Robert H Singer

Live-cell single mRNA imaging is a powerful tool but has been restricted in higher eukaryotes to artificial cell lines and reporter genes. We describe an approach that enables live-cell imaging of single endogenous labeled mRNA molecules transcribed in primary mammalian cells and tissue. We generated a knock-in mouse line with an MS2 binding site (MBS) cassette targeted to the 3′ untranslated region of the essential ββ-actin gene. As β-actin–MBS was ubiquitously expressed, we could uniquely address endogenous mRNA regulation in any tissue or cell type. We simultaneously followed transcription from the β-actin alleles in real time and observed transcriptional bursting in response to serum stimulation with precise temporal resolution. We tracked single endogenous labeled mRNA particles being transported in primary hippocampal neurons. The MBS cassette also enabled high-sensitivity fluorescence in situ hybridization (FISH), allowing detection and localization of single β-actin mRNA molecules in various mouse tissues.

DOI

Journal: Nature Methods

Driving a Macroscopic Oscillator with the Stochastic Motion of a Hydrogen Molecule

Christian Lotze, Martina Corso, Katharina J. Franke, Felix von Oppen, Jose Ignacio Pascual

Energy harvesting from noise is a paradigm proposed by the theory of stochastic resonances. We demonstrate that the random switching of a hydrogen (H2) molecule can drive the oscillation of a macroscopic mechanical resonator. The H2 motion was activated by tunneling electrons and caused fluctuations of the forces sensed by the tip of a noncontact atomic force microscope. The stochastic molecular noise and the periodic oscillation of the tip were coupled in a concerted dynamic that drives the system into self-oscillation. This phenomenon could be a way for enhancing the transfer of energy from incoherent sources into coherent dynamics of a molecular engine.

DOI

Journal: Science

Protein Folding Drives Disulfide Formation

Pallav Kosuri, Jorge Alegre-Cebollada, Jason Feng, Anna Kaplan, Alvaro Inglés-Prieto, Carmen L. Badilla, Brent R. Stockwell, Jose M. Sanchez-Ruiz, Arne Holmgren, Julio M. Fernández

PDI catalyzes the oxidative folding of disulfide-containing proteins. However, the sequence of reactions leading to a natively folded and oxidized protein remains unknown. Here we demonstrate a technique that enables independent measurements of disulfide formation and protein folding. We find that non-native disulfides are formed early in the folding pathway and can trigger misfolding. In contrast, a PDI domain favors native disulfides by catalyzing oxidation at a late stage of folding. We propose a model for cotranslational oxidative folding wherein PDI acts as a placeholder that is relieved by the pairing of cysteines caused by substrate folding. This general mechanism can explain how PDI catalyzes oxidative folding in a variety of structurally unrelated substrates.

DOI

Journal: Cell

Atomic Force Microscopy with Nanoscale Cantilevers Resolves Different Structural Conformations of the DNA Double Helix

Carl Leung, Aizhan Bestembayeva, Richard Thorogate, Jake Stinson, Alice Pyne, Christian Marcovich, Jinling Yang, Ute Drechsler, Michel Despont, Tilo Jankowski, Martin Tschöpe, and Bart W. Hoogenboom

Structural variability and flexibility are crucial factors for biomolecular function. Here we have reduced the invasiness and enhanced the spatial resolution of atomic force microscopy (AFM) to visualize, for the first time, different structural conformations of the two polynucleotide strands in the DNA double helix, for single molecules under near-physiological conditions. This is achieved by identifying and tracking the anomalous resonance behavior of nanoscale AFM cantilevers in the immediate vicinity of the sample.

DOI

Journal: Nano Letters