Florian C. Oberstrass, Louis E. Fernandes, Paul Lebel, and Zev Bryant
Changes in global DNA linking number can be accommodated by localized changes in helical structure. We have used single-molecule torque measurements to investigate sequence-specific strand separation and Z-DNA formation. By controlling the boundary conditions at the edges of sequences of interest, we have confirmed theoretical predictions of distinctive boundary-dependent backbending patterns in torque-twist relationships. Abrupt torque jumps are associated with the formation and collapse of DNA bubbles, permitting direct observations of DNA breathing dynamics.
DOI
Journal: Physical Review Letters
Kevin J. Freedman, S. Raza Haq, Joshua B. Edel, Per Jemth, and Min Jun Kim
Single molecule methods have provided a significantly new look at the behavior of biomolecules in both equilibrium and non-equilibrium conditions. Most notable are the stretching experiments performed by atomic force microscopes and laser tweezers. Here we present an alternative single molecule method that can unfold a protein domain, observed at electric fields greater than 106 V/m, and is fully controllable by the application of increasing voltages across the membrane of the pore. Furthermore this unfolding mechanism is characterized by measuring both the residence time of the protein within the nanopore and the current blockade. The unfolding data supports a gradual unfolding mechanism rather than the cooperative transition observed by classical urea denaturation experiments. Lastly it is shown that the voltage-mediated unfolding is a function of the stability of the protein by comparing two mutationally destabilized variants of the protein..
DOI
Journal: Scientific Reports
Rina Rosenzweig, Shoeib Moradi, Arash Zarrine-Afsar, John R. Glover, and Lewis E. Kay
HSP-100 protein machines, such as ClpB, play an essential role in reactivating protein aggregates that can otherwise be lethal to cells. Although the players involved are known, including the DnaK/DnaJ/GrpE chaperone system in bacteria, details of the molecular interactions are not well understood. Using methyl–transverse relaxation–optimized nuclear magnetic resonance spectroscopy, we present an atomic-resolution model for the ClpB-DnaK complex, which we verified by mutagenesis and functional assays. ClpB and GrpE compete for binding to the DnaK nucleotide binding domain, with GrpE binding inhibiting disaggregation. DnaK, in turn, plays a dual role in both disaggregation and subsequent refolding of polypeptide chains as they emerge from the aggregate. On the basis of a combined structural-biochemical analysis, we propose a model for the mechanism of protein aggregate reactivation by ClpB.
DOI
Journal: Science
Alan R. Fersht and Miriana Petrovich
DOI
Journal: Proceedings of the National Academy of Sciences
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
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
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
