Design of a single-chain polypeptide tetrahedron assembled from coiled-coil segments

Helena Gradišar, Sabina Božič, Tibor Doles, Damjan Vengust, Iva Hafner-Bratkovič, Alenka Mertelj, Ben Webb, Andrej Šali, Sandi Klavžar, and Roman Jerala

Protein structures evolved through a complex interplay of cooperative interactions, and it is still very challenging to design new protein folds de novo. Here we present a strategy to design self-assembling polypeptide nanostructured polyhedra based on modularization using orthogonal dimerizing segments. We designed and experimentally demonstrated the formation of the tetrahedron that self-assembles from a single polypeptide chain comprising 12 concatenated coiled coil–forming segments separated by flexible peptide hinges. The path of the polypeptide chain is guided by a defined order of segments that traverse each of the six edges of the tetrahedron exactly twice, forming coiled-coil dimers with their corresponding partners. The coincidence of the polypeptide termini in the same vertex is demonstrated by reconstituting a split fluorescent protein in the polypeptide with the correct tetrahedral topology. Polypeptides with a deleted or scrambled segment order fail to self-assemble correctly. This design platform provides a foundation for constructing new topological polypeptide folds based on the set of orthogonal interacting polypeptide segments.

DOI

Journal: Nature Chemical Biology

Torque Spectroscopy of DNA: Base-Pair Stability, Boundary Effects, Backbending, and Breathing Dynamics

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

Single molecule unfolding and stretching of protein domains inside a solid-state nanopore by electric field

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

Unraveling the Mechanism of Protein Disaggregation Through a ClpB-DnaK Interaction

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

Why phosphate is a bad buffer for guanidinium chloride titrations

Alan R. Fersht and Miriana Petrovich

DOI

Journal: Proceedings of the National Academy of Sciences

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