Highly Anisotropic Stability and Folding Kinetics of a Single Coiled Coil Protein under Mechanical Tension

Ying Gao, George Sirinakis, and Yongli Zhang



Coiled coils are one of the most abundant protein structural motifs and widely mediate protein interactions and force transduction or sensation. They are thus model systems for protein engineering and folding studies, particularly the GCN4 coiled coil. Major single-molecule methods have also been applied to this protein and revealed its folding kinetics at various spatiotemporal scales. Nevertheless, the folding energy and the kinetics of a single GCN4 coiled coil domain have not been well determined at a single-molecule level. Here we used high-resolution optical tweezers to characterize the folding and unfolding reactions of a single GCN4 coiled coil domain and their dependence on the pulling direction. In one axial and two transverse pulling directions, we observed reversible, two-state transitions of the coiled coil in real time. The transitions equilibrate at pulling forces ranging from 6 to 12 pN, showing different stabilities of the coiled coil in regard to pulling direction. Furthermore, the transition rates vary with both the magnitude and the direction of the pulling force by greater than 1000 folds, indicating a highly anisotropic and topology-dependent energy landscape for protein transitions under mechanical tension. We developed a new analytical theory to extract energy and kinetics of the protein transition at zero force. The derived folding energy does not depend on the pulling direction and is consistent with the measurement in bulk, which further confirms the applicability of the single-molecule manipulation approach for energy measurement. The highly anisotropic thermodynamics of proteins under tension should play important roles in their biological functions.

DOI

Journal: Journal of the American Chemical Society

Molecular Recognition of Insulin by a Synthetic Receptor

Jordan M. Chinai, Alexander B. Taylor, Lisa M. Ryno, Nicholas D. Hargreaves, Christopher A. Morris, P. John Hart, and Adam R. Urbach



The discovery of molecules that bind tightly and selectively to desired proteins continues to drive innovation at the interface of chemistry and biology. This paper describes the binding of human insulin by the synthetic receptor cucurbit[7]uril (Q7) in vitro. Isothermal titration calorimetry and fluorescence spectroscopy experiments show that Q7 binds to insulin with an equilibrium association constant of 1.5 × 10E6 M−1 and with 50−100-fold selectivity versus proteins that are much larger but lack an N-terminal aromatic residue, and with >1000-fold selectivity versus an insulin variant lacking the N-terminal phenylalanine (Phe) residue. The crystal structure of the Q7·insulin complex shows that binding occurs at the N-terminal Phe residue and that the N-terminus unfolds to enable binding. These findings suggest that site-selective recognition is based on the properties inherent to a protein terminus, including the unique chemical epitope presented by the terminal residue and the greater freedom of the terminus to unfold, like the end of a ball of string, to accommodate binding. Insulin recognition was predicted accurately from studies on short peptides and exemplifies an approach to protein recognition by targeting the terminus.

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Journal: Journal of the American Chemical Society

Histone fold modifications control nucleosome unwrapping and disassembly

Marek Simon, Justin A. North, John C. Shimko, Robert A. Forties, Michelle B. Ferdinand, Mridula Manohar, Meng Zhang, Richard Fishel, Jennifer J. Ottesen, and Michael G. Poirier



Nucleosomes are stable DNA–histone protein complexes that must be unwrapped and disassembled for genome expression, replication, and repair. Histone posttranslational modifications (PTMs) are major regulatory factors of these nucleosome structural changes, but the molecular mechanisms associated with PTM function remains poorly understood. Here we demonstrate that histone PTMs within distinct structured regions of the nucleosome directly regulate the inherent dynamic properties of the nucleosome. Precise PTMs were introduced into nucleosomes by chemical ligation. Single molecule magnetic tweezers measurements determined that only PTMs near the nucleosome dyad increase the rate of histone release in unwrapped nucleosomes. In contrast, FRET and restriction enzyme analysis reveal that only PTMs throughout the DNA entry–exit region increase unwrapping and enhance transcription factor binding to nucleosomal DNA. These results demonstrate that PTMs in separate structural regions of the nucleosome control distinct dynamic events, where the dyad regulates disassembly while the DNA entry–exit region regulates unwrapping. These studies are consistent with the conclusion that histone PTMs may independently influence nucleosome dynamics and associated chromatin functions.

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Journal:  Proceedings of the National Academy of Sciences

High-Speed Atomic Force Microscopy Reveals Rotary Catalysis of Rotorless F1-ATPase

Takayuki Uchihashi, Ryota Iino, Toshio Ando,and Hiroyuki Noji



F1 is an adenosine triphosphate (ATP)–driven motor in which three torque-generating β subunits in the α3β3 stator ring sequentially undergo conformational changes upon ATP hydrolysis to rotate the central shaft γ unidirectionally. Although extensive experimental and theoretical work has been done, the structural basis of cooperative torque generation to realize the unidirectional rotation remains elusive. We used high-speed atomic force microscopy to show that the rotorless F1 still “rotates”; in the isolated α3β3 stator ring, the three β subunits cyclically propagate conformational states in the counterclockwise direction, similar to the rotary shaft rotation in F1. The structural basis of unidirectionality is programmed in the stator ring. These findings have implications for cooperative interplay between subunits in other hexameric ATPases.



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Journal: Science

Pulsed Pressure Perturbations, an Extra Dimension in NMR Spectroscopy of Proteins

Werner Kremer , Martin Arnold , Claudia Elisabeth Munte , Rainer Hartl , Markus Beck Erlach , Joerg Koehler , Alexander Meier , and Hans Robert Kalbitzer


The introduction of the multidimensional NMR spectroscopy was a breakthrough in biological NMR me-thodology since it allowed the unequivocal correlation of different spin states of the system. The introduction of large pressure perturbations in the corresponding radio frequency (RF) pulse sequences adds an extra structural dimension into these experiments. We have developed a microprocessor controlled pressure jump unit that is able to introduce fast, strong pressure changes at any point in the pulse sequences. Repetitive pressure changes of 80 MPa in the sample tube are thus feasible in less than 30 ms. Two general forms of these experiments are proposed here, the pressure perturbation transient state spectroscopy (PPTSS) and the pressure perturbation state correlation spectroscopy (PPSCS). PPTSS can be used to measure the rate constants and the activation energies and activation volumes for the transition between different conformational states including the folded and unfolded state of proteins, for polymerisation-depolymerisation processes and for ligand binding at atomic resolution. PPSCS spectroscopy correlates the NMR parameters of different pressure induced states of the system thus allowing the measurement of properties of a given pressure induced state such as a folding intermediate in a different state e. g. the folded state. Selected examples for PPTSS and PPSCS spectroscopy are presented in this paper.


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Journal: Journal of the American Chemical Society

Protein Unfolding under Force: Crack Propagation in a Network

Adam M.R. de Graff, Gareth Shannon, Daniel W. Farrell, Philip M. Williams, and M.F. Thorpe

The mechanical unfolding of a set of 12 proteins with diverse topologies is investigated using an all-atom constraint-based model. Proteins are represented as polypeptides cross-linked by hydrogen bonds, salt bridges, and hydrophobic contacts, each modeled as a harmonic inequality constraint capable of supporting a finite load before breaking. Stereochemically acceptable unfolding pathways are generated by minimally overloading the network in an iterative fashion, analogous to crack propagation in solids. By comparing the pathways to those from molecular dynamics simulations and intermediates identified from experiment, it is demonstrated that the dominant unfolding pathways for 9 of the 12 proteins studied are well described by crack propagation in a network.


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Journal: Biophysical Journal

Single Molecule Detection of One, Two and Multiplex Proteins Involved in DNA/RNA Transaction

Yupeng Qiu and Sua Myong


Cellular processes involve complex arrangement of proteins engaged in a multitude of reactions, yet in a highly coordinated manner. The level of complexity, however, makes it difficult to investigate the role played by the individual protein constituent. Data taken from the conventional bulk solution methods suffer from ensemble averaging effect in which information from individual molecules is masked. The single molecule detection method overcomes this limitation by offering unique tools for monitoring the activity of individual molecules in isolation and in real-time dynamics. Included in this review are recent articles of single molecule studies representing a diverse array of experimental platforms which demonstrate the power and spectrum of single molecule detection.


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Journal: Cellular and Molecular Bioengineering