Self-Assembly from a Single-Molecule Perspective

2019 ◽  
pp. 147-155
Author(s):  
Kevin R. Pilkiewicz ◽  
Pratip Rana ◽  
Michael L. Mayo ◽  
Preetam Ghosh
Keyword(s):  
Single Molecule ◽  
Self Assembly

scholarly journals Large domain movements upon UvrD dimerization and helicase activation

2017 ◽  
Vol 114 (46) ◽  
pp. 12178-12183 ◽  
Author(s):  
Binh Nguyen ◽  
Yerdos Ordabayev ◽  
Joshua E. Sokoloski ◽  
Elizabeth Weiland ◽  
Timothy M. Lohman
Keyword(s):  
Escherichia Coli ◽  
Single Molecule ◽  
Self Assembly ◽  
Total Internal Reflection ◽  
Dna Helicase ◽  
Conformational State ◽  
Helicase Activity ◽  
Multiple Conformations ◽  
Dna Substrate

Escherichia coli UvrD DNA helicase functions in several DNA repair processes. As a monomer, UvrD can translocate rapidly and processively along ssDNA; however, the monomer is a poor helicase. To unwind duplex DNA in vitro, UvrD needs to be activated either by self-assembly to form a dimer or by interaction with an accessory protein. However, the mechanism of activation is not understood. UvrD can exist in multiple conformations associated with the rotational conformational state of its 2B subdomain, and its helicase activity has been correlated with a closed 2B conformation. Using single-molecule total internal reflection fluorescence microscopy, we examined the rotational conformational states of the 2B subdomain of fluorescently labeled UvrD and their rates of interconversion. We find that the 2B subdomain of the UvrD monomer can rotate between an open and closed conformation as well as two highly populated intermediate states. The binding of a DNA substrate shifts the 2B conformation of a labeled UvrD monomer to a more open state that shows no helicase activity. The binding of a second unlabeled UvrD shifts the 2B conformation of the labeled UvrD to a more closed state resulting in activation of helicase activity. Binding of a monomer of the structurally similar Escherichia coli Rep helicase does not elicit this effect. This indicates that the helicase activity of a UvrD dimer is promoted via direct interactions between UvrD subunits that affect the rotational conformational state of its 2B subdomain.

scholarly journals 3D DNA structural barcode copying and random access

2020 ◽  
Author(s):  
Filip Bošković ◽  
Alexander Ohmann ◽  
Ulrich F. Keyser ◽  
Kaikai Chen
Keyword(s):  
Data Storage ◽  
Single Molecule ◽  
Self Assembly ◽  
Large Scale ◽  
Three Dimensional ◽  
Random Access ◽  
Scale Production ◽  
Digital Information ◽  
Dna Nanostructures ◽  
Large Scale Production

AbstractThree-dimensional (3D) DNA nanostructures built via DNA self-assembly have established recent applications in multiplexed biosensing and storing digital information. However, a key challenge is that 3D DNA structures are not easily copied which is of vital importance for their large-scale production and for access to desired molecules by target-specific amplification. Here, we build 3D DNA structural barcodes and demonstrate the copying and random access of the barcodes from a library of molecules using a modified polymerase chain reaction (PCR). The 3D barcodes were assembled by annealing a single-stranded DNA scaffold with complementary short oligonucleotides containing 3D protrusions at defined locations. DNA nicks in these structures are ligated to facilitate barcode copying using PCR. To randomly access a target from a library of barcodes, we employ a non-complementary end in the DNA construct that serves as a barcode-specific primer template. Readout of the 3D DNA structural barcodes was performed with nanopore measurements. Our study provides a roadmap for convenient production of large quantities of self-assembled 3D DNA nanostructures. In addition, this strategy offers access to specific targets, a crucial capability for multiplexed single-molecule sensing and for DNA data storage.

scholarly journals Effects of Pathological Mutations on the Prion-Like Polymerisation of MyD88

2018 ◽  
Author(s):  
Ailís O’Carroll ◽  
Brieuc Chauvin ◽  
James Brown ◽  
Ava Meagher ◽  
Joanne Coyle ◽  
...  
Keyword(s):  
Single Molecule ◽  
Self Assembly ◽  
Point Mutations ◽  
Adaptor Protein ◽  
Full Length ◽  
Binary Response ◽  
Death Domain ◽  
Tir Domain ◽  
The Impact

AbstractA novel concept has emerged whereby the higher-order self-assembly of proteins provides a simple and robust mechanism for signal amplification. This appears to be a universal signalling mechanism within the innate immune system, where the recognition of pathogens or danger-associated molecular patterns need to trigger a strong, binary response within cells. Previously, multiple structural studies have been limited to single domains, expressed and assembled at high protein concentrations. We therefore set out to develop new in vitro strategies to characterise the behaviour of full-length proteins at physiological levels. In this study we focus on the adaptor protein MyD88, which contains two domains with different self-assembly properties: a TIR domain that can polymerise similarly to the TIR domain of Mal, and a Death Domain that has been shown to oligomerise with helical symmetry in the Myddosome complex. To visualize the behaviour of full-length MyD88 without purification steps, we use single-molecule fluorescence coupled to eukaryotic cell-free protein expression. These experiments demonstrate that at low protein concentration, only full-length MyD88 forms prion-like polymers. We also demonstrate that the metastability of MyD88 polymerisation creates the perfect binary response required in innate signalling: the system is silenced at normal concentrations but upstream signalling creates a “seed” that triggers polymerisation and amplification of the response. These findings pushed us to re-interpret the role of polymerisation in MyD88-related diseases and we studied the impact of disease-associated point mutations L93P, R196C and L252P/L265P at the molecular level. We discovered that all mutations completely block the ability of MyD88 to polymerise. We also confirm that L252P, a gain-of-function mutation, allows the MyD88 mutant to form extremely stable oligomers, even when expressed at low nanomolar concentrations. Thus, our results are consistent with and greatly add to the findings on the Myddosomes digital ‘all-or-none’ responses and the behaviour of the oncogenic mutation of MyD88.

Single-molecule magnets under dc field with an anion effect: self-assembly of pure dysprosium(iii) metallacycles

2021 ◽  
Vol 50 (1) ◽  
pp. 262-269
Author(s):  
Jianfeng Wu ◽  
Qianqian Yang ◽  
Haoyu Wang ◽  
Yan Ge ◽  
Jinkui Tang ◽  
...  
Keyword(s):  
Magnetic Properties ◽  
Single Molecule ◽  
Self Assembly ◽  
Structural Factors ◽  
Single Molecule Magnets ◽  
Anion Effect

The anion-adaptive self-assembly described here not only offers a facile approach to produce large single-molecule magnets but also provides an understanding of how structural factors affect the magnetic properties.

scholarly journals Identification and nanomechanical characterization of the fundamental single-strand protofilaments of amyloid α-synuclein fibrils

2018 ◽  
Vol 115 (28) ◽  
pp. 7230-7235 ◽  
Author(s):  
Francesco Simone Ruggeri ◽  
Fabrizio Benedetti ◽  
Tuomas P. J. Knowles ◽  
Hilal A. Lashuel ◽  
Sergey Sekatskii ◽  
...  
Keyword(s):  
Single Molecule ◽  
Self Assembly ◽  
Amyloid Fibrils ◽  
Molecular Assembly ◽  
Single Strand ◽  
Force Microscopy ◽  
Atomic Force ◽  
Presynaptic Protein ◽  
Polypeptide Chains

The formation and spreading of amyloid aggregates from the presynaptic protein α-synuclein in the brain play central roles in the pathogenesis of Parkinson’s disease. Here, we use high-resolution atomic force microscopy to investigate the early oligomerization events of α-synuclein with single monomer angstrom resolution. We identify, visualize, and characterize directly the smallest elementary unit in the hierarchical assembly of amyloid fibrils, termed here single-strand protofilaments. We show that protofilaments form from the direct molecular assembly of unfolded monomeric α-synuclein polypeptide chains. To unravel protofilaments’ internal structure and elastic properties, we manipulated nanomechanically these species by atomic force spectroscopy. The single-molecule scale identification and characterization of the fundamental unit of amyloid assemblies provide insights into early events underlying their formation and shed light on opportunities for therapeutic intervention at the early stages of aberrant protein self-assembly.

Enantioselective Self-Assembly of Triangular Dy3Clusters with Single-Molecule Magnet Behavior

2014 ◽  
Vol 9 (12) ◽  
pp. 3558-3564 ◽  
Author(s):  
Shuang-Yan Lin ◽  
Chao Wang ◽  
Lang Zhao ◽  
Jinkui Tang
Keyword(s):  
Single Molecule ◽  
Self Assembly ◽  
Single Molecule Magnet
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