A theoretical analysis of single molecule protein sequencing via weak binding spectra

AbstractWe propose and theoretically study an approach to massively parallel single molecule peptide sequencing, based on single molecule measurement of the kinetics of probe binding [1] to the N-termini of immobilized peptides. Unlike previous proposals, this method is robust to both weak and non-specific probe-target affinities, which we demonstrate by applying the method to a range of randomized affinity matrices consisting of relatively low-quality binders. This suggests a novel principle for proteomic measurement whereby highly non-optimized sets of low-affinity binders could be applicable for protein sequencing, thus shifting the burden of amino acid identification from biomolecular design to readout. Measurement of probe occupancy times, or of time-averaged fluorescence, should allow high-accuracy determination of N-terminal amino acid identity for realistic probe sets. The time-averaged fluorescence method scales well to extremely weak-binding probes. We argue that this method could lead to an approach with single amino acid resolution and the ability to distinguish many canonical and modified amino acids, even using highly non-optimized probe sets. This readout method should expand the design space for single molecule peptide sequencing by removing constraints on the properties of the fluorescent binding probes.Author summaryWe simplify the problem of single molecule protein sequencing by proposing and analyzing an approach that makes use of low-affinity, low-specificity binding reagents. This decouples the problem of protein sequencing from the problem of generating a high-quality library of binding reagents against each of the amino acids.

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Planar Boronic Graphene and Nitrogenized Graphene Heterostructure for Protein Stretch and Confinement

Biomolecules ◽

10.3390/biom11121756 ◽

2021 ◽

Vol 11 (12) ◽

pp. 1756

Author(s):

Xuchang Su ◽

Zhi He ◽

Lijun Meng ◽

Hong Liang ◽

Ruhong Zhou

Keyword(s):

Single Molecule ◽

Intrinsically Disordered Proteins ◽

Electron Tunneling ◽

Amyloid Β ◽

Protein Sequencing ◽

Disordered Proteins ◽

Force Microscopy ◽

Intrinsically Disordered ◽

Atomic Force ◽

Dynamics Simulations

Single-molecule techniques such as electron tunneling and atomic force microscopy have attracted growing interests in protein sequencing. For these methods, it is critical to refine and stabilize the protein sample to a “suitable mode” before applying a high-fidelity measurement. Here, we show that a planar heterostructure comprising boronic graphene (BC3) and nitrogenized graphene (C3N) sandwiched stripe (BC3/C3N/BC3) is capable of the effective stretching and confinement of three types of intrinsically disordered proteins (IDPs), including amyloid-β (1–42), polyglutamine (Q42), and α-Synuclein (61–95). Our molecular dynamics simulations demonstrate that the protein molecules interact more strongly with the C3N stripe than the BC3 one, which leads to their capture, elongation, and confinement along the center C3N stripe of the heterostructure. The conformational fluctuations of IDPs are substantially reduced after being stretched. This design may serve as a platform for single-molecule protein analysis with reduced thermal noise.

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ProtSeq: towards high-throughput, single-molecule protein sequencing via amino acid conversion into DNA barcodes

iScience ◽

10.1016/j.isci.2021.103586 ◽

2021 ◽

pp. 103586

Author(s):

Jessica M. Hong ◽

Michael Gibbons ◽

Ali Bashir ◽

Diana Wu ◽

Shirley Shao ◽

...

Keyword(s):

Amino Acid ◽

High Throughput ◽

Single Molecule ◽

Protein Sequencing ◽

Dna Barcodes

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Control of the Barrier in Cyanide Based Single Molecule Magnets Mn(III)2Mn(II)3: Theoretical Analysis

Journal of Chemical Theory and Computation ◽

10.1021/ct0500343 ◽

2005 ◽

Vol 1 (4) ◽

pp. 668-673 ◽

Cited By ~ 25

Author(s):

Boris S. Tsukerblat ◽

Andrew V. Palii ◽

Sergei M. Ostrovsky ◽

Sergei V. Kunitsky ◽

Sophia I. Klokishner ◽

...

Keyword(s):

Theoretical Analysis ◽

Single Molecule ◽

Single Molecule Magnets

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Single molecule kinetics. I. Theoretical analysis of indicators

The Journal of Chemical Physics ◽

10.1063/1.1785783 ◽

2004 ◽

Vol 121 (13) ◽

pp. 6361-6372 ◽

Cited By ~ 50

Author(s):

James B. Witkoskie ◽

Jianshu Cao

Keyword(s):

Theoretical Analysis ◽

Single Molecule

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Adaptive nanopores: A bioinspired label-free approach for protein sequencing and identification

Nano Research ◽

10.1007/s12274-020-3095-z ◽

2020 ◽

Vol 14 (1) ◽

pp. 328-333 ◽

Cited By ~ 2

Author(s):

Andrea Spitaleri ◽

Denis Garoli ◽

Moritz Schütte ◽

Hans Lehrach ◽

Walter Rocchia ◽

...

Keyword(s):

Amino Acids ◽

Single Molecule ◽

Dynamic Range ◽

Protein Sequencing ◽

High Fidelity ◽

Label Free ◽

Therapeutic Approaches ◽

Highly Sensitive ◽

Dynamics Simulations ◽

Structure Of Proteins

AbstractSingle molecule protein sequencing would tremendously impact in proteomics and human biology and it would promote the development of novel diagnostic and therapeutic approaches. However, its technological realization can only be envisioned, and huge challenges need to be overcome. Major difficulties are inherent to the structure of proteins, which are composed by several different amino-acids. Despite long standing efforts, only few complex techniques, such as Edman degradation, liquid chromatography and mass spectroscopy, make protein sequencing possible. Unfortunately, these techniques present significant limitations in terms of amount of sample required and dynamic range of measurement. It is known that proteins can distinguish closely similar molecules. Moreover, several proteins can work as biological nanopores in order to perform single molecule detection and sequencing. Unfortunately, while DNA sequencing by means of nanopores is demonstrated, very few examples of nanopores able to perform reliable protein-sequencing have been reported so far. Here, we investigate, by means of molecular dynamics simulations, how a re-engineered protein, acting as biological nanopore, can be used to recognize the sequence of a translocating peptide by sensing the “shape” of individual amino-acids. In our simulations we demonstrate that it is possible to discriminate with high fidelity, 9 different amino-acids in a short peptide translocating through the engineered construct. The method, here shown for fluorescence-based sequencing, does not require any labelling of the peptidic analyte. These results can pave the way for a new and highly sensitive method of sequencing.

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Publisher’s Note: “Theoretical analysis of spectral precision in spectroscopic single-molecule localization microscopy” [Rev. Sci. Instrum. 89, 123703 (2018)]

Review of Scientific Instruments ◽

10.1063/1.5113514 ◽

2019 ◽

Vol 90 (6) ◽

pp. 069901

Author(s):

Ki-Hee Song ◽

Biqin Dong ◽

Cheng Sun ◽

Hao F. Zhang

Keyword(s):

Theoretical Analysis ◽

Single Molecule ◽

Localization Microscopy ◽

Single Molecule Localization Microscopy

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Bottom-up fabrication of a multi-component nanopore sensor that unfolds, processes and recognizes single proteins

10.1101/2020.12.04.411884 ◽

2020 ◽

Author(s):

Shengli Zhang ◽

Gang Huang ◽

Roderick Versloot ◽

Bart Marlon Herwig ◽

Paulo Cesar Telles de Souza ◽

...

Keyword(s):

Single Molecule ◽

Design Principle ◽

Protein Sequencing ◽

Transmembrane Helices ◽

Single Molecule Sequencing ◽

E Coli ◽

Transmembrane Channels ◽

Single Molecule Analysis ◽

Nanopore Sensors ◽

Substrate Proteins

AbstractTransmembrane channels and pores have many biotechnological applications, notably in the single-molecule sequencing of DNA. Small synthetic nanopores have been designed using amphipathic peptides, or by assembling computationally designed transmembrane helices. The fabrication of more complex transmembrane devices has yet to be reported. In this work, we fabricated in two steps a multi-protein transmembrane device that addresses some of the main challenges in nanopore protein sequencing. In the first step, artificial nanopores are created from soluble proteins with toroid shapes. This design principle will allow fabricating a variety of nanopores for single-molecule analysis. In the second step one α-subuinit of the 20S proteasome from Thermoplasma acidophilum is genetically integrated into the artificial nanopore, and a 28-component nanopore-proteasome is co-assembled in E. coli cells. This multi-component molecular machine opens the door to two new approaches in protein sequencing, in which selected substrate proteins are unfolded, fed to into the proteasomal chamber and then identified by the nanopore sensor either as intact or fragmented polypeptides. The ability to integrate molecular devices directly onto a nanopore sensors allows creating next-generation protein sequencing devices, and will shed new lights on the fundamental processes of biological nanomachines.

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Application of Solid-State Nanopore in Protein Detection

International Journal of Molecular Sciences ◽

10.3390/ijms21082808 ◽

2020 ◽

Vol 21 (8) ◽

pp. 2808 ◽

Cited By ~ 2

Author(s):

Yuhan Luo ◽

Linlin Wu ◽

Jing Tu ◽

Zuhong Lu

Keyword(s):

Solid State ◽

Chemical Modification ◽

Single Molecule ◽

Protein Detection ◽

High Sensitivity ◽

Protein Sequencing ◽

Its Sequence ◽

Sequence Structure ◽

Single Molecule Level ◽

Protein Molecules

A protein is a kind of major biomacromolecule of life. Its sequence, structure, and content in organisms contains quite important information for normal or pathological physiological process. However, research of proteomics is facing certain obstacles. Only a few technologies are available for protein analysis, and their application is limited by chemical modification or the need for a large amount of sample. Solid-state nanopore overcomes some shortcomings of the existing technology, and has the ability to detect proteins at a single-molecule level, with its high sensitivity and robustness of device. Many works on detection of protein molecules and discriminating structure have been carried out in recent years. Single-molecule protein sequencing techniques based on solid-state nanopore are also been proposed and developed. Here, we categorize and describe these efforts and progress, as well as discuss their advantages and drawbacks.

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