scholarly journals A single-molecule electrical approach for amino acid detection and chirality recognition

2021 ◽  
Vol 7 (10) ◽  
pp. eabe4365
Author(s):  
Zihao Liu ◽  
Xingxing Li ◽  
Hiroshi Masai ◽  
Xinyi Huang ◽  
Susumu Tsuda ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Single Molecule ◽  
Theoretical Calculations ◽  
Protein Sequencing ◽  
Current Fluctuations ◽  
Point Contacts ◽  
Conductance Data ◽  
Single Molecule Junctions

One of the ultimate goals of analytic chemistry is to efficiently discriminate between amino acids. Here we demonstrate this ability using a single-molecule electrical methodology based on molecular nanocircuits formed from stable graphene-molecule-graphene single-molecule junctions. These molecular junctions are fabricated by covalently bonding a molecular machine featuring a permethylated-β-cyclodextrin between a pair of graphene point contacts. Using pH to vary the type and charge of the amino acids, we find distinct multimodal current fluctuations originating from the different host-guest interactions, consistent with theoretical calculations. These conductance data produce characteristic dwell times and shuttling rates for each amino acid, and allow accurate, statistical real-time, in situ measurements. Testing four amino acids and their enantiomers shows the ability to distinguish between them within a few microseconds, thus paving a facile and precise way to amino acid identification and even single-molecule protein sequencing.

scholarly journals Real-time dynamic single-molecule protein sequencing on an integrated semiconductor device

2022 ◽  
Author(s):  
Brian D Reed ◽  
Michael J Meyer ◽  
Valentin Abramzon ◽  
Omer Ad ◽  
Pat Adcock ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Dna Sequencing ◽  
Real Time ◽  
Single Molecule ◽  
Dynamic Range ◽  
Protein Sequencing ◽  
Peptide Sequence ◽  
Single Amino Acid ◽  
Functional Components

Proteins are the main structural and functional components of cells, and their dynamic regulation and post-translational modifications (PTMs) underlie cellular phenotypes. Next-generation DNA sequencing technologies have revolutionized our understanding of heredity and gene regulation, but the complex and dynamic states of cells are not fully captured by the genome and transcriptome. Sensitive measurements of the proteome are needed to fully understand biological processes and changes to the proteome that occur in disease states. Studies of the proteome would benefit greatly from methods to directly sequence and digitally quantify proteins and detect PTMs with single-molecule sensitivity and precision. However current methods for studying the proteome lag behind DNA sequencing in throughput, sensitivity, and accessibility due to the complexity and dynamic range of the proteome, the chemical properties of proteins, and the inability to amplify proteins. Here, we demonstrate single-molecule protein sequencing on a compact benchtop instrument using a dynamic sequencing by stepwise degradation approach in which single surface-immobilized peptide molecules are probed in real-time by a mixture of dye-labeled N-terminal amino acid recognizers and simultaneously cleaved by aminopeptidases. By measuring fluorescence intensity, lifetime, and binding kinetics of recognizers on an integrated semiconductor chip we are able to annotate amino acids and identify the peptide sequence. We describe the expansion of the number of recognizable amino acids and demonstrate the kinetic principles that allow individual recognizers to identify multiple amino acids in a highly information-rich manner that is sensitive to adjacent residues. Furthermore, we demonstrate that our method is compatible with both synthetic and natural peptides, and capable of detecting single amino acid changes and PTMs. We anticipate that with further development our protein sequencing method will offer a sensitive, scalable, and accessible platform for studies of the proteome.

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

2018 ◽  
Author(s):  
Samuel Rodriques ◽  
Adam Marblestone ◽  
Ed Boyden
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Single Molecule ◽  
Amino Acid Identity ◽  
Peptide Sequencing ◽  
Protein Sequencing ◽  
Single Amino Acid ◽  
Weak Binding ◽  
Binding Spectra ◽  
Accuracy Determination

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.

scholarly journals Roles of the C-Terminal Amino Acids of Non-Hexameric Helicases: Insights from Escherichia coli UvrD

2021 ◽  
Vol 22 (3) ◽  
pp. 1018
Author(s):  
Hiroaki Yokota
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Single Molecule ◽  
Underlying Mechanism ◽  
Amino Acid Sequences ◽  
E Coli ◽  
X Ray Crystallography ◽  
Terminal Amino

Helicases are nucleic acid-unwinding enzymes that are involved in the maintenance of genome integrity. Several parts of the amino acid sequences of helicases are very similar, and these quite well-conserved amino acid sequences are termed “helicase motifs”. Previous studies by X-ray crystallography and single-molecule measurements have suggested a common underlying mechanism for their function. These studies indicate the role of the helicase motifs in unwinding nucleic acids. In contrast, the sequence and length of the C-terminal amino acids of helicases are highly variable. In this paper, I review past and recent studies that proposed helicase mechanisms and studies that investigated the roles of the C-terminal amino acids on helicase and dimerization activities, primarily on the non-hexermeric Escherichia coli (E. coli) UvrD helicase. Then, I center on my recent study of single-molecule direct visualization of a UvrD mutant lacking the C-terminal 40 amino acids (UvrDΔ40C) used in studies proposing the monomer helicase model. The study demonstrated that multiple UvrDΔ40C molecules jointly participated in DNA unwinding, presumably by forming an oligomer. Thus, the single-molecule observation addressed how the C-terminal amino acids affect the number of helicases bound to DNA, oligomerization, and unwinding activity, which can be applied to other helicases.

scholarly journals Tuning Single-Molecule Conductance by Controlled Electric Field-Induced trans-to-cis Isomerisation

2021 ◽  
Vol 11 (8) ◽  
pp. 3317
Author(s):  
C.S. Quintans ◽  
Denis Andrienko ◽  
Katrin F. Domke ◽  
Daniel Aravena ◽  
Sangho Koo ◽  
...  
Keyword(s):  
Electric Field ◽  
Electric Fields ◽  
Single Molecule ◽  
Quantum Interference ◽  
Single Molecule Level ◽  
Wet Chemical Synthesis ◽  
Single Molecule Junctions ◽  
Tunnelling Microscopy ◽  
The Difference

External electric fields (EEFs) have proven to be very efficient in catalysing chemical reactions, even those inaccessible via wet-chemical synthesis. At the single-molecule level, oriented EEFs have been successfully used to promote in situ single-molecule reactions in the absence of chemical catalysts. Here, we elucidate the effect of an EEFs on the structure and conductance of a molecular junction. Employing scanning tunnelling microscopy break junction (STM-BJ) experiments, we form and electrically characterize single-molecule junctions of two tetramethyl carotene isomers. Two discrete conductance signatures show up more prominently at low and high applied voltages which are univocally ascribed to the trans and cis isomers of the carotenoid, respectively. The difference in conductance between both cis-/trans- isomers is in concordance with previous predictions considering π-quantum interference due to the presence of a single gauche defect in the trans isomer. Electronic structure calculations suggest that the electric field polarizes the molecule and mixes the excited states. The mixed states have a (spectroscopically) allowed transition and, therefore, can both promote the cis-isomerization of the molecule and participate in electron transport. Our work opens new routes for the in situ control of isomerisation reactions in single-molecule contacts.

A facile one pot route for the synthesis of imide tethered peptidomimetics

2016 ◽  
Vol 14 (2) ◽  
pp. 556-563 ◽  
Author(s):  
Veladi Panduranga ◽  
Girish Prabhu ◽  
Roopesh Kumar ◽  
Basavaprabhu Basavaprabhu ◽  
Vommina V. Sureshbabu
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Efficient Method ◽  
One Pot ◽  
Protected Amino Acid

A simple and efficient method for the synthesis of N,N’-orthogonally protected imide tethered peptidomimetics is presented. The imide peptidomimetics were synthesized by coupling the in situ generated selenocarboxylate of Nα-protected amino acids with Nα-protected amino acid azides in good yields.

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

iScience ◽  
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

scholarly journals A Chemically Soldered Polyoxometalate Single-Molecule Transistor

2020 ◽  
Author(s):  
Chuanli Wu ◽  
Xiaohang Qiao ◽  
Craig M. Robertson ◽  
Simon Higgins ◽  
Chenxin Cai ◽  
...  
Keyword(s):  
Single Molecule ◽  
Transport Mechanism ◽  
Electrochemical Behaviour ◽  
Oxidation States ◽  
Oxide Cluster ◽  
Quantum Tunnelling ◽  
Conductance Data ◽  
Single Molecule Junctions ◽  
Single Metal Oxide ◽  
Structural Versatility

Polyoxometalates have been proposed in the literature as promising components for nanoelectronic applications, where they could offer key advantages with their structural versatility and rich electrochemistry. Apart from a few studies on their ensemble behaviour (for instance, as monolayers or thin films) this potential remains largely unexplored. We synthesised a pyridyl-capped Anderson-Evans polyoxometalate and used it to fabricate single-molecule junctions, by using the organic termini to chemically “solder” a single metal oxide cluster to two nanoelectrodes through coordination bonds. Operating the device in an electrochemical environment allowed us to probe charge transport through different oxidation states of the polyoxometalate, and we report here an efficient three-state transistor behaviour. Conductance data fits a quantum tunnelling transport mechanism, with charge having different tunnelling probabilities through different oxidation states of the polyoxometalate. Our results show the promise of such compounds in nanoelectronics, and are, to our best knowledge, the first report on the single-entity electrochemical behaviour of polyoxometalates.<p></p>

Uptake and metabolism of amino acids by the dog liver perfused in situ

1962 ◽  
Vol 202 (3) ◽  
pp. 407-414 ◽  
Author(s):  
Rapier H. McMenamy ◽  
William C. Shoemaker ◽  
Jonas E. Richmond ◽  
David Elwyn
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Mixture ◽  
Acid Mixture ◽  
Sharp Rise ◽  
Dog Blood ◽  
Dog Liver ◽  
Amino Acid Concentrations ◽  
Uptake And Metabolism

Dog livers were perfused in situ for periods up to 6 hr with dog blood recycled through a pump-oxygenator. An amino acid mixture was administered for 90 min. Concentrations of amino acids were determined at intervals of 30 min or more. Rates of uptake and metabolism were calculated. After the start of perfusion, there is a fall in most plasma amino acid concentrations and a reciprocal rise in liver amino acids. Addition of amino acids causes a sharp rise in plasma amino acids. There is a rapid uptake of most of the amino acids by liver, although the concentrations of amino acids in liver fail to rise appreciably. Notable exceptions are valine, leucine, and isoleucine. Uptake of amino acids stimulates: a) an increase in the rate of synthesis of urea which ultimately accounts for 90% of the metabolized amino acids; b) a net synthesis of ornithine; and c) net noncatabolic metabolism of amino acids which may in part be protein synthesis. The results support the view that the liver temporarily stores a part of ingested amino acids as proteins, and subsequently makes them available to other organs.

Amino acid transport by juxtamedullary nephrons: distal reabsorption and recycling

1988 ◽  
Vol 255 (3) ◽  
pp. F397-F407 ◽  
Author(s):  
W. H. Dantzler ◽  
S. Silbernagl
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Transport ◽  
Acid Transport ◽  
Acidic Amino Acids ◽  
Vasa Recta ◽  
Collecting Ducts ◽  
Distal Site

Amino acid transport by juxtamedullary (JM) nephrons and its relationship to transport by superficial cortical (SC) nephrons and to function of vasa recta and collecting ducts were examined in vivo and in situ by free-flow micropuncture of Henle's loops, collecting ducts, and vasa recta and by continuous microinfusion of Henle's loops in exposed rat papillae. Fractional deliveries (FDs) of six neutral amino acids, two acidic amino acids, and taurine to tips of Henle's loops of JM nephrons could be substantially below those to early distal loops of SC nephrons, indicating that reabsorption before loop tips could be greater in JM than in SC nephrons. FDs to collecting ducts lower than to JM loop tips suggested reabsorption distal to loop tips. This was confirmed by continuous microinfusion of ascending limbs of Henle's loops. Distal site of reabsorption is unknown, but amino acids may move passively out of the thin ascending limb and be recycled into vasa recta and descending limb. Recycling of amino acids was supported by high FDs to tips of Henle's loops (sometimes greater than 1.0), higher concentrations in ascending than in descending vasa recta at same papilla level, and high mean concentrations in vasa recta.

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