scholarly journals A minimalist approach to protein identification

2017 ◽  
Author(s):  
G. Sampath
Keyword(s):  
Single Molecule ◽  
Protein Identification ◽  
Small Samples ◽  
Identification Rate ◽  
E Coli ◽  
Linear Sequence ◽  
Available Technology ◽  
H Pylori ◽  
Parameter Values ◽  
Non Destructive

AbstractComputations on proteome sequence databases show that most proteins can be identified from a protein’s isoelectric point (IEP) and digitized linear sequence volume (equal to the total volume of its residues). This is illustrated with four proteomes: H. pylori (1553 proteins), E. coli (4306 proteins), S. cerevisiae (6721 proteins), and H. sapiens (20207 proteins); the identification rate exceeds 90% in all four cases for appropriate parameter values. IEP can be obtained with 1-d gel electrophoresis (GE), whose accuracy is better than 0.01. Linear protein sequence volumes of unbroken proteins can be obtained with a sub-nanometer diameter nanopore that can measure residue volume with a resolution of 0.07-0.1 nm3 (Kennedy et al., Nature Nanotech., 2016, 11, 968-976; Dong et al., ACS Nano, 2017, doi: 10.1021/acsnano.6b08452); the blockade current due to a translocating protein is roughly proportional to the volume it excludes in the pore. There is no need to identify any of the residues. More than 90% of all the proteins have estimated translocation times higher than 1 μs, which is within the time resolution of available detectors. This is a minimalist proteolysis-free GE-and nanopore-based single-molecule approach requires very small samples, is non-destructive (the sample can be recovered for reuse), and can be translated with currently available technology into a portable device for possible use in the field, an academic lab, or a pre-screening step preceding conventional mass spectrometry.

scholarly journals A minimalist approach to protein identification

2017 ◽  
Author(s):  
G. Sampath
Keyword(s):  
Mass Spectrometry ◽  
Single Molecule ◽  
Protein Identification ◽  
Portable Device ◽  
Small Samples ◽  
Academic Setting ◽  
Minimalist Approach ◽  
Available Technology ◽  
Non Destructive

This work demonstrates a minimal single-molecule proteolysis-free approach that requires very small samples, is non-destructive, and can be translated with currently available technology into a portable device for possible use in the field or in an academic setting, or in a pre-screening step preceding conventional mass spectrometry.<br>

scholarly journals A minimalist approach to protein identification

2017 ◽  
Author(s):  
G. Sampath
Keyword(s):  
Mass Spectrometry ◽  
Single Molecule ◽  
Protein Identification ◽  
Portable Device ◽  
Small Samples ◽  
Academic Setting ◽  
Minimalist Approach ◽  
Available Technology ◽  
Non Destructive

This work demonstrates a minimal single-molecule proteolysis-free approach that requires very small samples, is non-destructive, and can be translated with currently available technology into a portable device for possible use in the field or in an academic setting, or in a pre-screening step preceding conventional mass spectrometry.<br>

scholarly journals Protein identification with a nanopore and a binary alphabet

2017 ◽  
Author(s):  
G. Sampath
Keyword(s):  
Single Molecule ◽  
High Speed ◽  
Protein Identification ◽  
Binary Sequences ◽  
False Detection Rate ◽  
False Detection ◽  
Post Translational Modifications ◽  
Identification Rate ◽  
Binary Alphabet ◽  
Protein Molecules

AbstractProtein sequences are recoded with a binary alphabet obtained by dividing the 20 amino acids into two subsets based on volume. A protein is identified from subsequences by database search. Computations on the Helicobacter pylori proteome show that over 93% of binary subsequences of length 20 are correct at a confidence level exceeding 90%. Over 98% of the proteins can be identified, most have multiple identifiers so the false detection rate is low. Binary sequences of unbroken protein molecules can be obtained with a nanopore from current blockade levels proportional to residue volume; only two levels, rather than 20, need be measured to determine a residue’s subset. This procedure can be translated into practice with a sub-nanopore that can measure residue volumes with ~0.07 nm3 resolution as shown in a recent publication. The high detector bandwidth required by the high speed of a translocating molecule can be reduced more than tenfold with an averaging technique, the resulting decrease in the identification rate is only 10%. Averaging also mitigates the homopolymer problem due to identical successive blockade levels. The proposed method is a proteolysis-free single-molecule method that can identify arbitrary proteins in a proteome rather than specific ones. This approach to protein identification also works if residue mass is used instead of mass; again over 98% of the proteins are identified by binary subsequences of length 20. The possibility of using this in mass spectrometry studies of proteins, in particular those with post-translational modifications, is under investigation.

scholarly journals E. coli Enterotoxin LtB Enhances Vaccine-Induced Anti-H. pylori Protection by Promoting Leukocyte Migration into Gastric Mucus via Inflammatory Lesions

Cells ◽  
2019 ◽  
Vol 8 (9) ◽  
pp. 982
Author(s):  
Xiaoyan Peng ◽  
Rongguang Zhang ◽  
Chen Wang ◽  
Feiyan Yu ◽  
Mingyang Yu ◽  
...  
Keyword(s):  
Cellular Immunity ◽  
Protective Efficacy ◽  
Gastric Injury ◽  
Oral Vaccines ◽  
Gastric Mucus ◽  
E Coli ◽  
Non Invasive ◽  
Considerable Impact ◽  
H Pylori

Current studies indicate that the anti-H. pylori protective efficacy of oral vaccines to a large extent depends on using mucosal adjuvants like E. coli heat-lable enterotoxin B unit (LtB). However, the mechanism by which Th17/Th1-driven cellular immunity kills H. pylori and the role of LtB remains unclear. Here, two L. lactis strains, expressing H. pylori NapA and LtB, respectively, were orally administrated to mice. As observed, the administration of LtB significantly enhanced the fecal SIgA level and decreased gastric H. pylori colonization, but also markedly aggravated gastric inflammatory injury. Both NapA group and NapA+LtB group had elevated splenocyte production of IL-8, IL-10, IL-12, IL-17, IL-23 and INF-γ. Notably, gastric leukocytes’ migration or leakage into the mucus was observed more frequently in NapA+LtB group than in NapA group. This report is the first that discusses how LtB enhances vaccine-induced anti-H. pylori efficacy by aggravating gastric injury and leukocytes’ movement into the mucus layer. Significantly, it brings up a novel explanation for the mechanism underlying mucosal cellular immunity destroying the non-invasive pathogens. More importantly, the findings suggest the necessity to further evaluate LtB’s potential hazards to humans before extending its applications. Thus, this report can provide considerable impact on the fields of mucosal immunology and vaccinology.

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 Fast and efficient Rmap assembly using the Bi-labelled de Bruijn graph

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Kingshuk Mukherjee ◽  
Massimiliano Rossi ◽  
Leena Salmela ◽  
Christina Boucher
Keyword(s):  
Single Molecule ◽  
De Bruijn Graph ◽  
Anabas Testudineus ◽  
E Coli ◽  
Genome Wide ◽  
A Genome ◽  
De Bruijn ◽  
Optical Maps ◽  
Definition Of ◽  
Numeric Representation

AbstractGenome wide optical maps are high resolution restriction maps that give a unique numeric representation to a genome. They are produced by assembling hundreds of thousands of single molecule optical maps, which are called Rmaps. Unfortunately, there are very few choices for assembling Rmap data. There exists only one publicly-available non-proprietary method for assembly and one proprietary software that is available via an executable. Furthermore, the publicly-available method, by Valouev et al. (Proc Natl Acad Sci USA 103(43):15770–15775, 2006), follows the overlap-layout-consensus (OLC) paradigm, and therefore, is unable to scale for relatively large genomes. The algorithm behind the proprietary method, Bionano Genomics’ Solve, is largely unknown. In this paper, we extend the definition of bi-labels in the paired de Bruijn graph to the context of optical mapping data, and present the first de Bruijn graph based method for Rmap assembly. We implement our approach, which we refer to as rmapper, and compare its performance against the assembler of Valouev et al. (Proc Natl Acad Sci USA 103(43):15770–15775, 2006) and Solve by Bionano Genomics on data from three genomes: E. coli, human, and climbing perch fish (Anabas Testudineus). Our method was able to successfully run on all three genomes. The method of Valouev et al. (Proc Natl Acad Sci USA 103(43):15770–15775, 2006) only successfully ran on E. coli. Moreover, on the human genome rmapper was at least 130 times faster than Bionano Solve, used five times less memory and produced the highest genome fraction with zero mis-assemblies. Our software, rmapper is written in C++ and is publicly available under GNU General Public License at https://github.com/kingufl/Rmapper.

scholarly journals Studying Binding, Conformational Transition and Assembly of E. Coli Cytolysin a Pore Forming Toxin by Single Molecule Fluorescence

2017 ◽  
Vol 112 (3) ◽  
pp. 524a
Author(s):  
Pradeep Sathyanarayana ◽  
Satyaghosh Maurya ◽  
Ganapathy Ayappa ◽  
Sandhya S. Visweswariah ◽  
Rahul Roy
Keyword(s):  
Single Molecule ◽  
Conformational Transition ◽  
Single Molecule Fluorescence ◽  
E Coli

scholarly journals Single-molecule protein identification by sub-nanopore sensors

2017 ◽  
Vol 13 (5) ◽  
pp. e1005356 ◽  
Author(s):  
Mikhail Kolmogorov ◽  
Eamonn Kennedy ◽  
Zhuxin Dong ◽  
Gregory Timp ◽  
Pavel A. Pevzner
Keyword(s):  
Single Molecule ◽  
Protein Identification ◽  
Nanopore Sensors

scholarly journals Development of an Internal Control for Evaluation and Standardization of a Quantitative PCR Assay for Detection of Helicobacter pylori in Drinking Water

2007 ◽  
Vol 73 (22) ◽  
pp. 7380-7387 ◽  
Author(s):  
Keya Sen ◽  
Nancy A. Schable ◽  
Dennis J. Lye
Keyword(s):  
Helicobacter Pylori ◽  
Drinking Water ◽  
Quantitative Pcr ◽  
Water Samples ◽  
Sodium Thiosulfate ◽  
Qpcr Assay ◽  
E Coli ◽  
H Pylori ◽  
Labeled Probe ◽  
Treated Drinking Water

ABSTRACT Due to metabolic and morphological changes that can prevent Helicobacter pylori cells in water from growing on conventional media, an H. pylori-specific TaqMan quantitative PCR (qPCR) assay was developed that uses a 6-carboxyfluorescein-labeled probe (A. E. McDaniels, L. Wymer, C. Rankin, and R. Haugland, Water Res. 39:4808-4816, 2005). However, proper internal controls are needed to provide an accurate estimate of low numbers of H. pylori in drinking water. In this study, the 135-bp amplicon described by McDaniels et al. was modified at the probe binding region, using PCR mutagenesis. The fragment was incorporated into a single-copy plasmid to serve as a PCR-positive control and cloned into Escherichia coli to serve as a matrix spike. It was shown to have a detection limit of five copies, using a VIC dye-labeled probe. A DNA extraction kit was optimized that allowed sampling of an entire liter of water. Water samples spiked with the recombinant E. coli cells were shown to behave like H. pylori cells in the qPCR assay. The recombinant E. coli cells were optimized to be used at 10 cells/liter of water, where they were shown not to compete with 5 to 3,000 cells of H. pylori in a duplex qPCR assay. Four treated drinking water samples spiked with H. pylori (100 cells) demonstrated similar cycle threshold values if the chlorine disinfectant was first neutralized by sodium thiosulfate.

scholarly journals Structures of the ATP-fueled ClpXP proteolytic machine bound to protein substrate

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Xue Fei ◽  
Tristan A Bell ◽  
Simon Jenni ◽  
Benjamin M Stinson ◽  
Tania A Baker ◽  
...  
Keyword(s):  
Single Molecule ◽  
Atp Hydrolysis ◽  
Functional Results ◽  
Structural Features ◽  
Specific Protein ◽  
Power Stroke ◽  
E Coli ◽  
Protein Substrates ◽  
Biophysical Studies ◽  
Sequential Models

ClpXP is an ATP-dependent protease in which the ClpX AAA+ motor binds, unfolds, and translocates specific protein substrates into the degradation chamber of ClpP. We present cryo-EM studies of the E. coli enzyme that show how asymmetric hexameric rings of ClpX bind symmetric heptameric rings of ClpP and interact with protein substrates. Subunits in the ClpX hexamer assume a spiral conformation and interact with two-residue segments of substrate in the axial channel, as observed for other AAA+ proteases and protein-remodeling machines. Strictly sequential models of ATP hydrolysis and a power stroke that moves two residues of the substrate per translocation step have been inferred from these structural features for other AAA+ unfoldases, but biochemical and single-molecule biophysical studies indicate that ClpXP operates by a probabilistic mechanism in which five to eight residues are translocated for each ATP hydrolyzed. We propose structure-based models that could account for the functional results.

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