scholarly journals Single Molecule Protein Sequencing Based on the Superspecificity of tRNA Synthetases

2020 ◽  
Author(s):  
G Sampath
Keyword(s):  
Single Molecule ◽  
De Novo ◽  
Protein Molecule ◽  
Electrolytic Cell ◽  
Protein Sequencing ◽  
Base Pairs ◽  
Trna Synthetases ◽  
Terminal Residue ◽  
Unfolded Protein ◽  
Intensity Measurements

Single molecule de novo protein sequencing based on the 'superspecificity' of amino-acyl tRNA synthetases (aaRS) is proposed. An unfolded protein molecule is threaded through a nanopore in an electrolytic cell (e-cell) to expose the terminal residue in the e-cell's trans chamber. After the residue is cleaved with a peptidase, a set of tRNAs, their aaRSs, and ATP are added to trans. An aaRS charges a cognate tRNA molecule with the residue. The charged tRNA (along with the other reactants) is transferred to an extended e-cell with N (20 ≤ N ≤ 61) pores in N individual cis chambers and a single trans chamber. Each pore holds an RNA molecule ending in a unique codon that is exposed in trans. The charged tRNA's anticodon base-pairs with the terminal codon of an RNA. If tRNAs and residues are fluorescently tagged with two different colors, the residue can be identified from the observed position of the color pair. As charging is 'superspecific' identification is unambiguous. The protein molecule in the first e-cell is advanced by one residue and the process repeated. In this approach there is no need for precise pore current or optical intensity measurements. Potential implementation issues are discussed. Several variations, including one in which the terminal residue is cleaved after charging, are also considered.

scholarly journals Single Molecule Protein Sequencing Based on the Superspecificity of tRNA Synthetases

2020 ◽  
Author(s):  
G Sampath
Keyword(s):  
Single Molecule ◽  
De Novo ◽  
Protein Molecule ◽  
Electrolytic Cell ◽  
Protein Sequencing ◽  
Base Pairs ◽  
Trna Synthetases ◽  
Terminal Residue ◽  
Unfolded Protein ◽  
Intensity Measurements

Single molecule de novo protein sequencing based on the 'superspecificity' of amino-acyl tRNA synthetases (aaRS) is proposed. An unfolded protein molecule is threaded through a nanopore in an electrolytic cell (e-cell) to expose the terminal residue in the e-cell's trans chamber. After the residue is cleaved with an exopeptidase, a set of tRNAs, their aaRSs, and ATP are added to trans. An aaRS charges a cognate tRNA molecule with the residue. The charged tRNA (along with the other reactants) is transferred to an extended e-cell with N (20 ≤ N ≤ 61) pores in N individual cis chambers and a single trans chamber. Each pore holds an RNA molecule ending in a unique codon that is exposed in trans. The charged tRNA's anticodon base-pairs with the terminal codon of an RNA. If tRNAs and residues are fluorescently tagged with two different colors, the residue can be identified from the observed position of the resulting color pair. As charging is 'superspecific' identification is unambiguous. The protein molecule in the first e-cell is advanced by one residue and the process repeated. In this approach there is no need for precise pore current or optical intensity measurements. Potential implementation issues are discussed. Other possibilities, including one in which the terminal residue is cleaved after charging, are also examined.

scholarly journals On Sequencing a Protein with Nanopores, tRNAs, and RNAs with Matching Terminal Codons

2019 ◽  
Author(s):  
G Sampath
Keyword(s):  
De Novo ◽  
A Priori ◽  
Protein Molecule ◽  
Electrolytic Cell ◽  
A Priori Information ◽  
Unfolded Protein ◽  
In Trans ◽  
Priori Information ◽  
The One ◽  
A Current

A method for sequencing a protein from a codon sequence is proposed. An unfolded protein molecule is threaded through a nano-sized pore in an electrolytic cell carboxyl end first and held with a voltage such that only the first residue is exposed in the trans chamber of the cell. A tRNA molecule in trans with matching anticodon for the residue binds itself to the latter in the presence of suitable catalysts. It is then cleaved and transferred to an extended electrolytic cell with N pores, 40 ≤ N ≤ 61, in N individual cis chambers and a single trans chamber. Each pore holds an RNA molecule ending in a unique codon that is held exposed in the trans chamber. In the presence of suitable catalysts the anticodon in the transferred tRNA binds with the codon of a matching RNA molecule. By reversing the voltages in the extended cell every RNA molecule except the one to which the transferred tRNA is bound retracts into its cis chamber, this identifies the residue unambiguously. The detected residue in the first cell is cleaved and the process repeated. Unlike in other nanopore-based methods, it suffices to detect the occurrence of a current blockade without having to measure the pore current precisely. A simplified but more time-consuming version that uses only the first cell is also described. In either case no a priori information about the protein is needed so de novo sequencing is possible. A feasibility analysis of the proposed scheme is presented.

scholarly journals De novo design of transmembrane β-barrels

2020 ◽  
Author(s):  
Anastassia A. Vorobieva ◽  
Paul White ◽  
Binyong Liang ◽  
Jim E Horne ◽  
Asim K. Bera ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Single Molecule ◽  
First Principles ◽  
Lipid Membranes ◽  
De Novo ◽  
Computational Design ◽  
Protein Sequencing ◽  
Naturally Occurring ◽  
Transmembrane Pores ◽  
Almost All

AbstractThe ability of naturally occurring transmembrane β-barrel proteins (TMBs) to spontaneously insert into lipid bilayers and form stable transmembrane pores is a remarkable feat of protein evolution and has been exploited in biotechnology for applications ranging from single molecule DNA and protein sequencing to biomimetic filtration membranes. Because it has not been possible to design TMBs from first principles, these efforts have relied on re-engineering of naturally occurring TMBs that generally have a biological function very different from that desired. Here we leverage the power of de novo computational design coupled with a “hypothesis, design and test” approach to determine principles underlying TMB structure and folding, and find that, unlike almost all other classes of protein, locally destabilizing sequences in both the β-turns and β-strands facilitate TMB expression and global folding by modulating the kinetics of folding and the competition between soluble misfolding and proper folding into the lipid bilayer. We use these principles to design new eight stranded TMBs with sequences unrelated to any known TMB and show that they insert and fold into detergent micelles and synthetic lipid membranes. The designed proteins fold more rapidly and reversibly in lipid membranes than the TMB domain of the model native protein OmpA, and high resolution NMR and X-ray crystal structures of one of the designs are very close to the computational model. The ability to design TMBs from first principles opens the door to custom design of TMBs for biotechnology and demonstrates the value of de novo design to investigate basic protein folding problems that are otherwise hidden by evolutionary history.One sentence summarySuccess in de novo design of transmembrane β-barrels reveals geometric and sequence constraints on the fold and paves the way to design of custom pores for sequencing and other single-molecule analytical applications.

scholarly journals Label-free amino acid identification for de novo protein sequencing via tRNA charging and current blockade in a nanopore

2020 ◽  
Author(s):  
G. Sampath
Keyword(s):  
Amino Acids ◽  
Free Amino ◽  
Prior Information ◽  
De Novo ◽  
Adenosine Monophosphate ◽  
Electrolytic Cell ◽  
Trna Synthetase ◽  
Protein Sequencing ◽  
Label Free ◽  
In Series

AbstractA label-free procedure to identify single amino acids (AAs) for de novo protein sequencing is developed in theory and simulated in part. A terminal AA cleaved from a protein/peptide, a tRNA, its cognate amino-acyl tRNA synthetase (AARS), and adenosine triphosphate (ATP) are brought together in a container where tRNA, if cognate, gets charged with AA and adenosine monophosphate (AMP) is released. The tRNAs (charged or not) are filtered out, deacylated, and transferred to an electrolytic cell (e-cell) with a nanopore. If a blockade occurs it can only be due to AA; this immediately identifies AA, the error rate is < 1/350 because of tRNA superspecificity. This procedure can be executed in parallel with 20 or more molecules of AA and 20 different tRNAs to identify AA in one cycle. (It can also be done in series if the sample size is small, with only a small number of molecules available.) In a second version the released AMP translocates with ATP and free AA into the cis chamber of an e-cell. If tRNA is charged an AMP blockade is observed, this identifies AA. This version is easier to implement and parallelize (or serialize, if only a few molecules are available). In the first method it suffices to distinguish a blockade from noise; in the second an AMP blockade must be differentiated from one due to ATP or any of the 20 AAs. Even so the error is < 10% when weighted over the human proteome. Both are de novo methods; no prior information about the protein is needed. Unlike most other nanopore-based methods they do not need to discriminate among the 20 proteinogenic AAs when measuring/observing blockades.

scholarly journals Third-generation sequencing and the future of genomics

2016 ◽  
Author(s):  
Hayan Lee ◽  
James Gurtowski ◽  
Shinjae Yoo ◽  
Maria Nattestad ◽  
Shoshana Marcus ◽  
...  
Keyword(s):  
Single Molecule ◽  
Structural Variation ◽  
De Novo ◽  
Third Generation ◽  
Base Pairs ◽  
Haplotype Phasing ◽  
Third Generation Sequencing ◽  
Second Generation Sequencing ◽  
High Quality Genome ◽  
Generation Sequencing

AbstractThird-generation long-range DNA sequencing and mapping technologies are creating a renaissance in high-quality genome sequencing. Unlike second-generation sequencing, which produces short reads a few hundred base-pairs long, third-generation single-molecule technologies generate over 10,000 bp reads or map over 100,000 bp molecules. We analyze how increased read lengths can be used to address longstanding problems in de novo genome assembly, structural variation analysis and haplotype phasing.

scholarly journals On the dynamics of some small structural motifs in rRNA upon ligand binding

2008 ◽  
Vol 73 (1) ◽  
pp. 41-53
Author(s):  
Aleksandra Rakic ◽  
Petar Mitrasinovic
Keyword(s):  
Molecular Dynamics ◽  
Conformational Changes ◽  
Binding Sites ◽  
De Novo ◽  
Reference Structure ◽  
Base Pairs ◽  
Rna Sequences ◽  
Conformational Model ◽  
Thermodynamic Variables ◽  
Dynamics Simulations

The present study characterizes using molecular dynamics simulations the behavior of the GAA (1186-1188) hairpin triloops with their closing c-g base pairs in large ribonucleoligand complexes (PDB IDs: 1njn, 1nwy, 1jzx). The relative energies of the motifs in the complexes with respect to that in the reference structure (unbound form of rRNA; PDB ID: 1njp) display the trends that agree with those of the conformational parameters reported in a previous study1 utilizing the de novo pseudotorsional (?,?) approach. The RNA regions around the actual RNA-ligand contacts, which experience the most substantial conformational changes upon formation of the complexes were identified. The thermodynamic parameters, based on a two-state conformational model of RNA sequences containing 15, 21 and 27 nucleotides in the immediate vicinity of the particular binding sites, were evaluated. From a more structural standpoint, the strain of a triloop, being far from the specific contacts and interacting primarily with other parts of the ribosome, was established as a structural feature which conforms to the trend of the average values of the thermodynamic variables corresponding to the three motifs defined by the 15-, 21- and 27-nucleotide sequences. From a more functional standpoint, RNA-ligand recognition is suggested to be presumably dictated by the types of ligands in the complexes.

scholarly journals A Truncated Granulocyte Colony-stimulating Factor Receptor (G-CSFR) Inhibits Apoptosis Induced by Neutrophil Elastase G185R Mutant

2017 ◽  
Vol 292 (8) ◽  
pp. 3496-3505 ◽  
Author(s):  
Yaling Qiu ◽  
Yangyang Zhang ◽  
Nan Hu ◽  
Fan Dong
Keyword(s):  
Unfolded Protein Response ◽  
Neutrophil Elastase ◽  
De Novo ◽  
Precursor Cells ◽  
Dominant Negative ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Unfolded Protein ◽  
Protein Response ◽  
Stimulating Factor

Mutations in ELANE encoding neutrophil elastase (NE) have been identified in the majority of patients with severe congenital neutropenia (SCN). The NE mutants have been shown to activate unfolded protein response and induce premature apoptosis in myeloid cells. Patients with SCN are predisposed to acute myeloid leukemia (AML), and progression from SCN to AML is accompanied by mutations in CSF3R encoding the granulocyte colony-stimulating factor receptor (G-CSFR) in ∼80% of patients. The mutations result in the expression of C-terminally truncated G-CSFRs that promote strong cell proliferation and survival. It is unknown why the CSF3R mutations, which are rare in de novo AML, are so prevalent in SCN/AML. We show here that a G-CSFR mutant, d715, derived from an SCN patient inhibited G-CSF-induced expression of NE in a dominant negative manner. Furthermore, G-CSFR d715 suppressed unfolded protein response and apoptosis induced by an SCN-derived NE mutant, which was associated with sustained activation of AKT and STAT5, and augmented expression of BCL-XL. Thus, the truncated G-CSFRs associated with SCN/AML may protect myeloid precursor cells from apoptosis induced by the NE mutants. We propose that acquisition of CSF3R mutations may represent a mechanism by which myeloid precursor cells carrying the ELANE mutations evade the proapoptotic activity of the NE mutants in SCN patients.

AStrap: identification of alternative splicing from transcript sequences without a reference genome

2018 ◽  
Vol 35 (15) ◽  
pp. 2654-2656 ◽  
Author(s):  
Guoli Ji ◽  
Wenbin Ye ◽  
Yaru Su ◽  
Moliang Chen ◽  
Guangzao Huang ◽  
...  
Keyword(s):  
Machine Learning ◽  
Alternative Splicing ◽  
Single Molecule ◽  
Reference Genome ◽  
De Novo ◽  
Supplementary Information ◽  
Model Organisms ◽  
Sequencing Data ◽  
Extensive Evaluation ◽  
Reference Genomes

Abstract Summary Alternative splicing (AS) is a well-established mechanism for increasing transcriptome and proteome diversity, however, detecting AS events and distinguishing among AS types in organisms without available reference genomes remains challenging. We developed a de novo approach called AStrap for AS analysis without using a reference genome. AStrap identifies AS events by extensive pair-wise alignments of transcript sequences and predicts AS types by a machine-learning model integrating more than 500 assembled features. We evaluated AStrap using collected AS events from reference genomes of rice and human as well as single-molecule real-time sequencing data from Amborella trichopoda. Results show that AStrap can identify much more AS events with comparable or higher accuracy than the competing method. AStrap also possesses a unique feature of predicting AS types, which achieves an overall accuracy of ∼0.87 for different species. Extensive evaluation of AStrap using different parameters, sample sizes and machine-learning models on different species also demonstrates the robustness and flexibility of AStrap. AStrap could be a valuable addition to the community for the study of AS in non-model organisms with limited genetic resources. Availability and implementation AStrap is available for download at https://github.com/BMILAB/AStrap. Supplementary information Supplementary data are available at Bioinformatics online.

scholarly journals Genomic Analysis of Sarcomyxa edulis Reveals the Basis of Its Medicinal Properties and Evolutionary Relationships

2021 ◽  
Vol 12 ◽  
Author(s):  
Fenghua Tian ◽  
Changtian Li ◽  
Yu Li
Keyword(s):  
Single Molecule ◽  
De Novo ◽  
Genomic Analysis ◽  
Single Copy ◽  
Whole Genome Sequence ◽  
Type I ◽  
Whole Genome ◽  
Uridine Diphosphate ◽  
Protein Coding ◽  
Medicinal Value

Yuanmo [Sarcomyxa edulis (Y.C. Dai, Niemelä &amp; G.F. Qin) T. Saito, Tonouchi &amp; T. Harada] is an important edible and medicinal mushroom endemic to Northeastern China. Here we report the de novo sequencing and assembly of the S. edulis genome using single-molecule real-time sequencing technology. The whole genome was approximately 35.65 Mb, with a G + C content of 48.31%. Genome assembly generated 41 contigs with an N50 length of 1,772,559 bp. The genome comprised 9,364 annotated protein-coding genes, many of which encoded enzymes involved in the modification, biosynthesis, and degradation of glycoconjugates and carbohydrates or enzymes predicted to be involved in the biosynthesis of secondary metabolites such as terpene, type I polyketide, siderophore, and fatty acids, which are responsible for the pharmacodynamic activities of S. edulis. We also identified genes encoding 1,3-β-glucan synthase and endo-1,3(4)-β-glucanase, which are involved in polysaccharide and uridine diphosphate glucose biosynthesis. Phylogenetic and comparative analyses of Basidiomycota fungi based on a single-copy orthologous protein indicated that the Sarcomyxa genus is an independent group that evolved from the Pleurotaceae family. The annotated whole-genome sequence of S. edulis can serve as a reference for investigations of bioactive compounds with medicinal value and the development and commercial production of superior S. edulis varieties.

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

Biomolecules ◽  
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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