Browse
Submit Data
Databases
API
Help

Dataset Information

26 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Controlled movement of ssDNA conjugated peptide through Mycobacterium smegmatis porin A (MspA) nanopore by a helicase motor for peptide sequencing application.

ABSTRACT: The lack of an efficient, low-cost sequencing method has long been a significant bottleneck in protein research and applications. In recent years, the nanopore platform has emerged as a fast and inexpensive method for single-molecule nucleic acid sequencing, but attempts to apply it to protein/peptide sequencing have resulted in limited success. Here we report a strategy to control peptide translocation through the MspA nanopore, which could serve as the first step toward strand peptide sequencing. By conjugating the target peptide to a helicase-regulated handle-ssDNA, we achieved a read length of up to 17 amino acids (aa) and demonstrated the feasibility of distinguishing between amino acid residues of different charges or between different phosphorylation sites. Further improvement of resolution may require engineering MspA-M2 to reduce its constriction zone's size and stretch the target peptide inside the nanopore to minimize random thermal motion. We believe that our method in this study can significantly accelerate the development and commercialization of nanopore-based peptide sequencing technologies.

SUBMITTER: Chen Z

PROVIDER: S-EPMC8654031 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Tailored Polymeric Membranes for Mycobacterium Smegmatis Porin A (MspA) Based Biosensors.

Project description:Nanopores based on protein channels inserted into lipid membranes have paved the way towards a wide-range of inexpensive biosensors, especially for DNA sequencing. A key obstacle in using these biological ion channels as nanodevices is the poor stability of lipid bilayer membranes. Amphiphilic block copolymer membranes have emerged as a robust alternative to lipid membranes. While previous efforts have shown feasibility, we demonstrate for the first time the effect of polymer composition on MspA protein functionality. We show that membrane-protein interaction depends on the hydrophobic-hydrophilic ratio (f-ratio) of the block copolymer. These effects are particularly pronounced in asymmetric protein pores like MspA compared to the cylindrical ?-Hemolysin pore. A key effect of membrane-protein interaction is the increased 1/f? noise. After first showing increases in 1/f? behaviour arise from increased substate activity, the noise power spectral density S(f) was used as a qualitative tool for understanding protein-membrane interactions in polymer membranes. Polymer compositions with f-ratios close to lipid membranes caused noise behaviour not observed in lipid membranes. However, by modifying the f-ratio using a modular synthetic approach, we were able to design a block copolymer exhibiting noise properties similar to a lipid membrane, albeit with better stability. Thus, by careful optimization, block copolymer membranes can emerge as a robust alternative for protein-pore based nano-biosensors.

| S-EPMC4582436 | biostudies-literature

Expression of the major porin gene mspA is regulated in Mycobacterium smegmatis.

Project description:MspA is the major porin of Mycobacterium smegmatis and is important for diffusion of small and hydrophilic solutes across its unique outer membrane. The start point of transcription of the mspA gene was mapped by primer extension and S1 nuclease experiments. The main promoter driving transcription of mspA was identified by single point mutations in lacZ fusions and resembled sigma(A) promoters of M. smegmatis. However, a 500-bp upstream fragment including P(mspA) in a transcriptional fusion with lacZ yielded only low beta-galactosidase activity, whereas activity increased 12-fold with a 700-bp fragment. Activation of P(mspA) by the 200-bp element was almost eliminated by increasing the distance by 14 bp, indicating binding of an activator protein. The chromosomal mspA transcript had a size of 900 bases and was very stable with a half-life of 6 minutes, whereas the stabilities of episomal mspA transcripts with three other 5' untranslated region (UTRs) were three- to sixfold reduced, indicating a stabilizing role of the native 5' UTR of mspA. Northern blot experiments revealed that the amount of mspA mRNA was increased under nitrogen limitation but reduced under carbon and phosphate limitation at 42 degrees C in stationary phase in the presence of 0.5 M sodium chloride, 18 mM hydrogen peroxide, and 10% ethanol and at acidic pH. These results show for the first time that M. smegmatis regulates porin gene expression to optimize uptake of certain nutrients and to protect itself from toxic solutes.

| S-EPMC1797333 | biostudies-literature

Investigating asymmetric salt profiles for nanopore DNA sequencing with biological porin MspA.

Project description:Nanopore DNA sequencing is a promising single-molecule analysis technology. This technique relies on a DNA motor enzyme to control movement of DNA precisely through a nanopore. Specific experimental buffer conditions are required based on the preferred operating conditions of the DNA motor enzyme. While many DNA motor enzymes typically operate in salt concentrations under 100 mM, salt concentration simultaneously affects signal and noise magnitude as well as DNA capture rate in nanopore sequencing, limiting standard experimental conditions to salt concentrations greater than ~100 mM in order to maintain adequate resolution and experimental throughput. We evaluated the signal contribution from ions on both sides of the membrane (cis and trans) by varying cis and trans [KCl] independently during phi29 DNA Polymerase-controlled translocation of DNA through the biological porin MspA. Our studies reveal that during DNA translocation, the negatively charged DNA increases cation selectivity through MspA with the majority of current produced by the flow of K+ ions from trans to cis. Varying trans [K+] has dramatic effects on the signal magnitude, whereas changing cis [Cl-] produces only small effects. Good signal-to-noise can be maintained with cis [Cl-] as small as 20 mM, if the concentration of KCl on the trans side is kept high. These results demonstrate the potential of using salt-sensitive motor enzymes (helicases, polymerases, recombinases) in nanopore systems and offer a guide for selecting buffer conditions in future experiments to simultaneously optimize signal, throughput, and enzyme activity.

| S-EPMC5531483 | biostudies-literature

Single molecule observation of hard-soft-acid-base (HSAB) interaction in engineered <i>Mycobacterium smegmatis</i> porin A (MspA) nanopores.

Project description:In the formation of coordination interactions between metal ions and amino acids in natural metalloproteins, the bound metal ion is critical either for the stabilization of the protein structure or as an enzyme co-factor. Though extremely small in size, metal ions, when bound to the restricted environment of an engineered biological nanopore, result in detectable perturbations during single channel recordings. All reported work of this kind was performed with engineered α-hemolysin nanopores and the observed events appear to be extremely small in amplitude (∼1-3 pA). We speculate that the cylindrical pore restriction of α-hemolysin may not be optimal for probing extremely small analytes. Mycobacterium smegmatis porin A (MspA), a conical shaped nanopore, was engineered to interact with Ca2+, Mn2+, Co2+, Ni2+, Zn2+, Pb2+ and Cd2+ and a systematically larger event amplitude (up to 10 pA) was observed. The measured rate constant suggests that the coordination of a single ion with an amino acid follows hard-soft-acid-base theory, which has never been systematically validated in the case of a single molecule. By adjusting the measurement pH from 6.8 to 8.0, the duration of a single ion binding event could be modified with a ∼46-fold time extension. The phenomena reported suggest MspA to be a superior engineering template for probing a variety of extremely small analytes, such as monatomic and polyatomic ions, small molecules or chemical intermediates, and the principle of hard-soft-acid-base interaction may be instructive in the pore design.

| S-EPMC8146584 | biostudies-literature

Rapid and multiplex preparation of engineered <i>Mycobacterium smegmatis</i> porin A (MspA) nanopores for single molecule sensing and sequencing.

Project description:Acknowledging its unique conical lumen structure, Mycobacterium smegmatis porin A (MspA) was the first type of nanopore that has successfully sequenced DNA. Recent developments of nanopore single molecule chemistry have also suggested MspA to be an optimum single molecule reactor. However, further investigations with this approach require heavy mutagenesis which is labor intensive and requires high end instruments for purifications. We here demonstrate an efficient and economic protocol which performs rapid and multiplex preparation of a variety of MspA mutants. The prepared MspA mutants were demonstrated in operations such as nanopore insertion, sequencing, optical single channel recording (oSCR), nanopore single molecule chemistry and nanopore rectification. The performance is no different from that of pores however prepared by other means. The time of all human operations and the cost for a single batch of preparation have been minimized to 40 min and 0.4$, respectively. This method is extremely useful in the screening of new MspA mutants, which has an urgent requirement in further investigations of new MspA nanoreactors. Its low cost and simplicity also enable efficient preparations of MspA nanopores for both industrial manufacturing and academic research.

| S-EPMC8278974 | biostudies-literature

Nanopore DNA sequencing with MspA.

Project description:Nanopore sequencing has the potential to become a direct, fast, and inexpensive DNA sequencing technology. The simplest form of nanopore DNA sequencing utilizes the hypothesis that individual nucleotides of single-stranded DNA passing through a nanopore will uniquely modulate an ionic current flowing through the pore, allowing the record of the current to yield the DNA sequence. We demonstrate that the ionic current through the engineered Mycobacterium smegmatis porin A, MspA, has the ability to distinguish all four DNA nucleotides and resolve single-nucleotides in single-stranded DNA when double-stranded DNA temporarily holds the nucleotides in the pore constriction. Passing DNA with a series of double-stranded sections through MspA provides proof of principle of a simple DNA sequencing method using a nanopore. These findings highlight the importance of MspA in the future of nanopore sequencing.

| S-EPMC2941267 | biostudies-literature

Hetero-oligomeric MspA pores in Mycobacterium smegmatis.

Project description:The porin MspA of Mycobacterium smegmatis is a biological nanopore used for DNA sequencing. The octameric MspA pore can be isolated from M. smegmatis in milligram quantities, is extremely stable against denaturation and rapidly inserts into lipid membranes. Here, we show that MspA pores composed of different Msp subunits are formed in M. smegmatis and that hetero-oligomers of different Msp monomers increase the heterogeneity of MspA pores designed for DNA sequencing. To improve the quality of preparations of mutant MspA proteins, all four msp genes were deleted from the M. smegmatis genome after insertion of an inducible porin gene from M. tuberculosis. In the msp quadruple mutant M. smegmatis ML712 no Msp porins were detected and mutant MspA proteins were produced at wild-type levels. Lipid bilayer experiments demonstrated that MspA pores isolated from ML712 formed functional channels and had a narrower conductance distribution than pores purified from M. smegmatis with background msp expression. Thus, the M. smegmatis msp quadruple mutant improves the homogeneity of MspA pores designed for DNA sequencing and might also facilitate the identification and functional characterization of other mycobacterial pore proteins.

| S-EPMC5975246 | biostudies-literature

Giant single molecule chemistry events observed from a tetrachloroaurate(III) embedded Mycobacterium smegmatis porin A nanopore.

Project description:Biological nanopores are capable of resolving small analytes down to a monoatomic ion. In this research, tetrachloroaurate(III), a polyatomic ion, is discovered to bind to the methionine residue (M113) of a wild-type α-hemolysin by reversible Au(III)-thioether coordination. However, the cylindrical pore geometry of α-hemolysin generates shallow ionic binding events (~5-6 pA) and may have introduced other undesired interactions. Inspired by nanopore sequencing, a Mycobacterium smegmatis porin A (MspA) nanopore, which possesses a conical pore geometry, is mutated to bind tetrachloroaurate(III). Subsequently, further amplified blockage events (up to ~55 pA) are observed, which report the largest single ion binding event from a nanopore measurement. By taking the embedded Au(III) as an atomic bridge, the MspA nanopore is enabled to discriminate between different biothiols from single molecule readouts. These phenomena suggest that MspA is advantageous for single molecule chemistry investigations and has applications as a hybrid biological nanopore with atomic adaptors.

| S-EPMC6906327 | biostudies-literature

Structural-profiling of low molecular weight RNAs by nanopore trapping/translocation using Mycobacterium smegmatis porin A.

Project description:Folding of RNA can produce elaborate tertiary structures, corresponding to their diverse roles in the regulation of biological activities. Direct observation of RNA structures at high resolution in their native form however remains a challenge. The large vestibule and the narrow constriction of a Mycobacterium smegmatis porin A (MspA) suggests a sensing mode called nanopore trapping/translocation, which clearly distinguishes between microRNA, small interfering RNA (siRNA), transfer RNA (tRNA) and 5 S ribosomal RNA (rRNA). To further profit from the acquired event characteristics, a custom machine learning algorithm is developed. Events from measurements with a mixture of RNA analytes can be automatically classified, reporting a general accuracy of ~93.4%. tRNAs, which possess a unique tertiary structure, report a highly distinguishable sensing feature, different from all other RNA types tested in this study. With this strategy, tRNAs from different sources are measured and a high structural conservation across different species is observed in single molecule.

| S-EPMC8185011 | biostudies-literature

Nanoscopic surfactant behavior of the porin MspA in aqueous media.

Project description:The mycobacterial porin MspA is one of the most stable channel proteins known to date. MspA forms vesicles at low concentrations in aqueous buffers. Evidence from dynamic light scattering, transmission electron microscopy and zeta-potential measurements by electrophoretic light scattering indicate that MspA behaves like a nanoscale surfactant. The extreme thermostability of MspA allows these investigations to be carried out at temperatures as high as 343 K, at which most other proteins would quickly denature. The principles of vesicle formation of MspA as a function of temperature and the underlying thermodynamic factors are discussed here. The results obtained provide crucial evidence in support of the hypothesis that, during vesicle formation, nanoscopic surfactant molecules, such as MspA, deviate from the principles underlined in classical surface chemistry.

| S-EPMC3678404 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data