Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Control of the conductance of engineered protein nanopores through concerted loop motions.

ABSTRACT: Protein nanopores have attracted much interest for nucleic acid sequencing, chemical sensing, and protein folding at the single molecule level. The outer membrane protein OmpG from E. coli stands out because it forms a nanopore from a single polypeptide chain. This property allows the separate engineering of each of the seven extracellular loops that control access to the pore. The longest of these loops, loop?6, has been recognized as the main gating loop that closes the pore at low pH values and opens it at high pH values. A method was devised to pin each of the loops to the embedding membrane and measure the single-pore conductances of the resulting constructs. The electrophysiological and complementary NMR measurements show that the pinning of individual loops alters the structure and dynamics of neighboring and distant loops in a correlated fashion. Pinning loop?6 generates a constitutively open pore and patterns of concerted loop motions control access to the OmpG nanopore.

SUBMITTER: Zhuang T

PROVIDER: S-EPMC4091679 | biostudies-literature | 2014 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Control of the conductance of engineered protein nanopores through concerted loop motions.

Zhuang Tiandi T Tamm Lukas K LK

Angewandte Chemie (International ed. in English) 20140428 23

Protein nanopores have attracted much interest for nucleic acid sequencing, chemical sensing, and protein folding at the single molecule level. The outer membrane protein OmpG from E. coli stands out because it forms a nanopore from a single polypeptide chain. This property allows the separate engineering of each of the seven extracellular loops that control access to the pore. The longest of these loops, loop 6, has been recognized as the main gating loop that closes the pore at low pH values a ...[more]

PMID: 24777684

Similar Datasets

Protein sensing with engineered protein nanopores.

Project description:The use of nanopores is a powerful new frontier in single-molecule sciences. Nanopores have been used effectively in exploring various biophysical features of small polypeptides and proteins, such as their folding state and structure, ligand interactions, and enzymatic activity. In particular, the ?-hemolysin (?HL) protein pore has been used extensively for the detection, characterization, and analysis of polypeptides because this protein nanopore is highly robust, versatile, and tractable under various experimental conditions. Inspired by the mechanisms of protein translocation across the outer membrane translocases of mitochondria, we have shown the ability to use nanopore-probe techniques in controlling a single protein using engineered ?HL pores. Here, we provide a detailed protocol for the preparation of ?HL protein nanopores. Moreover, we demonstrate that placing attractive electrostatic traps is instrumental in tackling single-molecule stochastic sensing of folded proteins.

| S-EPMC3708658 | biostudies-literature

Controlled translocation of individual DNA molecules through protein nanopores with engineered molecular brakes.

Project description:Protein nanopores may provide a cheap and fast technology to sequence individual DNA molecules. However, the electrophoretic translocation of ssDNA molecules through protein nanopores has been too rapid for base identification. Here, we show that the translocation of DNA molecules through the α-hemolysin protein nanopore can be slowed controllably by introducing positive charges into the lumen of the pore by site directed mutagenesis. Although the residual ionic current during DNA translocation is insufficient for direct base identification, we propose that the engineered pores might be used to slow down DNA in hybrid systems, for example, in combination with solid-state nanopores.

| S-EPMC3391008 | biostudies-literature

Protein concerted motions in the DNA-human topoisomerase I complex.

Project description:The collective motions of the core and C-terminal domains of human topoisomerase I (topo I) have been analysed by molecular dynamics simulation of the protein in covalent complex with a 22 bp DNA duplex. The analysis evidenced a great number of correlated movements of core subdomain I and II residues, and a central role for helix 5 in the protein-DNA communication, in particular with the scissile strand downstream of the cleavage site. The flow of information between these core subdomains and DNA suggests that subdomains I and II play an essential role in the DNA relaxation process. In core subdomain III the majority of DNA contacting residues do not communicate with protein regions far from DNA, suggesting that they have a structural role. However, selected core subdomain III residues, involved in the orientation of the active site region, show correlated movements with residues distant from DNA, indicating that the information concerning the catalytic event is also transmitted. The flexibility of two loops formed by residues 519-520 and 580-584 seems indispensable to the dynamic participation of core subdomain III to the DNA cleavage and religation steps. The motion of specific residues has also been found to explain the effect of single point mutations that make topo I resistant to the anticancer drug camptothecin.

| S-EPMC149835 | biostudies-literature

Detection of long-range concerted motions in protein by a distance covariance.

Project description:We asses the ability of a distance correlation coefficient (DiCC), calculated from distance covariance, for detecting long-range concerted motion in proteins. We establish a set of criteria for ideal correlation coefficient values based on the coefficient of determination in multi-dimension, R2. We compare in detail DiCC and conventional coefficients against these criteria. We demonstrate that in contrast to conventional correlation coefficients, which capture long-distance correlation adequately only with certain restrictions in multi-dimension, DiCC reflects appropriate correlation in both one- and multi-dimension. Finally we demonstrate the usefulness of DiCC for assessing long-distance correlated fluctuation in protein dynamics.

| S-EPMC3630994 | biostudies-literature

Molecular bases of cyclodextrin adapter interactions with engineered protein nanopores.

Project description:Engineered protein pores have several potential applications in biotechnology: as sensor elements in stochastic detection and ultrarapid DNA sequencing, as nanoreactors to observe single-molecule chemistry, and in the construction of nano- and micro-devices. One important class of pores contains molecular adapters, which provide internal binding sites for small molecules. Mutants of the alpha-hemolysin (alphaHL) pore that bind the adapter beta-cyclodextrin (betaCD) approximately 10(4) times more tightly than the wild type have been obtained. We now use single-channel electrical recording, protein engineering including unnatural amino acid mutagenesis, and high-resolution x-ray crystallography to provide definitive structural information on these engineered protein nanopores in unparalleled detail.

| S-EPMC2889592 | biostudies-literature

Aberrantly Large Single-Channel Conductance of Polyhistidine Arm-Containing Protein Nanopores.

Project description:There have been only a few studies reporting on the impact of polyhistidine affinity tags on the structure, function, and dynamics of proteins. Because of the relatively short size of the tags, they are often thought to have little or no effect on the conformation or activity of a protein. Here, using membrane protein design and single-molecule electrophysiology, we determined that the presence of a hexahistidine arm at the N-terminus of a truncated FhuA-based protein nanopore, leaving the C-terminus untagged, produces an unusual increase in the unitary conductance to ?8 nS in 1 M KCl. To the best of our knowledge, this is the largest single-channel conductance ever recorded with a monomeric ?-barrel outer membrane protein. The hexahistidine arm was captured by an anti-polyhistidine tag monoclonal antibody added to the side of the channel-forming protein addition, but not to the opposite side, documenting that this truncated FhuA-based protein nanopore inserts into a planar lipid bilayer with a preferred orientation. This finding is in agreement with the protein insertion in vivo, in which the large loops face the extracellular side of the membrane. The aberrantly large single-channel conductance, likely induced by a greater cross-sectional area of the pore lumen, along with the vectorial insertion into a lipid membrane, will have profound implications for further developments of engineered protein nanopores.

| S-EPMC5595664 | biostudies-other

A remote mutation affects the hydride transfer by disrupting concerted protein motions in thymidylate synthase.

Project description:The role of protein flexibility in enzyme-catalyzed activation of chemical bonds is an evolving perspective in enzymology. Here we examine the role of protein motions in the hydride transfer reaction catalyzed by thymidylate synthase (TSase). Being remote from the chemical reaction site, the Y209W mutation of Escherichia coli TSase significantly reduces the protein activity, despite the remarkable similarity between the crystal structures of the wild-type and mutant enzymes with ligands representing their Michaelis complexes. The most conspicuous difference between these two crystal structures is in the anisotropic B-factors, which indicate disruption of the correlated atomic vibrations of protein residues in the mutant. This dynamically altered mutant allows a variety of small thiols to compete for the reaction intermediate that precedes the hydride transfer, indicating disruption of motions that preorganize the protein environment for this chemical step. Although the mutation causes higher enthalpy of activation of the hydride transfer, it only shows a small effect on the temperature dependence of the intrinsic KIE, suggesting marginal changes in the geometry and dynamics of the H-donor and -acceptor at the tunneling ready state. These observations suggest that the mutation disrupts the concerted motions that bring the H-donor and -acceptor together during the pre- and re-organization of the protein environment. The integrated structural and kinetic data allow us to probe the impact of protein motions on different time scales of the hydride transfer reaction within a complex enzymatic mechanism.

| S-EPMC3490427 | biostudies-literature

SDS-assisted protein transport through solid-state nanopores.

Project description:Using nanopores for single-molecule sequencing of proteins - similar to nanopore-based sequencing of DNA - faces multiple challenges, including unfolding of the complex tertiary structure of the proteins and enforcing their unidirectional translocation through nanopores. Here, we combine molecular dynamics (MD) simulations with single-molecule experiments to investigate the utility of SDS (Sodium Dodecyl Sulfate) to unfold proteins for solid-state nanopore translocation, while simultaneously endowing them with a stronger electrical charge. Our simulations and experiments prove that SDS-treated proteins show a considerable loss of the protein structure during the nanopore translocation. Moreover, SDS-treated proteins translocate through the nanopore in the direction prescribed by the electrophoretic force due to the negative charge impaired by SDS. In summary, our results suggest that SDS causes protein unfolding while facilitating protein translocation in the direction of the electrophoretic force; both characteristics being advantageous for future protein sequencing applications using solid-state nanopores.

| S-EPMC5611827 | biostudies-literature

Controlling protein translocation through nanopores with bio-inspired fluid walls.

Project description:Synthetic nanopores have been used to study individual biomolecules in high throughput, but their performance as sensors does not match that of biological ion channels. Challenges include control of nanopore diameters and surface chemistry, modification of the translocation times of single-molecule analytes through nanopores, and prevention of non-specific interactions with pore walls. Here, inspired by the olfactory sensilla of insect antennae, we show that coating nanopores with a fluid lipid bilayer tailors their surface chemistry and allows fine-tuning and dynamic variation of pore diameters in subnanometre increments. Incorporation of mobile ligands in the lipid bilayer conferred specificity and slowed the translocation of targeted proteins sufficiently to time-resolve translocation events of individual proteins. Lipid coatings also prevented pores from clogging, eliminated non-specific binding and enabled the translocation of amyloid-beta (A?) oligomers and fibrils. Through combined analysis of their translocation time, volume, charge, shape and ligand affinity, different proteins were identified.

| S-EPMC3071889 | biostudies-literature

Protein Transport through Nanopores Illuminated by Long-Time-Scale Simulations.

Project description:The transport of molecules through nanoscale confined space is relevant in biology, biosensing, and industrial filtration. Microscopically modeling transport through nanopores is required for a fundamental understanding and guiding engineering, but the short duration and low replica number of existing simulation approaches limit statistically relevant insight. Here we explore protein transport in nanopores with a high-throughput computational method that realistically simulates hundreds of up to seconds-long protein trajectories by combining Brownian dynamics and continuum simulation and integrating both driving forces of electroosmosis and electrophoresis. Ionic current traces are computed to enable experimental comparison. By examining three biological and synthetic nanopores, our study answers questions about the kinetics and mechanism of protein transport and additionally reveals insight that is inaccessible from experiments yet relevant for pore design. The discovery of extremely frequent unhindered passage can guide the improvement of biosensor pores to enhance desired biomolecular recognition by pore-tethered receptors. Similarly, experimentally invisible nontarget adsorption to pore walls highlights how to improve recently developed DNA nanopores. Our work can be expanded to pressure-driven flow to model industrial nanofiltration processes.

| S-EPMC8291773 | 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