Protein Nanopores with Covalently Attached Molecular Adapters

Wu, Hai‐Chen; Astier, Yann; Maglia, Giovanni; Mikhailova, Ellina; Bayley, Hagan

doi:10.1021/ja0761840

Cited by 111 publications

(118 citation statements)

References 28 publications

Supporting

Mentioning

114

Contrasting

Order By: Relevance

“…The present work will now permit us to examine binding events at a known face of a CD. The work also provides information for designing new binding sites within the lumen of the αHL pore (37) or within other β barrel proteins (21,50) and for using molecular design to devise ways in which to covalently attach CDs within pores (13,51). These areas impact practical applications of nanopore technology including stochastic sensing (8), single-molecule DNA sequencing (9,12,13,52), the use of nanoreactors for the observation of single-molecule chemistry (10), and the construction of nano-and microdevices (11,53), as well as the design of CDs as therapeutic agents (23,24).…”

Section: Resultsmentioning

confidence: 99%

Molecular bases of cyclodextrin adapter interactions with engineered protein nanopores

Banerjee

Mikhailova

Cheley

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

104

View full text Add to dashboard Cite

Engineered protein pores have several potential applications in biotechnology: as sensor elements in stochastic detection and ultrarapid DNA sequencing, as nanoreactors to observe singlemolecule chemistry, and in the construction of nano-and microdevices. One important class of pores contains molecular adapters, which provide internal binding sites for small molecules. Mutants of the α-hemolysin (αHL) pore that bind the adapter β-cyclodextrin (βCD) ∼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 highresolution x-ray crystallography to provide definitive structural information on these engineered protein nanopores in unparalleled detail.alpha-hemolysin | single molecule | stochastic sensing | structure | unnatural amino acid M any research groups have used protein engineering to obtain enzymes and antibodies with new properties suited for specific tasks (1-6). Fewer groups have taken on the difficult problem of engineering membrane proteins (7). We have engineered the α-hemolysin protein pore, mindful of several potential applications in biotechnology, including its ability to act as a detector in stochastic sensing (8) and ultrarapid DNA sequencing (9), to serve as a nanoreactor for the observation of singlemolecule chemistry (10) and to act as a component for the construction of nano-and microdevices (11).An important breakthrough in this area, which enabled the stochastic sensing of organic molecules including the detection of DNA bases in the form of nucleoside monophosphates (12, 13), was the discovery of internal molecular adapters, a form of noncovalent protein modification (14). Most useful have been cyclodextrin (CD) adapters, which have until now been used in the absence of detailed structural information about how they work. The present paper is a definitive investigation, which provides such information through the application of a wide variety of technical approaches: single-channel electrical recording, protein engineering including unnatural amino acid mutagenesis, and x-ray crystallography. The studies employing mutagenesis show that the striking interactions seen in the crystal structures also occur in individual pores in lipid bilayers.We reveal that the tight-binding αHL mutants (15) M113N 7 and M113F 7 bind βCD in different orientations within the heptameric pore. In the case of M113N 7 , the top (primary hydroxyls) of the CD ring faces the trans entrance of the pore. In the case of M113F 7 , the bottom (secondary hydroxyls) of the CD ring faces the trans entrance, while the top of the ring is bonded to the pore through remarkable CH-π interactions. Another tight-binding mutant, M113V 7 , can bind the CD in both orientations. These results illustrate the exquisite level of engineering that can be achieved with protein nanopores, which is, for example, far beyond what is possible with solid-state pores. The work also provides information valuable for the design of ...

show abstract

Section: Resultsmentioning

confidence: 99%

Molecular bases of cyclodextrin adapter interactions with engineered protein nanopores

Banerjee

Mikhailova

Cheley

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

104

View full text Add to dashboard Cite

show abstract

“…The nanopores are situated in individual array wells, and single DNA molecules are introduced into the wells and progressively digested by exonuclease. The released single-nucleotide bases enter the nanopore and alter the electrical current, creating a characteristic current change for each individual base (95,96 ). Although the technology is seemingly futuristic, considerable NHGRI funding is being directed toward a variety of nanopore technologies under development as part of the goal of achieving the $1000 genome.…”

Section: Technologies On the Horizonmentioning

confidence: 99%

Next-Generation Sequencing: From Basic Research to Diagnostics

2009

View full text Add to dashboard Cite

Background: For the past 30 years, the Sanger method has been the dominant approach and gold standard for DNA sequencing. The commercial launch of the first massively parallel pyrosequencing platform in 2005 ushered in the new era of high-throughput genomic analysis now referred to as next-generation sequencing (NGS). Content: This review describes fundamental principles of commercially available NGS platforms. Although the platforms differ in their engineering configurations and sequencing chemistries, they share a technical paradigm in that sequencing of spatially separated, clonally amplified DNA templates or single DNA molecules is performed in a flow cell in a massively parallel manner. Through iterative cycles of polymerase-mediated nucleotide extensions or, in one approach, through successive oligonucleotide ligations, sequence outputs in the range of hundreds of megabases to gigabases are now obtained routinely. Highlighted in this review are the impact of NGS on basic research, bioinformatics considerations, and translation of this technology into clinical diagnostics. Also presented is a view into future technologies, including real-time single-molecule DNA sequencing and nanopore-based sequencing. Summary: In the relatively short time frame since 2005, NGS has fundamentally altered genomics research and allowed investigators to conduct experiments that were previously not technically feasible or affordable. The various technologies that constitute this new paradigm continue to evolve, and further improvements in technology robustness and process streamlining will pave the path for translation into clinical diagnostics.

show abstract

“…2a, left). The wild-type (WT) pore is a 'blank slate' for protein engineering with properties similar to those of an electrolyte-filled tube; it is weakly rectifying and weakly anion selective and gates only at extreme applied potentials of either polarity 12 .aHL has been modified by mutagenesis or targeted chemical modification to form pores with a wide range of properties [13][14][15][16] , but none has exhibited sufficient rectification for our purpose. We had, however, noticed that aHL pores with positively charged side chains projecting into the lumen of the transmembrane b barrel tended to gate (open and close) at negative potentials.…”

mentioning

confidence: 99%

“…aHL has been modified by mutagenesis or targeted chemical modification to form pores with a wide range of properties [13][14][15][16] , but none has exhibited sufficient rectification for our purpose. We had, however, noticed that aHL pores with positively charged side chains projecting into the lumen of the transmembrane b barrel tended to gate (open and close) at negative potentials.…”

mentioning

confidence: 99%

Droplet networks with incorporated protein diodes show collective properties

et al. 2009

Self Cite

View full text Add to dashboard Cite

Recently, we demonstrated that submicrolitre aqueous droplets submerged in an apolar liquid containing lipid can be tightly connected by means of lipid bilayers [1][2][3][4][5] to form networks [4][5][6] . Droplet interface bilayers have been used for rapid screening of membrane proteins 7,8 and to form asymmetric bilayers with which to examine the fundamental properties of channels and pores 9 . Networks, meanwhile, have been used to form microscale batteries and to detect light 4 . Here, we develop an engineered protein pore with diode-like properties that can be incorporated into droplet interface bilayers in droplet networks to form devices with electrical properties including those of a current limiter, a half-wave rectifier and a full-wave rectifier. The droplet approach, which uses unsophisticated components (oil, lipid, salt water and a simple pore), can therefore be used to create multidroplet networks with collective properties that cannot be produced by droplet pairs.To obtain directional ionic current flows in droplet networks ( Fig. 1), we constructed a diode-like pore from staphylococcal a-haemolysin (aHL). aHL forms a heptameric protein pore 10 that inserts vectorially into lipid bilayers 11 . The crystal structure of the pore reveals a 14-stranded transmembrane b barrel capped by an extramembraneous domain, which contains a roughly spherical cavity 10 ( Fig. 2a, left). The wild-type (WT) pore is a 'blank slate' for protein engineering with properties similar to those of an electrolyte-filled tube; it is weakly rectifying and weakly anion selective and gates only at extreme applied potentials of either polarity 12 .aHL has been modified by mutagenesis or targeted chemical modification to form pores with a wide range of properties [13][14][15][16] , but none has exhibited sufficient rectification for our purpose. We had, however, noticed that aHL pores with positively charged side chains projecting into the lumen of the transmembrane b barrel tended to gate (open and close) at negative potentials. Therefore, in an attempt to obtain a fully rectifying pore, we tested an extreme version of aHL in which seven residues were replaced with arginines (7R-aHL) to yield a heptameric pore in which 49 additional positively charged side chains were located within the barrel (Fig. 2a, right). In 1 M KCl, 25 mM Tris HCl at pH 8.0, 100 mM, in planar lipid bilayers, the 7R-aHL pore has a unitary conductance of 0.95 + 0.01 nS (þ50 mV, n ¼ 8). The conductance of the WT pore under the same conditions is similar (0.99 + 0.02 nS, n ¼ 4), which suggests, surprisingly, that the drastically altered 7R-aHL pore is properly formed. The current-voltage (I-V) characteristics of 7R-aHL in 1 M KCl, however, showed virtually complete current rectification (Fig. 2b,c). At positive applied potentials, 7R-aHL remained in an open form with a stable steady-state current and infrequent short-lived closures of less than 10 ms. By contrast, at negative applied potentials, the pore was closed, with occasional brief current spikes ascriba...

show abstract

Protein Nanopores with Covalently Attached Molecular Adapters

Cited by 111 publications

References 28 publications

Molecular bases of cyclodextrin adapter interactions with engineered protein nanopores

Molecular bases of cyclodextrin adapter interactions with engineered protein nanopores

Next-Generation Sequencing: From Basic Research to Diagnostics

Droplet networks with incorporated protein diodes show collective properties

Contact Info

Product

Resources

About