Engineering a Rigid Protein Tunnel for Biomolecular Detection

Mohammad, Mohammad M.; Iyer, Raghuvaran; Howard, Khalil R.; McPike, Mark P.; Borer, Philip N.; Movileanu, Liviu

doi:10.1021/ja3043646

Cited by 114 publications

(160 citation statements)

References 44 publications

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“…In addition to their biological interest they are increasingly relevant to biotechnology, serving as scaffolds for bacterial surface display (2,3) and atomically precise pores for nanopore-based DNA sequencing. Although the suitability of natural β-barrel membrane proteins for biotechnology has been improved by protein engineering (3)(4)(5)(6)(7)(8)(9)(10), the ability to design membrane proteins de novo would deliver tools customized to meet the demands of each application.…”

mentioning

confidence: 99%

Computational redesign of the lipid-facing surface of the outer membrane protein OmpA

Stapleton

Whitehead

Nanda

2015

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

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Advances in computational design methods have made possible extensive engineering of soluble proteins, but designed β-barrel membrane proteins await improvements in our understanding of the sequence determinants of folding and stability. A subset of the amino acid residues of membrane proteins interact with the cell membrane, and the design rules that govern this lipid-facing surface are poorly understood. We applied a residue-level depth potential for β-barrel membrane proteins to the complete redesign of the lipid-facing surface of Escherichia coli OmpA. Initial designs failed to fold correctly, but reversion of a small number of mutations indicated by backcross experiments yielded designs with substitutions to up to 60% of the surface that did support folding and membrane insertion. T he β-barrel membrane proteins comprise one of the two structural classes of integral membrane proteins. They are found within the outer membranes of bacteria, mitochondria, and chloroplasts, where they perform a range of structural, transport, and catalytic functions (1). In addition to their biological interest they are increasingly relevant to biotechnology, serving as scaffolds for bacterial surface display (2, 3) and atomically precise pores for nanopore-based DNA sequencing. Although the suitability of natural β-barrel membrane proteins for biotechnology has been improved by protein engineering (3-10), the ability to design membrane proteins de novo would deliver tools customized to meet the demands of each application.De novo design provides a stringent test of our understanding of the determinants of protein folding and stability. Protein design software [e.g., Rosetta (11,12)] has made tremendous strides in addressing the design problem for small water-soluble proteins (13-15), and design of simplified model α-helical membrane proteins including single transmembrane helices and small bundles (16)(17)(18)(19)(20) has also been accomplished. In contrast, a designed β-barrel membrane protein has yet to be reported, perhaps as a consequence of the unique design challenges presented by the folding pathway and architecture of these proteins. Unlike the α-helical membrane proteins, nascent β-barrel membrane proteins must transit the periplasm to the outer membrane, where folding and membrane insertion are thought to occur in concert (21,22). An extensive network of chaperones maintains the solubility of the unfolded barrel and guides membrane insertion. The C-terminal β-strand is known to interact with the BAM chaperone complex (23-25), which assists the folding of all β-barrel membrane proteins. However, despite recent progress (26-30), we do not fully understand how interactions between chaperones and transiting membrane proteins are directed by sequence-encoded information.Further complicating design is the inside-out architecture of β-barrel membrane proteins. In place of a hydrophobic core is either a central water-filled pore or a solid core composed of polar side chains. The lipid bilayer becomes increasingly hydrophobic...

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mentioning

confidence: 99%

Computational redesign of the lipid-facing surface of the outer membrane protein OmpA

Stapleton

Whitehead

Nanda

2015

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

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“…Although several new types of nanopores have been developed over the past several years [32][33][34][35][36][37][38][39][40][41] , biological protein pores and SS-nanopores are still the two mainstays in nanopore research. Protein pores have the advantages of uniform pore size, high reproducibility and the capacity for precise modification by site-directed mutagenesis, chemical modification and incorporation of unnatural amino acids, whereas SS-nanopores possess intrinsically high durability and tunable pore size and shape.…”

Section: Comparison With Other Methodsmentioning

confidence: 99%

“…The past decade has witnessed a burst of new nanopores made from different materials. These include biological nanopores such as Mycobacterium smegmatis porin A (MspA) 30 , phi29 connectors 31 , ferric hydroxamate uptake component A (FhuA) 32 and cytolysin A 33 ; carbon nanotubes [34][35][36] ; DNA nanostructures 37,38 ; organic macromolecules [39][40][41] ; and atomically thin 2D materials such as graphene [42][43][44] , boron nitride 45 and molybdenum disulfide 46 . Currently, most of the new nanopores are focused on nucleic acid analysis.…”

Section: Introductionmentioning

confidence: 99%

Fabrication of nanopores with ultrashort single-walled carbon nanotubes inserted in a lipid bilayer

Liu

Xie

et al. 2015

Nat Protoc

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We describe a protocol for the insertion of ultrashort single-walled carbon nanotubes (SWCNTs) to form nanopores in a Montal-Mueller lipid bilayer. The SWCNTs are designed to bind to a specific analyte of interest; binding will result in the reduction of current in single-channel recording experiments. The first stage of the PROCEDURE is to cut and separate the SWCNTs. We cut long, purified SWCNTs with sonication in concentrated sulfuric acid/nitric acid (3/1). Isolation of ultrashort SWCNTs is carried out by size-exclusion HPLC separation. The second stage is to insert these short SWCNTs into the lipid bilayer. This step requires a microinjection probe made from a glass capillary. The setup for protein nanopore research can be adopted for the single-channel recording experiments without any special treatment. The obtained current traces are of very high quality, showing stable baselines and little background noise. Example procedures are shown for investigating ion transport and DNA translocation through these SWCNT nanopores. This nanopore has potential applications in molecular sensing, nanopore DNA sequencing and early disease diagnosis. For example, we have selectively detected modified 5-hydroxymethylcytosine in single-stranded DNA (ssDNA), which may have implications in screening specific genomic DNA sequences. The protocol takes ∼15 d, including SWCNT purification, cutting and separation, as well as the formation of SWCNT nanopores for DNA analyses.

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“…Die passende Selektivität für ein bestimmtes Biomolekül lässt sich über die Abmessungen der Nanopore festlegen. Dementsprechend wurden verschiedene biologische Membranproteine wie Areolysin, [2] Porin A aus Mycobacterium smegmatis (MspA), [3] ClyA, [4] FhuA, [5] membranadaptiertes phi29-Motorprotein [6] und SP1 [7] in nanoporenbasierten Analysesystemen eingesetzt. Eine andere Art von künstlichen Nanoporen sind Festkçrpernanoporen.…”

Section: Einführungunclassified

Nanoporentechniken zur Sequenzierung und Detektion von Nukleinsäuren

et al. 2013

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Nanoporentechniken, die die Funktionen natürlicher Ionenkanäle nachahmen, sind nützliche Methoden für den Einzelmolekülnachweis. Das Verfahren erlaubt die selektive Analyse von Nukleinsäuren in Echtzeit und im Hochdurchsatz. Zwei Arten von Nanoporen finden Anwendung: biologische Nanoporen und Festkörpernanoporen. Dieser Kurzaufsatz beleuchtet die Grundlagen und jüngsten Fortschritte bei der nanoporenbasierten Sequenzierung und Detektion von Nukleinsäuren. Außerdem wird eine neuartige, erst kürzlich eingeführte Nanoporentechnik für die Analyse von Biomolekülen vorgestellt.

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Engineering a Rigid Protein Tunnel for Biomolecular Detection

Cited by 114 publications

References 44 publications

Computational redesign of the lipid-facing surface of the outer membrane protein OmpA

Computational redesign of the lipid-facing surface of the outer membrane protein OmpA

Fabrication of nanopores with ultrashort single-walled carbon nanotubes inserted in a lipid bilayer

Nanoporentechniken zur Sequenzierung und Detektion von Nukleinsäuren

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