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Chemical synthesis of switchable peptide-based nanopores: from ion channels to bio-inspired materials

Mueller, Lena (2019):
Chemical synthesis of switchable peptide-based nanopores: from ion channels to bio-inspired materials.
Darmstadt, Technische Universität,
DOI: 10.25534/tuprints-00011495,
[Ph.D. Thesis]

Abstract

The generation of different types of peptide-based nanopores is elucidated in this work. First, biological nanopores are investigated. Ion channels and pore-forming proteins represent an ultra-selective and ultra-sensitive category of nanopores. Eukaryotic potassium channels are structures of enormous size while prokaryotic potassium channels are comparable in their overall gating behavior but some are extremely reduced in their size. Within this work, the total chemical synthesis of the viral potassium channel KcvNTS is shown using Solid Phase Peptide Synthesis (SPPS) and Native Chemical Ligation (NCL) towards product formation. The main attention is on overcoming the challenging key steps including the substantial insolubility of the extensively hydrophobic fragments. Secondly, hybrid nanopores are generated. Nanopores composed of solid-state materials can be used as a robust and stable scaffold to integrate a selective and sensitive peptide moiety to generate a hybrid nanopore for a sensing application. In this work, the immobilization of the Amino Terminal Cu(II)- and Ni(II) binding motif (ATCUN) to conically shaped PET-based solid state nanopores is undertaken. The generated sensor is tuned (pH 6.5) to only bind Cu(II) leading to the design of a selective and sensitive Cu(II)-sensor (limit of detection in solution 13.5 nM using fluorescence titration; hybrid system in fM range using I-V measurements). Furthermore, the motif is investigated substantially regarding the mandatory histidine moiety. Mutants are designed and examined towards their binding behavior. Additionally, the DNA scission ability of the ATCUN motif and its mutants is investigated towards several plasmids paving the way for the design of a sequencing device using the presented polymer-based hybrid nanopore system.

Item Type: Ph.D. Thesis
Erschienen: 2019
Creators: Mueller, Lena
Title: Chemical synthesis of switchable peptide-based nanopores: from ion channels to bio-inspired materials
Language: English
Abstract:

The generation of different types of peptide-based nanopores is elucidated in this work. First, biological nanopores are investigated. Ion channels and pore-forming proteins represent an ultra-selective and ultra-sensitive category of nanopores. Eukaryotic potassium channels are structures of enormous size while prokaryotic potassium channels are comparable in their overall gating behavior but some are extremely reduced in their size. Within this work, the total chemical synthesis of the viral potassium channel KcvNTS is shown using Solid Phase Peptide Synthesis (SPPS) and Native Chemical Ligation (NCL) towards product formation. The main attention is on overcoming the challenging key steps including the substantial insolubility of the extensively hydrophobic fragments. Secondly, hybrid nanopores are generated. Nanopores composed of solid-state materials can be used as a robust and stable scaffold to integrate a selective and sensitive peptide moiety to generate a hybrid nanopore for a sensing application. In this work, the immobilization of the Amino Terminal Cu(II)- and Ni(II) binding motif (ATCUN) to conically shaped PET-based solid state nanopores is undertaken. The generated sensor is tuned (pH 6.5) to only bind Cu(II) leading to the design of a selective and sensitive Cu(II)-sensor (limit of detection in solution 13.5 nM using fluorescence titration; hybrid system in fM range using I-V measurements). Furthermore, the motif is investigated substantially regarding the mandatory histidine moiety. Mutants are designed and examined towards their binding behavior. Additionally, the DNA scission ability of the ATCUN motif and its mutants is investigated towards several plasmids paving the way for the design of a sequencing device using the presented polymer-based hybrid nanopore system.

Place of Publication: Darmstadt
Divisions: 07 Department of Chemistry
07 Department of Chemistry > Fachgebiet Biochemie
Date Deposited: 02 Jun 2020 07:31
DOI: 10.25534/tuprints-00011495
Official URL: https://tuprints.ulb.tu-darmstadt.de/11495
URN: urn:nbn:de:tuda-tuprints-114951
Referees: Tietze, Asst. Prof Alesia A. ; Thiel, Prof. Dr. Gerhard
Refereed / Verteidigung / mdl. Prüfung: 27 February 2020
Alternative Abstract:
Alternative abstract Language

Die vorliegende Arbeit beschäftigt sich mit der Herstellung unterschiedlicher Arten von Nanoporen. Zuerst wird der Fokus auf biologische Nanoporen, zu denen Ionenkanäle und andere Poren-formende Proteine gehören, gelegt. Eukaryotische Kaliumkanäle sind Strukturen von enormer Größe. Einige prokaryotische Kaliumkanäle hingegen, sind in ihrer Größe extrem reduziert, während sie in der Funktion und ihrem gating Verhalten vergleichbar sind. In dieser Arbeit wird die chemische Synthese von KcvNTS dargestellt. Hierzu wird die Festphasenpeptidsynthesis (SPPS) und Native Chemische Ligation (NCL) verwendet. Dabei liegt der Fokus auf der Bewältigung der Schwierigkeiten in der SPPS, NCL und Analytik, die mit der extremen Hydrophobizität der membran-spannenden Fragmente einhergeht. Im nächsten Teil liegt das Hauptaugenmerk auf der Herstellung eines Sensors, basierend auf Hybrid-Nanoporen. Hierbei wird ein ATCUN Motif (Amino Terminal Cu(II)- and Ni(II) Binding Motif) mit sensorischen Eigenschaften in konische Nanoporen einer PET Folie immobilisiert. Mit diesem Sensor ist es möglich, durch eine Einstellung des pH (pH 6,5), eine selektive Cu(II) Bindung zu erreichen, um Cu(II) sensitiv nachweisen zu können (limit of detection 13,5 nM über Titration, im fM Bereich über I-V Messungen). Darüber hinaus werden Mutanten dieses Motifs hergestellt, die nicht mehr das verbindliche Histidin enthalten. Diese werden bezüglich ihrer Bindungseigenschaften untersucht. Weiterhin ist bekannt, dass ATCUN Peptide DNA schneiden und linearisieren können. Diese Eigenschaft wird für das ATCUN motif, sowie die Mutanten untersucht mit dem Ziel, ein Sequenzierungsgerät auf Grundlage der zuvor dargestellten Hybridnanopore herzustellen.

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