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Protein Origami with Split Armadillo Repat Proteins

English title Protein Origami with Split Armadillo Repat Proteins
Applicant Zerbe Oliver
Number 184744
Funding scheme Project funding (Div. I-III)
Research institution Institut für Chemie Universität Zürich
Institution of higher education University of Zurich - ZH
Main discipline Organic Chemistry
Start/End 01.01.2020 - 31.12.2023
Approved amount 268'509.00
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All Disciplines (2)

Discipline
Organic Chemistry
Biochemistry

Keywords (4)

supramolecular chemistry; proximity-related effects; protein engineering; protein origami

Lay Summary (German)

Lead
Das Projekt will ein System schaffen, das es erlaubt, Moleküle in einer genau definierten Entfernung und geometrischen Beziehung zueinander anzuordnen. Es wird für diesen Zweck Protein-Origami-Techniken verwenden. Es nutzt eine frühere Entdeckung aus meiner Gruppe, dass Armadillo-Repeat-Proteine Peptide binden können, und dass dieser Erkennungsprozess so durchgeführt werden kann, dass das Armadillo-Repeat-Protein in zwei komplementären Hälften geliefert wird, die nur dann das Protein der vollen Länge bilden, wenn ein Peptid hinzugefügt wird.Das System erstellt ein innovatives System im Baukastenprinzip, das als Reaktion auf hinzugefügte Komponenten montiert oder demontiert wird.
Lay summary

Ziel des Projekts ist es, makromolekulare Komplexe auf kontrollierte Weise so zu erstellen, dass Proteine oder andere Makromoleküle in einer bestimmten geometrischen Beziehung zueinander stehen. Viele Prozesse in der Biochemie oder Chemie hängen von der Nähe der Moleküle zueinander ab. Die Chemiker haben viele Lösungen, die Moleküle in exakten Abständen zueinander platzieren können, aber im Allgemeinen erfordern die zugehörigen Gerüste komplexe chemische Synthesen. Darüber hinaus sind die Systeme meistens statisch, d.h. zu Beginn der Synthese müssen alle geometrischen Variablen definiert werden und können im Folgenden nicht mehr angepasst werden.

Wir versuchen ein auf Proteinen basierendes System aufzubauen, um ein dynamisches Gerüst zu schaffen. Das System stellt eine Leiter dar, in der sowohl Leiterholme als auch Leiterstufen aus Proteinen bestehen. Die Leiterholme sind aus einem Coploymer aus Armadillo-Repeat-Proteinen und Coiled-Coil-Polypeptiden aufgebaut. Die Leiterstufen werden mit der DARPIN-Technologie hergestellt, bei der es sich um Proteine handelt, die verschiedene Peptide, Proteine oder andere Moleküle binden können. DARPINs an ihrem Ende sind mit Erkennungssequenzen fusioniert, die an die Armadillo-Repeatproteine in den Leiterholmen binden können und dadurch zur Vernetzung der Holme dienen. Beim Mischen von Leiterholmen und Leiterstufenelementen wird eine Leiter konstruiert, in der die Leiterstufen Proteine enthalten, die selbst wiederum spezifische Cargos binden können. Durch das Hinzufügen der Cargos werden diese dann genau wie die Perlen einer Schnur in einem bestimmten Abstand zueinander positioniert.

Das System kann so hergestellt werden, dass es sich nur als Reaktion auf ein hinzugefügtes Peptid zusammensetzt, und so dass es auch wieder zerlegt werden kann.

Direct link to Lay Summary Last update: 15.04.2019

Responsible applicant and co-applicants

Employees

Name Institute

Associated projects

Number Title Start Funding scheme
141832 Structure-based design of Armadillo Repeat Proteins as a modular recognition system for the sequence-specific binding of peptides 01.08.2012 Sinergia

Abstract

The creation of macromolecular structures has inspired chemists for a long time. Such structures can be used to create macromolecules with defined shape. Ideally, the system is created from pre-defined building blocks to reduce the synthetic work. Macromolecular structures can in addition to shape also possess function resulting in small molecular machines like motors for example. The systems that were created by chemists, while clearly presenting sophisticated molecular machines, require substantial synthetic effort. Alternatively, macromolecular assemblies can be built from biomolecules. An example is the assembly of viral coatings that is built from identical copies of proteins with suitable structure and properties. An example where bio-molecules can be polymerized into defined shapes using nucleic acids has become famous as DNA origami. The beauty of that method is that a few simple building blocks together with a suitable chemistry (solid-phase nucleic acid chemistry) can be used to rapidly assemble well-defined macromolecular shapes. Most of these methods share the common disadvantage that the created shapes are static, and usually the shape determines the synthetic strategy early on and cannot be changed easily.Herein, I suggest developing a system, consisting of protein building blocks and connectors, in the following referred to as hubs, from which switchable macromolecular assemblies can be created. It is based on a previous project in my group, in which we discovered that Armadillo repeat proteins (ArmRP) can be reconstituted from their complementary fragments (Watson et al., Structure 2014). When these protein fragments are mixed they form a complex with high affinity, and the structure of the complex is virtually indistinguishable from the parent single chain protein. We further developed the system such that the two complementary protein fragments only interact in presence of a peptide ligand (Michel et al., Angew. Chem. 2018). I now propose to covalently link the two protein fragments onto different hubs and use their conditional assembly, triggered by presence of a specific peptide sequence, to pull larger systems together.Since the binding affinity of the peptide ligands to the single-chain proteins is higher, the structures can be dis-assembled upon addition of the un-split protein. In the original proposal I have aimed at using both organic molecules and proteins as hubs. I still believe that both hubs have their specific advantages and disadvantages, and that it is worthwhile to follow up on both systems. However, in order to focus resources and to best exploit my higher expertise in biochemistry when compared to organic chemistry, I now suggest focusing on proteins as hubs. The principle here is to use a protein that is genetically fused to recognition elements. The latter can bind to Armadillo repeat proteins. An additional useful feature when using proteins as hubs is that they can be modified to incorporate binders for other proteins, such that the hubs can be used to place molecules in a defined distance and geometry relative to one another. As a proof of principle I aim to create a ladder, in which the ladder holms are formed by Armadillo repeat proteins fused to coiled coils, and the ladder steps are created by ankyrin proteins. The ankyrins are connected to the ladder holm via recognition sequences at their termini that bind with high affinity and specificity to the ArmRPs. The system can be modified to bring two different proteins in proximity in response to addition of a peptide.The project aims to create a set of building blocks that can assemble, potentially in a dynamic manner, into defined structures, thereby allows placing molecules in defined geometric relations relative to each other, and has the potential in future to also put catalytically or biologically active molecules in proximity. As described below such a system can be used to tackle many biologically relevant questions or create biomaterials with interesting properties. It would clearly advance the field of supramolecular chemistry in that it aims at creating dynamic systems and systems with function.
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