directed-assembly-elastin-like-proteins-into-defined-supramolecular

Directed Assembly of Elastin-like Proteins into defined Supramolecular Structures and Cargo Encapsulation In Vitro

Tailored proteinaceous building blocks are versatile candidates for the assembly of supramolecular structures such as minimal cells, drug delivery ...

University of Freiburg

The access to defined protein-based material systems is a major challenge in bionanotechnology and regenerative medicine. Exact control over sequence composition and modification is an important requirement for the intentional design of structure and function. Herein structural- and matrix proteins provide a great potential, but their large repetitive sequences pose a major challenge in their assembly. Here we introduce an integrative "one-vector-toolbox-platform" (OVTP) approach which is fast, efficient and reliable. The OVTP allows for the assembly, multimerization, intentional arrangement and direct translation of defined molecular DNA-tecton libraries, in combination with the selective functionalization of the yielded protein-tecton libraries. The diversity of the generated tectons ranges from elastine-, resilin, silk- to epitope sequence elements. OVTP comprises the expandability of modular biohybrid-materials via the assembly of defined multi-block domain genes and genetically encoded unnatural amino acids (UAA) for site-selective chemical modification. Thus, allowing for the modular combination of the protein-tecton library components and their functional expansion with chemical libraries via UAA functional groups with bioorthogonal reactivity. OVTP enables access to multitudes of defined protein-based biohybrid-materials for self-assembled superstructures such as nanoreactors and nanobiomaterials, e.g. for approaches in biotechnology and individualized regenerative medicine. 

Introducing a combinatorial DNA-toolbox platform constituting defined protein-based biohybrid-materials.

Nanoscale biological materials formed by the assembly of defined block-domain proteins control the formation of cellular compartments such as organelles. Here, we introduce an approach to intentionally 'program' the de novo synthesis and self-assembly of genetically encoded amphiphilic proteins to form cellular compartments, or organelles, in Escherichia coli. These proteins serve as building blocks for the formation of artificial compartments in vivo in a similar way to lipid-based organelles. We investigated the formation of these organelles using epifluorescence microscopy, total internal reflection fluorescence microscopy and transmission electron microscopy. The in vivo modification of these protein-based de novo organelles, by means of site-specific incorporation of unnatural amino acids, allows the introduction of artificial chemical functionalities. Co-localization of membrane proteins results in the formation of functionalized artificial organelles combining artificial and natural cellular function. Adding these protein structures to the cellular machinery may have consequences in nanobiotechnology, synthetic biology and materials science, including the constitution of artificial cells and bio-based metamaterials.

Designer amphiphilic proteins as building blocks for the intracellular formation of organelle-like compartments.

The control over the defined assembly of nano-objects with nm-precision is important to create systems and materials with enhanced properties, for example, metamaterials. In nature, the precise assembly of inorganic nano-objects with unique features, for example, magnetosomes, is accomplished by efficient and reliable recognition schemes involving protein effectors. Here we present a molecular approach using protein-based 'adaptors/connectors' with genetically encoded interaction sites to guide the assembly and functionality of different plasmonically active gold nanoparticle architectures (AuNP). The interaction of the defined geometricaly shaped protein adaptors with the AuNP induces the self-assembly of nanoarchitectures ranging from AuNP encapsulation to one-dimensional chain-like structures, complex networks and stars. Synthetic biology and bionanotechnology are applied to co-translationally encode unnatural amino acids as additional site-specific modification sites to generate functionalized biohybrid nanoarchitectures. This protein adaptor-based nano-object assembly approach might be expanded to other inorganic nano-objects creating biohybrid materials with unique electronic, photonic, plasmonic and magnetic properties. 

Molecular protein adaptor with genetically encoded interaction sites guiding the hierarchical assembly of plasmonically active nanoparticle architectures.

The molecular structuring of complex architectures and the enclosure of space are essential requirements for technical and living systems. Self-assembly of supramolecular structures with desired shape, size, and stability remains challenging since it requires precise regulation of physicochemical and conformational properties of the components. Here a general platform for controlled self-assembly of tailored amphiphilic elastin-like proteins into desired supramolecular protein assemblies ranging from spherical coacervates over molecularly defined twisted fibers to stable unilamellar vesicles is introduced. The described assembly protocols efficiently yield protein membrane-based compartments (PMBC) with adjustable size, stability, and net surface charge. PMBCs demonstrate membrane fusion and phase separation behavior and are able to encapsulate structurally and chemically diverse cargo molecules ranging from small molecules to naturally folded proteins. The ability to engineer tailored supramolecular architectures with defined fusion behavior, tunable properties, and encapsulated cargo paves the road for novel drug delivery systems, the design of artificial cells, and confined catalytic nanofactories.

Self-Assembly Toolbox of Tailored Supramolecular Architectures Based on an Amphiphilic Protein Library.

Life in its molecular context is characterized by the challenge of orchestrating structure, energy and information processes through compartmentalization and chemical transformations amenable to mimicry of protocell models. Here we present an alternative protocell model incorporating dynamic membranes based on amphiphilic elastin-like proteins (ELPs) rather than phospholipids. For the first time we demonstrate the feasibility of combining vesicular membrane formation and biocatalytic activity with molecular entities of a single class: proteins. The presented self-assembled protein-membrane-based compartments (PMBCs) accommodate either an anabolic reaction, based on free DNA ligase as an example of information transformation processes, or a catabolic process. We present a catabolic process based on a single molecular entity combining an amphiphilic protein with tobacco etch virus (TEV) protease as part of the enclosure of a reaction space and facilitating selective catalytic transformations. Combining compartmentalization and biocatalytic activity by utilizing an amphiphilic molecular building block with and without enzyme functionalization enables new strategies in bottom-up synthetic biology, regenerative medicine, pharmaceutical science and biotechnology.

Minimalist Protocell Design: A Molecular System Based Solely on Proteins that Form Dynamic Vesicular Membranes Embedding Enzymatic Functions.

The nature of the first prebiotic compartments and their possible minimal molecular composition is of great importance in the origin of life scenarios. Current protocell model membranes are proposed to be lipid-based. This paradigm has several shortcomings such as limited membrane stability of monoacyl lipid-based membranes (e.g., fatty acids), missing pathways to synthesize protocell membrane components (e.g., phospholipids) under early earth conditions, and the requirement for different classes of molecules for the formation of compartments and the catalysis of reactions. Amino acids on the other hand are known to arise and persist with remarkable abundance under early earth conditions since the fundamental Miller-Urey experiments. They were also postulated early to form protocellular structures, for example, proteinoid capsules. Here, we present a protocell model constituted by membranes assembled from amphiphilic proteins based on prebiotic amino acids. Self-assembled dynamic protein membrane-based compartments (PMBCs) are impressively stable and compatible with prevalent cellular membrane constituents forming protein-only or protein-lipid hybrid membranes. They can embed processes essential for extant living cells, such as enclosure of molecules, membrane fusion, phase separation, and complex biosynthetic elements from modern cells demonstrating "upward" compatibility. Our findings suggest that prebiotic PMBCs represent a new type of protocell as a possible ancestor of current lipid-based cells. The presented prebiotic PMBC model can be used to design artificial cells, important for the study of structural, catalytic, and evolutionary pathways related to the emergence of life.

Article	Year
Introducing a combinatorial DNA-toolbox platform constituting defined protein-based biohybrid-materials. Biomaterials\| PubMed ID: 25078434	2014
Designer amphiphilic proteins as building blocks for the intracellular formation of organelle-like compartments. Nature materials\| PubMed ID: 25362355	2015
Molecular protein adaptor with genetically encoded interaction sites guiding the hierarchical assembly of plasmonically active nanoparticle architectures. Nature communications\| PubMed ID: 25813537	2015
Self-Assembly Toolbox of Tailored Supramolecular Architectures Based on an Amphiphilic Protein Library. Small (Weinheim an der Bergstrasse, Germany)\| PubMed ID: 31173449	2019
Minimalist Protocell Design: A Molecular System Based Solely on Proteins that Form Dynamic Vesicular Membranes Embedding Enzymatic Functions. Chembiochem : a European journal of chemical biology\| PubMed ID: 31183952	2019
Prebiotic Protocell Model Based on Dynamic Protein Membranes Accommodating Anabolic Reactions. Langmuir : the ACS journal of surfaces and colloids\| PubMed ID: 31287709	2019

Andreas Schreiber

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