TP2 Engineering von biologischen Poren

© Copyright: Jan Michael Hosan, Hessen schafft Wissen

Project area 2

Engineering of Biological Pores

In project area 2 proteins are modified by genetic and protein engineering so that they have new sensor and switching characteristics. Cooperation between project areas 1 and 2 should lead to a stable integration of biological pores into the solid-state pores and /or that protein components for switching or sensing can be used in solid-state pores.

Project 2.1

Small ion channels with new switching properties as selective pores in solid-state polymer films

Principal Investigator: Gerhard Thiel

Der viralen Kaliumkanals KcvNTS
The viral potassium channel KcvNTS. The four subunits of the tetramer are shown in blue and yellow. The typical features of viral potassium channels such as the pore- and filter region and the outer transmembrane domain (TMD1) and the inner transmembrane domain (TMD2) of the two opposite sub-units are shown on the right.

Gerhard Thiel’s (Biology) is working on very small and extremely robust biological pores (ion channels) that offer themselves as ideal building blocks for nanosensors. Their suitability is based on high heat resistance, high ion conductivity, susceptibility to genetic manipulation and the very simple structure. These robust channel proteins are to be integrated into miniaturized membrane bilayers that are stretched over nanopores in polymers and tested for stability. Using protein engineering, these channel proteins can be equipped with new switching and sensor properties. In the long term switchable elements can be created which can be used in technical micro-devices for the analysis of medically relevant molecules.

Project 2.2

Coupling of modified binding proteins in biological and artificial nanopores

Principal Investigator: Bodo Laube

Bodo Laube’s group (Biology) investigates the ability of binding proteins (BP) to recognize individual analyte molecules specifically (extreme selectivity) and their enhancing effects (high sensitivity). Binding proteins represent a large protein superfamily of bacterial receptors that determine the selective binding of ligands such as carbohydrates, amino acids, peptides, anions, heavy metal cations etc. and they fulfill all requirements for a biosensor. The remarkable evolutionary adaptability and functional linkage of this superfamily in a variety of biological receptors is probably due to the positioning of the binding site at the interface between two domains and the resulting ligand-mediated conformational change. This allows the functional linkage of binding proteins to other protein domains by confirmative coupling. This structure-function principle of binding proteins can be found in eukaryotic proteins as a selective sensor for complex molecules and in information processing neural systems. The diversity of ligand binding properties and the functional adaptability of the BP superfamily are used to generate new biosensors with new features in the medical /pharmaceutical field and in environmental analysis.

Generierung von spezifischen Bindeproteinen (BP) und Rezeptoren für den Einsatz in Medizin und Umweltanalytik mit anschließender Kopplung an biologische und artifizielle Nanoporen. a) Gezielte Modifikation der Bindungsspezifitäten von BP und Liganden Bindedomänen (LBD) durch computergestütztes Design. b) Kopplung von BP an biologische Nanoporen: Änderung der Bindungsspezifität ionotroper Glutamatrezeptoren (iGluR) durch Substitution der LBD durch natürlich vorkommende BP. Direkte Kopplung von c) Rezeptoren und d) BP an artifizielle Nanoporen. e) Insertion von Rezeptoren in Lipid-Nanodiscs als „natürliche Membranumgebung“ mit anschließender Kopplung an artifizielle Nanoporen.
Generation of specific binding proteins (BP) and receptors for use in medical and environmental analysis with subsequent coupling of biological and synthetic nanopores. a) Targeted modification of the binding specificities of BP and ligand binding domains (LBD) by computer-aided design. b) Coupling of BP to biological nanopores: change of the binding specificity of ionotropic glutamate receptors (iGluR) by substituting the LBD by naturally occurring BP. Direct coupling of c) receptors and d) BP to synthetic nanopores. e) Insertion of receptors in lipid-nanodiscs as “natural membrane environment” with subsequent coupling to synthetic nanopores.

Project 2.3

In silico design and optimization of composite ion channels

Principal Investigator: Kay Hamacher

(a) Example of correlates between mechanical function and evolutionary signatures of coevolution. (b) Identification of functionally relevant interactions between transmembrane domains in a viral Kcv channel using coarse-grained molecular models.
(a) Example of correlates between mechanical function and evolutionary signatures of coevolution. (b) Identification of functionally relevant interactions between transmembrane domains in a viral Kcv channel using coarse-grained molecular models.

Kay Hamacher’s group (Biology) operates in the large-scale analysis of structure-function relationship of proteins and their complexes. Therefore, coarse-graining models, especially elastic network models, are performed for systematic investigations of dynamic-functional effects of mutations on ion channels. The small viral ion channels, which are analyzed in Gerhard Thiel’s group (Project 2.1), are very well suited as a starting point for directed, engineering-oriented design of new features and switching characteristics in ion channels. However, by merging domains which have had no interaction before, can lead to undesirable effects and reduce or even prevent the function. The elucidation of the mechanistic processes is an important step for deriving rational rules for the targeted design of new elements.

Junior research groups

Project 2.4

Chemical synthesis of switchable protein-based nanopore

Principal Investigator: Alesia Tietze

Functional pore in polymer-based scaffold
Functional pore in polymer-based scaffold

The aim of Alesia Tietze’s group is the chemical synthesis of switchable (pH, ion-type, ligand binding) protein-based nanopores mimicking biological ion channels and peptides forming helix structures. The Fmoc-based solid-phase peptide synthesis (SPPS) of pore-forming membrane proteins (28 – 90 AA) will be accomplished applying novel methods developed in the group. Chemical modifications of polymer facing residues anchoring to polymer-based scaffold will be integrated into the peptide/protein structure by variation of side-chain length and variety of chemical modifications.

Project 2.5

Protein engineering of ion conducting nanopores

Principal Investigator: Viktor Stein

Ion transport is mediated by highly efficient and selective protein channels across cellular membranes. In many cases, their mechanism of action is well understood. Yet, harnessing their many useful properties for biotechnological applications remains largely unexplored. The goal of the AG Stein is to re-engineer naturally occurring protein-based ion channels into highly specific biosensors that can detect biotechnologically and biomedically relevant molecules. Protein-based ion channels with custom response functions are constructed using a combination of structure-guided protein engineering and high-throughput screening. This is followed up by their detailed biophysical characterization to understand the molecular basis of their sensor function.