In Vesiculo Synthesis of Peptide Membrane Precursors for Autonomous Vesicle Growth

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an ...

Technische Universität München

AFM-based single-molecule force spectroscopy has been used to study the effect of Hofmeister salts and protein hydrophobicity on the adhesion of recombinant spider silk proteins onto solid substrates. Therefore, a molecular probe consisting of a spider silk protein and an AFM tip has been developed, which (i) is a well-defined, small system that can be simulated by molecular dynamics simulations, (ii) allows access to the whole soluble concentration range for ions, and (iii) provides the distribution of desorption forces rather than just ensemble-averaged mean values. The measured desorption forces follow the Hofmeister series for anions (H2PO4-, Cl-, I-) with a stabilizing energy of more than 15 kBT for 5 M NaH2PO4. Moreover, this effect is influenced by the hydrophobicity of the spider silk protein, indicating that hydrophobic and Hofmeister effects are closely related.

Hydrophobic and Hofmeister effects on the adhesion of spider silk proteins onto solid substrates: an AFM-based single-molecule study.

Spectroscopie de force de mol&eacute;cule unique ax&eacute;e sur l&#39;AFM a &eacute;t&eacute; utilis&eacute;e pour &eacute;tudier l&#39;effet des sels Hofmeister et l&#39;hydrophobicit&eacute; de prot&eacute;ine sur l&#39;adh&eacute;rence de prot&eacute;ines de soie araign&eacute;e recombinante sur des substrats solides. Par cons&eacute;quent, une sonde mol&eacute;culaire consistant en une prot&eacute;ine en soie d&#39;araign&eacute;e et une pointe d&#39;AFM a &eacute;t&eacute; d&eacute;velopp&eacute;e, qui (i) est un syst&egrave;me bien d&eacute;fini, petit qui peut &ecirc;tre simul&eacute; par des simulations de dynamique mol&eacute;culaire, (ii) permet d&#39;acc&eacute;der &agrave; la plage de concentration ensemble soluble pour les ions et (iii) indique la r&eacute;partition des forces de d&eacute;sorption plut&ocirc;t que juste moyennes sur l&#39;ensemble des valeurs moyennes. Les forces de d&eacute;sorption mesur&eacute;es suivent la s&eacute;rie de Hofmeister anions (H2PO4-, Cl-, j&#39;ai-) avec une &eacute;nergie stabilisante de plus de 15 kBT pour 5 M NaH2PO4. En outre, que cet effet est influenc&eacute; par l&#39;hydrophobie de la prot&eacute;ine en soie d&#39;araign&eacute;e, indiquant que hydrophobe et Hofmeister effets sont &eacute;troitement li&eacute;es.

Effets hydrophobes et Hofmeister sur l&#39;adh&eacute;rence de prot&eacute;ines de soie araign&eacute;e sur des substrats solides : une &eacute;tude ax&eacute;e sur l&#39;AFM de mol&eacute;cules simples.

Spettroscopia AFM-base forza singola molecola &egrave; stata usata per studiare l&#39;effetto dei sali Hofmeister e idrofobicit&agrave; proteina sull&#39;adesione di proteine ricombinanti ragno della seta su substrati solidi. Pertanto, una sonda molecolare costituito da una proteina di seta di ragno e una punta AFM &egrave; stata sviluppata, che (i) &egrave; un sistema ben definito, piccolo che pu&ograve; essere simulato da simulazioni di dinamica molecolare, (ii) permette l&#39;accesso all&#39;intervallo di concentrazione completamente solubili per gli ioni e (iii) fornisce la distribuzione di desorbimento forze piuttosto che i valori medi solo una media di ensemble. Le forze misurate desorbimento seguono la serie di Hofmeister per gli anioni (H2PO4-, Cl-, I-) con un energia stabilizzante di kBT oltre 15 per 5 M NaH2PO4. Inoltre, che questo effetto &egrave; influenzato dalla idrofobicit&agrave; di proteine della seta del ragno, che indica che idrofobo e Hofmeister effetti sono strettamente correlati.

Idrofobico e Hofmeister effetti sull&#39;adesione di proteine della seta ragno su substrati solidi: uno studio di singola molecola basata su AFM.

The adhesion of polypeptides and proteins at interfaces is important for a wide variety of systems--from the adhesion onto vessels during their production process over functional coatings to protein-membrane interactions in single cells. Herein, we apply an atomic-force-microscopy-based single-molecule method and poly-D-tyrosine to determine the adhesion strength and location of polypeptides at interfaces. Surprisingly, the support (solid, liquid or gas) hardly influences the adhesion in an aqueous environment, while the addition of ethanol to the solvent cuts the adhesion in half. These findings allow us to propose that the adsorbed polypeptide spans over both the depletion layer and the hydrophobic hydration layer to facilitate a compensation mechanism between dispersive and hydration forces.

Adsorption mechanism of polypeptides and their location at hydrophobic interfaces.

Atomic force microscopy (AFM) based single polymer force spectroscopy allows to detect the interaction (energy) between single polymers and interfaces in aqueous environment. We use this method to delineate the effect of ions, pH, co-solutes and temperature on the adhesion of biopolymers onto solid substrates.

Measuring the interaction between ions, biopolymers and interfaces--one polymer at a time.

Fast and efficient transport of molecular cargoes along tracks or on supramolecular platforms is an important prerequisite for the development of future nanorobotic systems and assembly lines. Here, we study the diffusive transport of DNA cargo strands bound to a supramolecular DNA origami structure via an extended tether arm. For short distances (on the order of a few nanometers), transport from a start to a target site is found to be less efficient than for direct transfer without tether. For distances on the scale of the origami platform itself, however, cargo transfer mediated by a rigid tether arm occurs very fast and robust, whereas a more flexible, hinged tether is found to be considerably less efficient. Our results suggest diffusive motion on a molecular tether as a highly efficient mechanism for fast transfer of cargoes over long distances. 

Diffusive transport of molecular cargo tethered to a DNA origami platform.

Nanoscale plasmonic waveguides composed of metallic nanoparticles are capable of guiding electromagnetic energy below the optical diffraction limit. Signal feed-in and readout typically require the utilization of electronic effects or near-field optical techniques, whereas for their fabrication mainly lithographic methods are employed. Here we developed a switchable plasmonic waveguide assembled from gold nanoparticles (AuNPs) on a DNA origami structure that facilitates a simple spectroscopic excitation and readout. The waveguide is specifically excited at one end by a fluorescent dye, and energy transfer is detected at the other end via the fluorescence of a second dye. The transfer distance is beyond the multicolor FRET range and below the Abbé limit. The transmittance of the waveguide can also be reversibly switched by changing the position of a AuNP within the waveguide, which is tethered to the origami platform by a thermoresponsive peptide. High-yield fabrication of the plasmonic waveguides in bulk was achieved using silica particles as solid supports. Our findings enable bulk solution applications for plasmonic waveguides as light-focusing and light-polarizing elements below the diffraction limit.

Self-Assembled Active Plasmonic Waveguide with a Peptide-Based Thermomechanical Switch.

Membrane compartmentalization and growth are central aspects of living cells, and are thus encoded in every cell's genome. For the creation of artificial cellular systems, genetic information and production of membrane building blocks will need to be coupled in a similar manner. However, natural biochemical reaction networks and membrane building blocks are notoriously difficult to implement in vitro. Here, we utilized amphiphilic elastin-like peptides (ELP) to create self-assembled vesicular structures of about 200 nm diameter. In order to genetically encode the growth of these vesicles, we encapsulate a cell-free transcription-translation system together with the DNA template inside the peptide vesicles. We show in vesiculo production of a functioning fluorescent RNA aptamer and a fluorescent protein. Furthermore, we implement in situ expression of the membrane peptide itself and finally demonstrate autonomous vesicle growth due to the incorporation of this ELP into the membrane.

Towards synthetic cells using peptide-based reaction compartments.

In nature, compartmentalized and spatially organized enzyme cascades are utilized to increase the efficiency of enzymatic reactions. From a technologically relevant perspective, synthetic enzyme systems have to be optimized with emphasis on enzyme activity, productivity, scalability, and ease of use. But the underlying principles and relevant parameters that lead to an enhancement of the activity of enzyme cascades through spatial organization are still under debate. Here, we report on the 10-fold activity enhancement of the GOx-HRP enzyme cascade for the oxidation of luminol, when the enzymes are colocalized on micron-scaled solid scaffolds. Both enzymes were initially assembled and concentrated on DNA origami rectangles and finally further concentrated on the surface of silica particles. We show that each particular component of the designed system contributes to the activity enhancement. Furthermore, we measured an influence of the silica particle length scale on the total productivity by a factor of 5-10, but to a lesser extent on the maximum enzyme activity. Our findings demonstrate that micrometer-sized scaffolds can be used to enhance the efficiency of enzyme-cascades by at least a magnitude and that solid-phase scaffolds enable scalability for technological applications.

Tobias Pirzer

Physics of Synthetic Systems - E14,
Physics-Department and ZNN,
Physics of Synthetic Systems - E14, Physics-Department and ZNN

Technische Universität München

In Vesiculo Synthesis of Peptide Membrane Precursors for Autonomous Vesicle Growth

Tobias Pirzer

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Physics of Synthetic Systems - E14,Physics-Department and ZNN,Physics of Synthetic Systems - E14, Physics-Department and ZNN

Technische Universität München

In Vesiculo Synthesis of Peptide Membrane Precursors for Autonomous Vesicle Growth

Physics of Synthetic Systems - E14,
Physics-Department and ZNN,
Physics of Synthetic Systems - E14, Physics-Department and ZNN