Martina Rangl

AFM imaging of functionalized carbon nanotubes on biological membranes

Multifunctional carbon nanotubes are promising for biomedical applications as their nano-size, together with their physical stability, gives access into the cell and various cellular compartments including the nucleus. However, the direct and label-free detection of carbon nanotube uptake into cells is a challenging task. The atomic force microscope (AFM) is capable of resolving details of cellular surfaces at the nanometer scale and thus allows following of the docking of carbon nanotubes to biological membranes. Here we present topographical AFM images of non-covalently functionalized single walled (SWNT) and double walled carbon nanotubes (DWNT) immobilized on different biological membranes, such as plasma membranes and nuclear envelopes, as well as on a monolayer of avidin molecules. We were able to visualize DWNT on the nuclear membrane while at the same time resolving individual nuclear pore complexes. Furthermore, we succeeded in localizing individual SWNT at the border of incubated cells and in identifying bundles of DWNT on cell surfaces by AFM imaging.

Single‐Molecule Analysis of the Recognition Forces Underlying Nucleo‐Cytoplasmic Transport

To move molecules across the nuclear envelope they have to overcome the selective barrier of the nuclear pore which is formed by nucleoporins with FG repeats. For this, they are chaperoned by shuttling receptors that interact with FG nups thereby passing the barrier with an unresolved mechanism. We explored the molecular binding and dissociation of this process using single molecule force spectroscopy showing that no energetic cost is required for translocation.

Increased imaging speed and force sensitivity for bio-applications with small cantilevers using a conventional AFM setup

Highlights ► Development of small cantilever. ► Speed increase by a factor of ten using small cantilevers on a commercial AFM. ► Force sensitivity increase by a factor of five using small cantilever prototypes for force spectroscopy measurements.

Molecular Recognition Force Spectroscopy: A New Tool to Tailor Targeted Nanoparticles

The density of targeting moieties in a nanoparticle-based gene-delivery system has been shown to play a fundamental role in its vectoring performance. Here, molecular recognition force spectroscopy is proposed as a novel screening tool to optimize the density of targeting moieties of functionalized nanoparticles towards attaining cell-specific interaction. By tailoring the nanoparticle formulation, the unbinding event probability between nanoparticles tethered to an atomic force microscopy tip and neuronal cells is directly correlated to the nanoparticle gene-vectoring capacity. Additionally, new insights into protein-receptor interaction are revealed. This novel approach opens new avenues in the field of nanomedicine.

Modification of the loops in the ligand-binding site turns avidin into a steroid-binding protein

BackgroundEngineered proteins, with non-immunoglobulin scaffolds, have become an important alternative to antibodies in many biotechnical and therapeutic applications. When compared to antibodies, tailored proteins may provide advantageous properties such as a smaller size or a more stable structure.ResultsAvidin is a widely used protein in biomedicine and biotechnology. To tailor the binding properties of avidin, we have designed a sequence-randomized avidin library with mutagenesis focused at the loop area of the binding site. Selection from the generated library led to the isolation of a steroid-binding avidin mutant (sbAvd-1) showing micromolar affinity towards testosterone (Kd ~ 9 μM). Furthermore, a gene library based on the sbAvd-1 gene was created by randomizing the loop area between β-strands 3 and 4. Phage display selection from this library led to the isolation of a steroid-binding protein with significantly decreased biotin binding affinity compared to sbAvd-1. Importantly, differential scanning calorimetry and analytical gel-filtration revealed that the high stability and the tetrameric structure were preserved in these engineered avidins.ConclusionsThe high stability and structural properties of avidin make it an attractive molecule for the engineering of novel receptors. This methodology may allow the use of avidin as a universal scaffold in the development of novel receptors for small molecules.

Stable, Non-Destructive Immobilization of Native Nuclear Membranes to Micro-Structured PDMS for Single-Molecule Force Spectroscopy

In eukaryotic cells the nucleus is separated from the cytoplasm by a double-membraned nuclear envelope (NE). Exchange of molecules between the two compartments is mediated by nuclear pore complexes (NPCs) that are embedded in the NE membranes. The translocation of molecules such as proteins and RNAs through the nuclear membrane is executed by transport shuttling factors (karyopherines). They thereby dock to particular binding sites located all over the NPC, the so-called phenylalanine-glycin nucleoporines (FG Nups). Molecular recognition force spectroscopy (MRFS) allows investigations of the binding at the single-molecule level. Therefore the AFM tip carries a ligand for example, a particular karyopherin whereas the nuclear membrane with its receptors is mounted on a surface. Hence, one of the first requirements to study the nucleocytoplasmatic transport mechanism using MRFS is the development of an optimized membrane preparation that preserves structure and function of the NPCs. In this study we present a stable non-destructive preparation method of Xenopus laevis nuclear envelopes. We use micro-structured polydimethylsiloxane (PDMS) that provides an ideal platform for immobilization and biological integrity due to its elastic, chemical and mechanical properties. It is a solid basis for studying molecular recognition, transport interactions, and translocation processes through the NPC. As a first recognition system we investigate the interaction between an important transport shuttling factor, importin beta, and its binding sites on the NPC, the FG-domains.

Molecular recognition force spectroscopy

Atomic force microscopy (AFM), developed in the late eighties to explore atomic details on hard material surfaces, has evolved to an imaging method capable of achieving fine structural details on biological samples. Its particular advantage in biology is that the measurements can be carried out in aqueous and physiological environment, which opens the possibility to study the dynamics of biological processes in vivo. The additional potential of the AFM to measure ultra-low forces at high lateral resolution has paved the way for measuring inter- and intra-molecular forces of bio-molecules on the single molecule level. Molecular recognition studies using AFM open the possibility to detect specific ligand—receptor interaction forces and to observe molecular recognition of a single ligand—receptor pair. Applications include biotin—avidin, antibody—antigen, NTA nitrilotriacetate—hexahistidine 6, and cellular proteins, either isolated or in cell membranes.

Investigating the binding behaviour of two avidin-based testosterone binders using molecular recognition force spectroscopy

Molecular recognition force spectroscopy, a biosensing atomic force microscopy technique allows to characterise the dissociation of ligand–receptor complexes at the molecular level. Here, we used molecular recognition force spectroscopy to study the binding capability of recently developed testosterone binders. The two avidin‐based proteins called sbAvd‐1 and sbAvd‐2 are expected to bind both testosterone and biotin but differ in their binding behaviour towards these ligands. To explore the ligand binding and dissociation energy landscape of these proteins, we tethered biotin or testosterone to the atomic force microscopy probe while the testosterone‐binding protein was immobilized on the surface. Repeated formation and rupture of the ligand–receptor complex at different pulling velocities allowed determination of the loading rate dependence of the complex‐rupturing force. In this way, we obtained the molecular dissociation rate (koff) and energy landscape distances (xβ) of the four possible complexes: sbAvd‐1‐biotin, sbAvd‐1‐testosterone, sbAvd‐2‐biotin and sbAvd‐2‐testosterone. It was found that the kinetic off‐rates for both proteins and both ligands are similar. In contrast, the xβ values, as well as the probability of complex formations, varied considerably. In addition, competitive binding experiments with biotin and testosterone in solution differ significantly for the two testosterone‐binding proteins, implying a decreased cross‐reactivity of sbAvd‐2. Unravelling the binding behaviour of the investigated testosterone‐binding proteins is expected to improve their usability for possible sensing applications. Copyright

Single-Molecule Analysis of the Recognition Forces Underlying Nucleo-Cytoplasmic Transport

Macromolecular exchange between the nucleus and cytoplasm of cells is gated at nuclear pores by a family of intrinsically-disordered nucleoporins (nups). These feature phenylalanine-glycine repeats in ‘cohesive’ domains (FG domains) that interact to form a physical barrier. Through unknown mechanisms, karyopherins (importins, exportins, transportins, NTRs) penetrate this barrier to facilitate the movement of large proteins and RNPs across without paying an external energetic cost, simply by interacting with FG domains. To address the molecular binding and dissociation mechanisms involved in this coupled gating-translocation process, single molecule force spectroscopy was used here to measure the interaction force between nup FG repeats, and between importin beta and nup FG repeats. As predicted, cohesive FG domains bound each other through multiple FG repeat interactions. In contrast importin bound only relaxed coil multiple FG repeats simultaneously, whereas just one FG binding site was assessed in collapsed coil multiple FG domains. Most importantly, the interaction forces and fast dissociation rate constants measured between two FG repeats, and between importin and one FG repeat, were almost identical. This suggests that the force needed to separate interactions between FG repeats of nups at the NPC (i.e. for kaps to penetrate the gate and translocate across) could be provided in full by the enthalpy gained through the formation of karyopherin-FG repeat interactions.