Ralf Biehl

Amphiphilic PEG multiblock copolymers can be readily synthesized by polycondensation reactions. Their multiblock structure leads to the formation of hydrogels consisting of highly interconnected micelles. Here, we investigate as model systems copolyesters consisting of alternating hydrophobic alkyldicarboxylic acids (Cm) and hydrophilic polyethylene glycols (EGn). We investigate the structure of the formed micelles and hydrogels over a wide range of concentrations, temperatures, and block lengths using small-angle X-ray scattering (SAXS), small-angle neutron scattering (SANS), cryo-TEM, differential scanning calorimetry (DSC), and rheology. We find that the multiblock copolymers form hydrogels already at low concentrations. With increasing concentration, we observe for all copolymers the formation of a highly ordered body-centered cubic (BCC) phase and a crystalline lamellar phase. The increase of the alkyl and PEG chain lengths systematically increases the BCC unit cell size. We observe that the PEG-blocks significantly penetrate into the hydrophobic micellar cores, thereby increasing micellar exchange rates. This leads to the formation of transient networks with high mechanical moduli, which are of interest for self-healing, robotics and medical applications.

Oppositely charged polyelectrolytes are known to form polyelectrolyte complexes. Polyelectrolyte complexes are typically structurally disordered. In recent years, complexes of oppositely charged polypeptides have received increasing attention. Polypeptides provide additional interaction motifs such as hydrogen bonds and salt bridges that could stabilize ordered assemblies. Under specific conditions, the formation of secondary structures has been reported. So far, it has been difficult to obtain long‐range ordered assemblies of polypeptide complexes. Here we show that liquid‐solid phase separation of oppositely charged homopolypeptides spontaneously leads to the formation of extended polypeptide helices that form highly ordered hexagonally or square‐packed helix arrays with helix–helix distances of 1.6–2.0 nm. The helices exhibit an additional periodic staggering along their axis with a periodicity of 15 – 30 nm, equal to the length of the extended helices. This hierarchical helix assembly was demonstrated for polyarginine assembled with polyaspartic acid or polyglutamic acid as examples. The materials can be shear‐aligned to achieve macroscopic uniaxial orientation of the helices. Crosslinking leads to solid elastic materials. Upon heating to temperatures above 40°C, the structure disorders due to a reversible helix‐coil transition. The structural and mechanical properties suggest a new route to synthetic peptide‐based materials.

The growing number of approved nucleic acid therapeutics illustrates the potential to treat diseases by targeting their genetic blueprints in vivo. The 10-23 DNAzyme is capable of cleaving a wide range of target RNA with high selectivity. However, its poor performance in vivo restricts its therapeutic application as gene silencing agent. Studies on ribozymes have shown that the crowded environment in cells and associated effects can impact ribozyme folding and thermostability, resulting in a change in activity. This opens up the question whether DNAzymes are also affected by molecular crowding. Here, we investigate the functional and structural influence of molecular crowding conditions on the 10-23 DNAzyme. The stability and activity of a PrP-specific 10-23 DNAzyme were examined in presence of PEG, dextran, and osmolytes. Our results indicate that osmolytes decrease DNAzyme activity in a concentration-dependent manner, while certain PEG and dextran concentrations promote activity. To rationalize our observations, we studied the cosolutes effect on physicochemical solution properties and the structure of the DNAzyme:RNA complex using FCS and SAXS. The data reveal that enhanced activity is observed under conditions where a combination of physiochemical properties matches an optimum that seems to be dependent on the metal ion cofactor. Structural influence under such conditions is indicated less. We propose that a certain degree of molecular crowding is required to favor a state, which allows for higher catalytic turnover. In addition, we show that the requirement for magnesium and manganese as a cofactor remains unchanged under the conditions applied. Our work contributes to a better understanding of how the cellular environment affects DNAzyme structure and function.

A fundamental feature of the antibody structure is the flexible linker between the 3 fragments that allows great flexibility and simultaneous binding to epitopes of antigens and receptors. Combining dynamic light scattering, neutron spin-echo spectroscopy and PFG-NMR we determine characteristic internal fragment dynamics on top of translational and rotational diffusion under crowding conditions. Short-time and long-time translational diffusion show an effective hard sphere like behavior within a colloidal picture. Internal fragment motions are characterized as “attack” and “search” motions complemented by rotational fragment motions. We find that the “attack” motions exposing the binding domain are highly preserved from low to physiologically relevant concentrations and higher, while “search” motions and overall rotational diffusion are suppressed under crowding conditions. Hydrodynamic interactions change the friction between fragments determining relaxation times while interparticle interactions influence the strength of the entropic spring between fragments. The strategic redesign of the linker region to facilitate “attack” motions and fragment rotation has the potential to enhance the therapeutic efficacy of mAbs.

Transportation of active macromolecules through cell membranes is an essential biological process. However, for hydrophilic macromolecules, the hydrophobic interior of lipid bilayers suppresses the passive translocation, and there are only few cases reported. We use alternating amphiphilic polymers (AAPs) in which the sizes of the hydrophilic and hydrophobic units can be varied over a broad range, keeping the polymers water-soluble. For small units, the macromolecules show a homopolymer-like character. Pulse field gradient NMR and neutron reflectivity measurements show that the chains have a high solubility in the membrane hydrophobic interior that allows the chains to passively translocate. Increasing the length of the hydrophilic units leads to more polar AAPs with low membrane solubility and a reduced translocation speed. If hydrophilic and hydrophobic moieties are increased in size, the AAPs have a strong amphiphilic character and adsorb to lipid membranes only with their hydrophobic units, have a high membrane concentration, and have an overall fast translocation kinetics.

The small-angle neutron scattering (SANS) diffractometer KWS-2 of the Jülich Centre for Neutron Science at the Heinz Maier-Leibnitz Zentrum, Garching, Germany, is a high-flux instrument based on the combination of a specially developed neutron guide system, which allows delivery of a high neutron intensity for a wide wavelength range λ between 2.8 and 20 Å to the instrument, and a versatile velocity selector, which allows an easy choice of wavelength spread Δλ/λ depending on whether the specific scientific goal is either improved resolution, i.e. Δλ/λ = 10%, or high intensity, i.e. Δλ/λ = 20%. Here we report an evaluation of KWS-2 performance by measurements and McStas simulations under the condition that only the thermal neutron source (TNS) is available at the FRM II reactor. According to the simulations, a flux decrease by a factor of 2.5 for λ = 2.8–3 Å and by a factor of 10 for λ ≥ 4.5 Å is expected if the reactor is operated only with the TNS compared with the established performance with cold neutrons provided by the cold neutron source. The flux decrease in TNS operation can be mitigated for λ ≥ 4.5 Å by using a low-resolution velocity selector which provides Δλ/λ = 20% for standard positioning and Δλ/λ = 35% when tilted at an angle ξ_i = −10° to the beam axis. According to measurements, this degradation of resolution for increasing intensity does not seem to be critical for the structural characterization of small biological morphologies, which would be one of the most investigated topics if only thermal neutrons were available at the FRM II. However, ordering effects in the scattering characteristics of lamellar soft or biophysical systems are barely observable when the resolution is relaxed to Δλ/λ ≥ 20%, which defines the performance limits of the experimental approach. Furthermore, by using MgF₂ focusing lenses when working with large samples, an intensity gain on the sample of up to 12-fold is achieved while keeping the same Q _min resolution as with the standard pinhole mode, restoring the flux loss expected with thermal neutrons for λ ≥ 7 Å.

Polyelectrolytes (PE) are polymeric macromolecules in aqueous solutions characterized by their chain topology and intrinsic charge in a neutralizing fluid. Structure and dynamics are related to several characteristic screening length scales determined by electrostatic, excluded volume, and hydrodynamic interactions. We examine PE dynamics in dilute to semidilute conditions using dynamic light scattering, neutron spinecho spectroscopy, and pulse field gradient NMR spectroscopy. We connect macroscopic diffusion to segmental chain dynamics, revealing a decoupling of local chain dynamics from interchain interactions. Collective diffusion is described within a colloidal picture, including electrostatic and hydrodynamic interactions. Chain dynamics is characterized by the classical Zimm model of a neutral chain retarded by internal friction. We observe that hydrodynamic interactions are not fully screened between chains and that the internal friction within the chain increases with an increase in ion condensation on the chain.

G-quadruplexes (G4s) are helical four-stranded structures forming from guanine-rich nucleic acid sequences, which are thought to play a role in cancer development and malignant transformation. Most current studies focus on G4 monomers, yet under suitable and biologically relevant conditions, G4s undergo multimerization. Here, we investigate the stacking interactions and structural features of telomeric G4 multimers by means of a novel low-resolution structural approach that combines small-angle X-ray scattering (SAXS) with extremely coarse-grained (ECG) simulations. The degree of multimerization and the strength of the stacking interaction are quantitatively determined in G4 self-assembled multimers. We show that self-assembly induces a significant polydispersity of the G4 multimers with an exponential distribution of contour lengths, consistent with a step-growth polymerization. On increasing DNA concentration, the strength of the stacking interaction between G4 monomers increases, as well as the average number of units in the aggregates. We utilized the same approach to explore the conformational flexibility of a model single-stranded long telomeric sequence. Our findings indicate that its G4 units frequently adopt a beads-on-a-string configuration. We also observe that the interaction between G4 units can be significantly affected by complexation with benchmark ligands. The proposed methodology, which identifies the determinants that govern the formation and structural flexibility of G4 multimers, may be an affordable tool aiding in the selection and design of drugs that target G4s under physiological conditions.

Guanylate binding proteins (GBPs) are soluble dynamin-like proteins that undergo a conformational transition for GTP-controlled oligomerization and disrupt membranes of intracellular parasites to exert their function as part of the innate immune system of mammalian cells. We apply neutron spin echo, X-ray scattering, fluorescence, and EPR spectroscopy as techniques for integrative dynamic structural biology to study the structural basis and mechanism of conformational transitions in the human GBP1 (hGBP1). We mapped hGBP1’s essential dynamics from nanoseconds to milliseconds by motional spectra of sub-domains. We find a GTP-independent flexibility of the C-terminal effector domain in the µs-regime and resolve structures of two distinct conformers essential for an opening of hGBP1 like a pocket knife and for oligomerization. Our results on hGBP1’s conformational heterogeneity and dynamics (intrinsic flexibility) deepen our molecular understanding relevant for its reversible oligomerization, GTP-triggered association of the GTPase-domains and assembly-dependent GTP-hydrolysis.