Fatty acid binding to the human transport proteins FABP3, FABP4, and FABP5 from a Ligand's perspective.

Fatty acid binding proteins (FABPs) are a family of amphiphilic transport proteins with high diversity in terms of their amino acid sequences and binding preferences. Beyond their main biological role as cytosolic fatty acid transporters, many aspects regarding their binding mechanism and functional specializations in human cells remain unclear. In this work, the binding properties and thermodynamics of FABP3, FABP4, and FABP5 were analyzed under various physical conditions. For this purpose, the FABPs were loaded with fatty acids bearing fluorescence or spin probes as model ligands, comparing their binding affinities via microscale thermophoresis (MST) and continuous-wave electron paramagnetic resonance (CW EPR) spectroscopy. The CW EPR spectra of non-covalently bound 5- and 16-DOXYL stearic acid (5/16-DSA) deliver in-depth information about the dynamics and chemical environments of ligands inside the binding pockets of the FABPs. EPR spectral simulations allow the construction of binding curves, revealing two different binding states ('intermediately' and 'strongly' bound). The proportion of bound 5/16-DSA depends strongly on the FABP concentration and the temperature but with remarkable differences between the three isoforms. Additionally, the more dynamic state ('intermediately bound') seems to dominate at body temperature with thermodynamic preference. The ligand binding studies were supplemented by aggregation studies via dynamic light scattering and bioinformatic analyses. Beyond the remarkably fine-tuned binding properties exhibited by each FABP, which were discernible with our EPR-centered approach, the results of this work attest to the power of simple spectroscopic experiments to provide new insights into the ligand binding mechanisms of proteins in general on a molecular level.

Characterization of the active site in the thiocyanate-forming protein from Thlaspi arvense (TaTFP) using EPR spectroscopy.

Glucosinolates are plant thioglucosides, which act as chemical defenses. Upon tissue damage, their myrosinase-catalyzed hydrolysis yields aglucones that rearrange to toxic isothiocyanates. Specifier proteins such as thiocyanate-forming protein from Thlaspi arvense (TaTFP) are non-heme iron proteins, which capture the aglucone to form alternative products, e.g. nitriles or thiocyanates. To resolve the electronic state of the bound iron cofactor in TaTFP, we applied continuous wave electron paramagnetic resonance (CW EPR) spectroscopy at X-and Q-band frequencies (∼9.4 and ∼34 GHz). We found characteristic features of high spin and low spin states of a d 5 electronic configuration and local rhombic symmetry during catalysis. We monitored the oxidation states of bound iron during conversion of allylglucosinolate by myrosinase and TaTFP in presence and absence of supplemented Fe2+. Without added Fe2+, most high spin features of bound Fe3+ were preserved, while different g'-values of the low spin part indicated slight rearrangements in the coordination sphere and/or structural geometry. We also examined involvement of the redox pair Fe3+/Fe2 in samples with supplemented Fe2+. The absence of any EPR signal related to Fe3+ or Fe2+ using an iron-binding deficient TaTFP variant allowed us to conclude that recorded EPR signals originated from the bound iron cofactor.

Tuning the Internal Compartmentation of Single-Chain Nanoparticles as Fluorescent Contrast Agents.

Controlling the internal structures of single-chain nanoparticles (SCNPs) is an important factor for their targeted chemical design and synthesis, especially in view of nanosized compartments presenting different local environments as a main feature to control functionality. We here design SCNPs bearing near-infrared fluorescent dyes embedded in hydrophobic compartments for use as contrast agents in pump-probe photoacoustic (PA) imaging, displaying improved properties by the location of the dye in the hydrophobic particle core. Compartment formation is controlled via single-chain collapse and subsequent crosslinking of an amphiphilic polymer using external crosslinkers in reaction media of adjustable polarity. Different SCNPs with hydrodynamic diameters of 6-12 nm bearing adjustable label densities are synthesized. It is found that the specific conditions for single-chain collapse have a major impact on the formation of the desired core-shell structure, in turn adjusting the internal nanocompartments together with the formation of excitonic dye couples, which in turn increase their fluorescence lifetime and PA signal generation. SCNPs with the dye molecules accumulate at the core also show a nonlinear PA response as a function of pulse energy-a property that can be exploited as a contrast mechanism in molecular PA tomography.

Dipolar Interactions and Protein Hydration in Highly Concentrated Antibody Formulations.

Molecular interaction mechanisms in high-concentrated protein systems are of fundamental importance for the rational development of biopharmaceuticals such as monoclonal antibody (mAb) formulations. In such high-concentrated protein systems, the intermolecular distances between mAb molecules are reduced to the size of the protein diameter (approx. 10 nm). Thus, protein-protein interactions are more pronounced at high concentrations; so a direct extrapolation of physicochemical properties obtained from measurements at a low protein concentration of the corresponding properties at a high protein concentration is highly questionable. Besides the charge-charge interaction, the effects of molecular crowding, dipolar interaction, changes in protein hydration, and self-assembling tendency become more relevant. Here, protein hydration, protein dipole moment, and protein-protein interactions were studied in protein concentrations up to 200 mg/mL (= 1.3 mM) in different formulations for selected mAbs using dielectric relaxation spectroscopy (DRS). These data are correlated with the second virial coefficient, A2, the diffusion interaction parameter, kD, the elastic shear modulus, G', and the dynamic viscosity, η. When large contributions of dipolar protein-protein interactions were observed, the tendency of self-assembling and an increase in solution viscosity were detected. These effects were examined using specific buffer conditions. Furthermore, different types of protein-water interactions were identified via DRS, whereby the effect of high protein concentration on protein hydration was investigated for different high-concentrated liquid formulations (HCLFs).

Macro- and Nanoscale Effect of Ethanol on Bovine Serum Albumin Gelation and Naproxen Release.

We report extended ethanol-induced gelation procedures of bovine serum albumin (BSA) at 37 °C and investigate the release behavior of a spin-labeled naproxen derivative (SL-NPX) from these hydrogels. The macroscopic mechanical properties of these gels during formation were studied using rheology, while a nanoscopic, more molecular view was obtained by analyzing the secondary structure of the protein during gelation via infrared (ATR-IR) spectroscopy. To evaluate the potential use of BSA hydrogels in controlled drug delivery, SL-NPX-BSA interaction was investigated in detail by continuous-wave electron paramagnetic resonance (CW EPR) spectroscopy, which provides information on the interaction of the small drug molecules and the hydrogel. In addition to CW EPR spectroscopy, dynamic light scattering (DLS), which provides insight into the size and nature of released components, was applied to characterize the combined influence of incubation time, ethanol, SL-drug, and BSA concentration on release behavior. It was found that the alteration of initial drug loading percentage, hydrogel incubation time as well as BSA and alcohol concentrations affect and thus tune the release rate of SL-NPX from BSA hydrogels. These results lead to the conclusion that BSA hydrogels as controlled release systems offer a remarkable fine-tuning capability for pharmaceutical applications due to the variety of gelation parameters.

Incorporation of ß-Alanine in Cu(II) ATCUN Peptide Complexes Increases ROS Levels, DNA Cleavage and Antiproliferative Activity.

Redox-active Cu(II) complexes are able to form reactive oxygen species (ROS) in the presence of oxygen and reducing agents. Recently, Faller et al. reported that ROS generation by Cu(II) ATCUN complexes is not as high as assumed for decades. High complex stability results in silencing of the Cu(II)/Cu(I) redox cycle and therefore leads to low ROS generation. In this work, we demonstrate that an exchange of the α-amino acid Gly with the ß-amino acid ß-Ala at position 2 (Gly2→ß-Ala2) of the ATCUN motif reinstates ROS production (• OH and H2 O2 ). Potentiometry, cyclic voltammetry, EPR spectroscopy and DFT simulations were utilized to explain the increased ROS generation of these ß-Ala2-containing ATCUN complexes. We also observed enhanced oxidative cleavage activity towards plasmid DNA for ß-Ala2 compared to the Gly2 complexes. Modifications with positively charged Lys residues increased the DNA affinity through electrostatic interactions as determined by UV/VIS, fluorescence, and CD spectroscopy, and consequently led to a further increase in nuclease activity. A similar trend was observed regarding the cytotoxic activity of the complexes against several human cancer cell lines where ß-Ala2 peptide complexes had lower IC50 values compared to Gly2. The higher cytotoxicity could be attributed to an increased cellular uptake as determined by ICP-MS measurements.

Nanoscopic Dynamics Dictate the Phase Separation Behavior of Intrinsically Disordered Proteins.

Many intrinsically disordered proteins (IDPs) in nature may undergo liquid-liquid phase separation to assemble membraneless organelles with varied liquid-like properties and stability/dynamics. While solubility changes underlie these properties, little is known about hydration dynamics in phase-separating IDPs. Here, by studying IDP polymers of similar composition but distinct liquid-like dynamics and stability upon separation, namely, thermal hysteresis, we probe at a nanoscopic level hydration/dehydration dynamics in IDPs as they reversibly switch between phase separation states. Using continuous-wave electron paramagnetic resonance (CW EPR) spectroscopy, we observe distinct backbone and amino acid side-chain hydration dynamics in these IDPs. This nanoscopic view reveals that side-chain rehydration creates a dynamic water shield around the main-chain backbone that effectively and counterintuitively prevents water penetration and governs IDP solubility. We find that the strength of this superficial water shell is a sequence feature of IDPs that encodes for the stability of their phase-separated assemblies. Our findings expose and offer an initial understanding of how the complexity of nanoscopic water-IDP interactions dictate their rich phase separation behavior.

Nanoscale Model System for the Human Myelin Sheath.

Neurodegenerative disorders are among the most common diseases in modern society. However, the molecular bases of diseases such as multiple sclerosis or Charcot-Marie-Tooth disease remain far from being fully understood. Research in this field is limited by the complex nature of native myelin and by difficulties in obtaining good in vitro model systems of myelin. Here, we introduce an easy-to-use model system of the myelin sheath that can be used to study myelin proteins in a native-like yet well-controlled environment. To this end, we present myelin-mimicking nanodiscs prepared through one of the amphiphilic copolymers styrene/maleic acid (SMA), diisobutylene/maleic acid (DIBMA), and styrene/maleimide sulfobetaine (SMA-SB). These nanodiscs were tested for their lipid composition using chromatographic (HPLC) and mass spectrometric (MS) methods and, utilizing spin probes within the nanodisc, their comparability with liposomes was studied. In addition, their binding behavior with bovine myelin basic protein (MBP) was scrutinized to ensure that the nanodiscs represent a suitable model system of myelin. Our results suggest that both SMA and SMA-SB are able to solubilize the myelin-like (cytoplasmic) liposomes without preferences for specific lipid headgroups or fatty acyl chains. In nanodiscs of both SMA and SMA-SB (called SMA(-SB)-lipid particles, short SMALPs or SMA-SBLPs, respectively), the polymers restrict the lipids' motion in the hydrophobic center of the bilayer. The headgroups of the lipids, however, are sterically less hindered in nanodiscs when compared with liposomes. Myelin-like SMALPs are able to bind bovine MBP, which can stack the lipid bilayers like in native myelin, showing the usability of these simple, well-controlled systems in further studies of protein-lipid interactions of native myelin.

Nanoscale structure and dynamics of thermoresponsive single-chain nanoparticles investigated by EPR spectroscopy.

We characterize temperature-dependent macroscopic and nanoscopic phase transitions and nanoscopic pre-transitions of water-soluble single chain nanoparticles (SCNPs). The studied SCNPs are based on polymers displaying lower-critical solution temperature (LCST) behavior and show nanoscale compartmentation. SCNPs are amenable to continuous wave electron paramagnetic resonance (CW EPR) spectroscopy to study how amphiphilic, non-covalently added nitroxide spin probes or covalently attached spin labels sample their environment concerning nanoscale structures (polarity, hydrophilicity/-phobicity) and dynamics. These SCNPs are formed through single-chain collapse and have been shown to have nanosized compartments that are rigidified during the crosslinking process. We analyze the temperature-dependent phase transitions of spin-labeled SCNPs by rigorous spectral simulations of a series of multicomponent EPR-spectra that derive from the nanoinhomogeneities (1) that are due to the single-chain compartmentation in SCNPs and (2) the transformation upon temperature change due to the LCST behavior. These transitions of the SCNPs and their respective polymer precursors can be monitored and understood on the nanoscale by following EPR-spectroscopic parameters like hyperfine couplings that depend on the surrounding solvent molecules or Heisenberg spin exchange between small molecule spin probes or covalently attached spin labels in the nanocompartments. In particular, for one SCNP, we find an interesting behavior that we ascribe to the properties of the nanosized inner core with continuous effects before and jump-like changes after the macroscopic thermal collapse, indicating highly efficient desolvation and compaction upon an increase in temperature and aggregation of individual nanoparticles above the collapse temperature.

Expanding the toolbox for predictive parameters describing antibody stability considering thermodynamic and kinetic determinants.

PURPOSE: Introduction of the activation energy (Ea) as a kinetic parameter to describe and discriminate monoclonal antibody (mAb) stability. METHODS: Ea is derived from intrinsic fluorescence (IF) unfolding thermograms. An apparent irreversible three-state fit model based on the Arrhenius integral is developed to determine Ea of respective unfolding transitions. These activation energies are compared to the thermodynamic parameter of van´t Hoff enthalpies (∆Hvh). Using a set of 34 mAbs formulated in four different formulations, both the apparent thermodynamic and kinetic parameters together with apparent melting temperatures are correlated collectively with each other to storage stabilities to evaluate its predictive power with respect to long-term effects potentially reflected in shelf-life. RESULTS: Ea allows for the discrimination of (i) different parent mAbs, (ii) different variants that originate from parent mAbs, and (iii) different formulations. Interestingly, we observed that the Ea of the CH2 unfolding transition shows strongest correlations with monomer and aggregate content after storage at accelerated and stress conditions when collectively compared to ∆Hvh and Tm of the CH2 transition. Moreover, the predictive parameters determined for the CH2 domain show generally stronger correlations with monomer and aggregate content than those derived for the Fab. Qualitative assessment by ranking Ea of the Fab domain showed good agreement with monomer content in storage stabilities of individual mAb sub-sets. CONCLUSION: Ea from IF unfolding transitions can be used in addition to other commonly used thermodynamic predictive parameters to discriminate and characterize thermal stability of different mAbs in different formulations. Hence, it shows great potential for antibody engineering and formulation scientists.

Influence of a single ether bond on assembly, orientation, and miscibility of phosphocholine lipids at the air-water interface.

How does a small change in the structure of a phospholipid affect its supramolecular assembly? In aqueous suspensions, the substitution of one ester linkage in DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) by an ether linkage alters its phase behaviour completely. To unravel the effect of replacing a phospholipid's ester linkage by an ether linkage in lipid monolayers, we characterized pure monolayers of the model lipid DPPC and its sn-2 ether analogue PHPC (1-palmitoyl-2-O-hexadecyl-sn-glycero-3-phosphocholine) as well as mixtures of both by measurements of surface pressure-molecular area (π-Amol) isotherms. In addition, we used infrared reflection absorption spectroscopy (IRRAS) to study lipid condensation, lipid chain orientation, headgroup hydration, and lipid miscibility in all samples. Mixed monolayers consisting of DPPC and PHPC were studied further using epifluorescence microscopy. Our results indicate a strong influence of the sn-2 ether linkage on headgroup hydration and ordering effects in the regions of the apolar chains and the headgroups. Both effects could originate from changes in glycerol conformation. Furthermore, we observed a second plateau in the π-Amol isotherms of DPPC/PHPC mixtures and analysis of the mixed π-Amol isotherms reveals a non-ideal mixing behaviour of both lipids which may be caused by conformational differences in their headgroups.

Access to New Cytotoxic Triterpene and Steroidal Acid-TEMPO Conjugates by Ugi Multicomponent-Reactions.

Multicomponent reactions, especially the Ugi-four component reaction (U-4CR), provide powerful protocols to efficiently access compounds having potent biological and pharmacological effects. Thus, a diverse library of betulinic acid (BA), fusidic acid (FA), cholic acid (CA) conjugates with TEMPO (nitroxide) have been prepared using this approach, which also makes them applicable in electron paramagnetic resonance (EPR) spectroscopy. Moreover, convertible amide modified spin-labelled fusidic acid derivatives were selected for post-Ugi modification utilizing a wide range of reaction conditions which kept the paramagnetic center intact. The nitroxide labelled betulinic acid analogue 6 possesses cytotoxic effects towards two investigated cell lines: prostate cancer PC3 (IC50 7.4 ± 0.7 µM) and colon cancer HT29 (IC50 9.0 ± 0.4 µM). Notably, spin-labelled fusidic acid derivative 8 acts strongly against these two cancer cell lines (PC3: IC50 6.0 ± 1.1 µM; HT29: IC50 7.4 ± 0.6 µM). Additionally, another fusidic acid analogue 9 was also found to be active towards HT29 with IC50 7.0 ± 0.3 µM (CV). Studies on the mode of action revealed that compound 8 increased the level of caspase-3 significantly which clearly indicates induction of apoptosis by activation of the caspase pathway. Furthermore, the exclusive mitochondria targeting of compound 18 was successfully achieved, since mitochondria are the major source of ROS generation.

Fluorescent and Water Dispersible Single-Chain Nanoparticles: Core-Shell Structured Compartmentation.

Single-chain nanoparticles (SCNPs) are highly versatile structures resembling proteins, able to function as catalysts or biomedical delivery systems. Based on their synthesis by single-chain collapse into nanoparticular systems, their internal structure is complex, resulting in nanosized domains preformed during the crosslinking process. In this study we present proof of such nanocompartments within SCNPs via a combination of electron paramagnetic resonance (EPR) and fluorescence spectroscopy. A novel strategy to encapsulate labels within these water dispersible SCNPs with hydrodynamic radii of ≈5 nm is presented, based on amphiphilic polymers with additional covalently bound labels, attached via the copper catalyzed azide/alkyne "click" reaction (CuAAC). A detailed profile of the interior of the SCNPs and the labels' microenvironment was obtained via electron paramagnetic resonance (EPR) experiments, followed by an assessment of their photophysical properties.

An Azidolipid Monolayer - Transitions, Miscibility, and UV Reactivity Studied by Infrared Reflection Absorption Spectroscopy.

In this study, we characterized monolayers of an azide-modified lipid at the air-water interface, pure and in its mixtures with the model lipid DPPC, with the aim of proving its potential to be applied for photo-cross-linking with other molecules. We chose a phospholipid bearing a terminal azide group in one of its hydrophobic tails to study its monolayer characteristics with the Langmuir film balance technique. Furthermore, we performed infrared reflection absorption spectroscopy (IRRAS) to get detailed insights into the organization of those monolayers as well as high-resolution mass spectrometry (HRMS) to see the effects of UV-irradiation on the lipids' chemical structure and organization. Our results suggest that in expanded monolayers of pure azide-modified membrane lipids, the azido-terminated chain folds back toward the air-water interface. Above the LE/LC (liquid-expanded/liquid-condensed) phase transition, the chains stretched, and thus, the azide group detaches from the interface. From temperature-dependent monolayer compressions, we evaluated all relevant thermodynamic parameters of the monolayers, such as the phase transition pressure, the critical temperature, and the triple point, and compare them to those of model lipids. For future applications, we studied the miscibility of the azide-modified lipid with DPPC in monolayers and found at least a certain miscibility over all investigated mixing ratios ranging from 10 to 75% of the azidolipid. Finally, we irradiated the azidolipid monolayer with UV light at 305 nm and measured photodissociation of the azide, leading to chemical cross-linking with other lipids, which shows the potential to be used as a cross-linking agent within self-assembled lipid or lipid/protein layers.

Thermodynamic Unfolding and Aggregation Fingerprints of Monoclonal Antibodies Using Thermal Profiling.

PURPOSE: Predicting thermal protein stability is of major interest in the development of protein-based biopharmaceuticals. Therefore, this study provides a predictive tool for determining transition enthalpies, which can be used for ranking different proteins according to their thermal stability. METHODS: Unfolding and aggregation profiles of eight different therapeutic monoclonal antibodies (mAbs) of type G, isotype 1 were investigated. The unfolding profiles were determined by intrinsic fluorescence (IF) spectroscopy and differential scanning calorimetry (DSC). A three-state unfolding fitting model was used to determine thermodynamic parameters for macromolecular multi-domain mAbs in IF experiments, like the van't Hoff enthalpy change (∆Hvh) and the entropy change (∆S) of the unfolding event. The derived values were compared to thermodynamic parameters obtained directly by calorimetry. Moreover, differences in the Fab enthalpies were used to predict aggregation behavior and protein thermal stabilities. To do so, the liquid-formulated mAbs were investigated exemplarily by size exclusion chromatography (SEC) after accelerated thermal-induced stress conditions. RESULTS: Comparing the thermodynamic parameters derived from IF spectroscopy and DSC resulted in similar values. Data generated by thermal-induced stress at 40°C show similar stability ranking as postulated through the Fab enthalpies for mAbs in two different formulations, while at 25°C a meaningful ranking is not possible, because distinct differences in the thermal stability cannot be observed. The additional consideration of Fab enthalpies to predict the 40 °C SEC ranking seems to be more reliable compared to the use of exclusively the melting temperatures or aggregation onset temperatures and times. CONCLUSION: We show that thermodynamic profiling can help predicting unfolding and aggregation properties of therapeutic mAbs at 40°C. Therefore, analyzing thermodynamic unfolding parameters is a useful and supportive tool discriminating thermal stability profiles of mAbs for further pharmaceutical development and clinical studies.

The Effect of Ethanol on Gelation, Nanoscopic, and Macroscopic Properties of Serum Albumin Hydrogels.

Serum albumin has shown great potential in the development of new biomaterials for drug delivery systems. Different methods have been proposed to synthesis hydrogels out of serum albumin. It has been observed that ethanol can also act as a trigger for serum albumin denaturation and subsequent gelation. In this study, we focus on basic mechanisms of the albumin gelation process at 37 °C when using the chemical denaturant ethanol. The temperature of 37 °C was chosen to resemble human body temperature, and as under physiological conditions, albumin is in a non-denatured N conformation. As established in our previous publication for the triggers of pH and temperature (and time), we here explore the conformational and physical properties space of albumin hydrogels when they are ethanol-induced and show that the use of ethanol can be advisable for certain gel properties on the nanoscopic and macroscopic scale. To this end, we combine spectroscopic and mechanically (rheology) based data for characterizing the gels. We also study the gels' binding capacities for fatty acids with electron paramagnetic resonance (EPR) spectroscopy, which implies observing the effects of bound stearic acids on gelation. Ethanol reduces the fraction of the strongly bound FAs in bovine serum albumin (BSA) hydrogels up to 52% and induces BSA hydrogels with a maximum storage modulus of 5000 Pa. The loosely bound FAs in ethanol-based hydrogels, besides their relatively weak mechanical properties, introduce interesting new materials for fast drug delivery systems and beyond.

Probing the Y2 Receptor on Transmembrane, Intra- and Extra-Cellular Sites for EPR Measurements.

The function of G protein-coupled receptors is intrinsically linked to their conformational dynamics. In conjugation with site-directed spin labeling, electron paramagnetic resonance (EPR) spectroscopy provides powerful tools to study the highly dynamic conformational states of these proteins. Here, we explored positions for nitroxide spin labeling coupled to single cysteines, introduced at transmembrane, intra- and extra-cellular sites of the human neuropeptide Y2 receptor. Receptor mutants were functionally analyzed in cell culture system, expressed in Escherichia coli fermentation with yields of up to 10 mg of purified protein per liter expression medium and functionally reconstituted into a lipid bicelle environment. Successful spin labeling was confirmed by a fluorescence assay and continuous wave EPR measurements. EPR spectra revealed mobile and immobile populations, indicating multiple dynamic conformational states of the receptor. We found that the singly mutated positions by MTSL ((1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl) methyl methanesulfonothioate) have a water exposed immobilized conformation as their main conformation, while in case of the IDSL (bis(1-oxyl-2,2,5,5-tetramethyl-3-imidazolin-4-yl) disulfide) labeled positions, the main conformation are mainly of hydrophobic nature. Further, double cysteine mutants were generated and examined for potential applications of distance measurements by double electron-electron resonance (DEER) pulsed EPR technique on the receptor.

Ligand-Binding Cooperativity Effects in Polymer-Protein Conjugation.

We present an electron paramagnetic resonance (EPR) spectroscopic characterization of structural and dynamic effects that stem from post-translational modifications of bovine serum albumin (BSA), an established model system for polymer-protein conjugation. Beyond the typical drug delivery and biocompatibility aspect of such systems, we illustrate the causes that alter internal dynamics and therefore functionality in terms of ligand-binding to the BSA protein core. Uptake of the paramagnetic fatty acid derivative 16-doxyl stearic acid by several BSA-based squaric acid macroinitiators and polymer-protein conjugates was studied by EPR spectroscopy, aided by dynamic light scattering (DLS) and zeta potential measurements. The conjugates were grafted from oligo(ethylene glycol) methyl ether methacrylate (OEGMA), forming an overall core-shell-like structure. It is found that ligand-binding and associated parameters such as binding affinity, cooperativity, and the number of binding sites of BSA change drastically with the extent of surface modification. In the course of processing BSA, the ligands also change their preference for individual binding sites, as observed from a comparative view of their spatial alignments in double electron electron resonance (DEER) experiments. The protein-attached polymers constitute a diffusion barrier that significantly hamper ligand uptake. Moreover, zeta potentials (ζ) decrease linearly with the degree of surface modification in protein macroinitiators and an effective dielectric constant can be estimated for the polymer layer in the conjugates. All this suggests that ligand uptake characteristics in BSA can be fine-tuned by the extent and nature of such post-translational modifications (PTMs). We show that EPR spectroscopy is suitable for quantifying these subtle PTM-based functional effects from self-assembly of substrate and ligand.

Tuning the shape anisotropy of loosely bound colloid-like ionic clusters in solution.

We characterize the influence of the ionic ratio on the dynamic self-assembly process involving a macrocyclic tetraimidazolium molecular box and small dianionic salts into highly defined, colloid-like ionic clusters in solution, called ionoids. Based on our studies utilizing dynamic light scattering (DLS) and continuous wave electron paramagnetic resonance (CW EPR) spectroscopy, we determine a region of privileged ionic ratios, which allow the formation of monodisperse, spheroidal structures of loosely bound ions in solution with adjustable (i) hydrodynamic radii between 6 nm and 12 nm and (ii) shape anisotropy. Inspired by Hertzsprung-Russell diagrams (HRDs) used in astrophysics to describe the fate of stars, we construct ionoid evolution diagrams (IEDs). IEDs are essential for grasping and describing the highly complex temporal development of these dynamically self-assembled structures in solution from the level of the individual ionic building blocks to stable clusters with a minimum lifetime of months, and thus aid in crafting future globular ionoids and anisotropic ionic clusters.

Modulation of amyloid ß peptide aggregation by hydrophilic polymers.

A substantial number of diseases leading to loss of neurologic functions such as Morbus Alzheimer, Morbus Parkinson, or Chorea Huntington are related to the fibrillation of particular amyloidogenic peptides. In vitro amyloid fibrillation strongly depends on admixture with other proteins and peptides, lipids, nanoparticles, surfactants and polymers. We investigated amyloid-beta 1-40 peptide (Aß1-40) fibrillation in mixture with thermoresponsive poly(oligo(ethylene glycol)macrylates), in which the polymer's hydrophobicity is tuned by variation of the number of ethylene glycol-units in the side chain (m = 1-9), the end groups (B = butoxy; C = carboxy; D = dodecyl; P = pyridyldisulfide) and the degree of polymerization (n) of the polymers. The polymers were prepared via RAFT-polymerization, obtaining a broad range of molecular masses (Mn = 700 to 14 600 g mol-1 kDa-1, polydispersity indices PDI = 1.10 to 1.25) and tunable cloud point temperatures (Tcp), ranging from 42.4 °C to 80 °C, respectively. Proper combination of hydrophobic end groups with hydrophilic side chains of the polymer allowed to alter the hydrophilicity/hydrophobicity of these polymers, which is shown to enhance Aß1-40 aggregation significantly in case of the endgroup D (with n = 16, 23, 56). We observed that the less hydrophilic polymers (m = 1-2) were able to both decrease and elongate the lag (tlag) and characteristic times (tchar) of Aß1-40 fibril formation in dependence of their end groups, molecular mass and hydrophilicity. On the other hand, highly hydrophilic polymers (m = 3, 5, 9) either decreased, or only marginally influenced the lag and characteristic times of Aß1-40 fibrillation, in all cases forming ß-sheet rich fibrils as observed by TEM and CD-spectroscopy. Our results support that balanced hydrophobic and hydrophilic interactions of a polymer with Aß1-40 is important for inhibiting amyloid-formation pathways.