Matthias J. N. Junk

Use of X-Ray Diffraction, Molecular Simulations, and Spectroscopy to Determine the Molecular Packing in a Polymer-Fullerene Bimolecular Crystal

The molecular packing in a polymer: fullerene bimolecular crystal is determined using X-ray diffraction (XRD), molecular mechanics (MM) and molecular dynamics (MD) simulations, 2D solid-state NMR spectroscopy, and IR absorption spectroscopy. The conformation of the electron-donating polymer is significantly disrupted by the incorporation of the electron-accepting fullerene molecules, which introduce twists and bends along the polymer backbone and 1D electron-conducting fullerene channels.

EPR Spectroscopic Characterization of Local Nanoscopic Heterogeneities during the Thermal Collapse of Thermoresponsive Dendronized Polymers

Despite these efforts,a molecular-scale picture of what happens when thermores-ponsive polymers start to dehydrate at a certain temperature,subsequently collapse, and then assemble to mesoglobules,does not exist. This absence severely hampers rationalmaterials design.In an exploratory research effort aimed at detectingunusual properties of dendronized polymers,

Effect of ionic liquids on the solution structure of human serum albumin.

The effect of several ionic liquids (ILs) on the solution structure of human serum albumin (HSA) is revealed by continuous wave electron paramagnetic resonance (EPR) spectroscopy and nanoscale distance measurements with double electron-electron resonance (DEER) spectroscopy. HSA, the most abundant protein in human blood, is able to bind and transport multiple fatty acids (FAs). Using spin-labeled FA, the uptake of the FA by the protein and their spatial distribution in the protein can be monitored. The FA distribution provides an indirect yet effective way to characterize the structure of the protein in solution. Addition of imidazolium-based ILs to an aqueous solution of HSA/FA conjugates is accompanied by significant destabilization and unfolding of the proteins tertiary structure. In contrast, HSA maintains its tertiary structure when choline dihydrogenphosphate (dhp) is added. The comparison of FA distance distributions in HSA with and without choline dhp surprisingly revealed that with this IL, the FA anchoring units are in better agreement with the crystallographic data. Furthermore, the FA entry point distribution appears widened and more asymmetric than in pure buffer. These results indicate that choline dhp as a cosolvent may selectively stabilize HSA conformations closer to the crystal structure out of the overall conformational ensemble.

EPR spectroscopy reveals nanoinhomogeneities in the structure and reactivity of thermoresponsive hydrogels.

The dynamic and chemical behavior of solute molecules inside new thermoresponsive hydrogels (photocrosslinked poly(N-isopropylacrylamide) (pNiPAAm) copolymers) is studied by continuous-wave electron paramagnetic resonance spectroscopy. Via addition of paramagnetic tracer molecules (so-called spin probes) a picture is obtained of the thermally induced collapse on the molecular scale, which proceeds over a substantially broader temperature range than indicated by the sharp macroscopic volume transition. The sampling of hydrophilic and hydrophobic environments suggests a discontinuous collapse mechanism with a coexistence of collapsed and expanded network regions. These structural inhomogeneities on the nanoscale also lead to an inhomogeneity in chemical reactivity. The hydrophilic regions form nanoreactors, which strongly accelerate the reaction while the hydrophobic regions act as nanoshelters, in which enclosed spin probes are protected from the decay. The results show that the system consisting of a statistical binary or tertiary copolymer displays remarkably complex behavior that mimics spatial and chemical inhomogeneities observed in functional biopolymers such as enzymes.

Formation of a Mesoscopic Skin Barrier in Mesoglobules of Thermoresponsive Polymers

With the combination of molecular scale information from electron paramagnetic resonance (EPR) spectroscopy and meso-/macroscopic information from various other characterization techniques, the formation of mesoglobules of thermoresponsive dendronized polymers is explained. Apparent differences in the EPR spectra in dependence of the heating rate, the chemical nature of the dendritic substructure of the polymer, and the concentration are interpreted to be caused by the formation of a dense polymeric layer at the periphery of the mesoglobule. This skin barrier is formed in a narrow temperature range of ~4 K above T(C) and prohibits the release of molecules that are incorporated in the polymer aggregate. In large mesoglobules, formed at low heating rates and at high polymer concentrations, a considerable amount of water is entrapped that microphase-separates from the collapsed polymer chains at high temperatures. This results in the aggregates possessing an aqueous core and a corona consisting of collapsed polymer chains. A fast heating rate, a low polymer concentration, and hydrophobic subunits in the dendritic polymer side chains make the entrapment of water less favorable and lead to a higher degree of vitrification. This may bear consequences for the design and use of thermoresponsive polymeric systems in the fast growing field of drug delivery.

Molecular pockets derived from cholic acid as chemosensors for metal ions.

Molecular pockets in the form of a tripod made of cholic acid were found to be able to solubilize pyrene in polar media as a result of the facial amphiphilicity of bile acids. The trimer containing 1,2,3-triazole groups can complex with heavy metal ions, as clearly shown by electron paramagnetic resonance spectroscopy. Both the metal cation and the pyrene molecule can be brought close together in the cavity formed by the cholic acid trimer, resulting in significantly improved efficiency for fluorescence quenching of pyrene. The decrease of fluorescence intensity can be used for the detection of heavy metal ions, and the detection limit is about 1 microM in water, suggesting the usefulness of such molecules as chemosensors for such metal ions. A different trimer without the coordinating triazole groups is shown to shield pyrene away from metal ions, causing a much reduced fluorescence quenching.

DEER in biological multispin-systems: A case study on the fatty acid binding to human serum albumin

In this study, self-assembled systems of human serum albumin (HSA) and spin-labeled fatty acids are characterized by double electron-electron resonance (DEER). HSA, being the most important transport protein of the human blood, is capable to host up to seven paramagnetic fatty acid derivatives. DEER measurements of these self-assembled multispin clusters are strongly affected by correlations of more than two spins, the evaluation of the latter constituting the central topic of this paper. While the DEER modulation depth can be used to obtain qualitative information of the number of coupled spins, the quantitative analysis is hampered by the occurrence of cluster mixtures with different numbers of coupled spins and contributions from unbound spin-labeled material. Applying flip angle dependent DEER measurements, unwanted multispin correlations were found to lead not only to a broadening of the distance peaks but also to cause small distances to be overestimated and large distances to be suppressed. It is thus favorable to use spin-diluted systems with an average of two paramagnetic molecules per spin cluster when a quantitative analysis of the distance distribution is sought.

Atomic Force Spectroscopy of Thermoresponsive Photo-Cross-Linked Hydrogel Films

Responsive hydrogel thin films are interesting materials as responsive adhesives or as an active matrix in actuators and sensing applications, and thus, knowledge about their structural and micromechanical properties is of high relevance. Using atomic force spectroscopy, temperature-induced structural and adhesive changes of thermoresponsive hydrogel layers with micrometer thickness based on photo-cross-linked N-isopropylacrylamide (NiPAAm) were investigated in the temperature range of 18-50 degrees C. Grafted onto flat surfaces, these hydrogel layers are restricted to a highly anisotropic swelling and deswelling predominantly perpendicular to the substrate surface, which was monitored and evaluated by force spectroscopy during vertical tip approach and retraction. Analyses of the tip penetration depth yielded quantitative information about the degree of swelling. As a second feature, the critical temperature was found to decrease with increasing cross-linking density. Temperature-dependent measurements with hydrophobic and hydrophilic atomic force microscopy (AFM) tips revealed a strong adhesion to the hydrogel layer in the swollen state, which was reduced upon the layer volume collapse. These observations on the micrometer-thick gel network layers are in contrast to previous reports on ultrathin pNiPAAm brushes and monolayers, which show no adhesion in the swollen state but only in the collapsed state. Furthermore, it was found that the hydrophobicity of the hydrogel probed with a hydrophobic tip continuously increases with temperature over a broad range of at least 30 K.

Nanoscale Inhomogeneities in Thermoresponsive Polymers

This article highlights the occurrence and nature of nanoscale inhomogeneities in thermoresponsive polymers and focuses on different experimental techniques for their observation and characterization. Such inhomogeneities can be regarded as nanoscopic domains of collapsed polymer segments (or of a small number of unimers), which provide a nonpolar, hydrophobic interior. Continuous wave (CW) electron paramagnetic resonance (EPR) spectroscopy on amphiphilic reporter molecules (spin probes) as an intrinsically local technique is particularly emphasized. In combination with different ensemble-averaging methods, it provides a holistic understanding of the often inhomogeneous nanoscale processes during the temperature-induced collapse of a thermoresponsive polymer.

Structural Insight into Proteorhodopsin Oligomers

Oligomerization has important functional implications for many membrane proteins. However, obtaining structural insight into oligomeric assemblies is challenging, as they are large and resist crystallization. We focus on proteorhodopsin (PR), a protein with seven transmembrane α-helices that was found to assemble to hexamers in densely packed lipid membrane, or detergent-solubilized environments. Yet, the structural organization and the subunit interface of these PR oligomers were unknown. We used site-directed spin-labeling together with electron spin-resonance lineshape and Overhauser dynamic nuclear polarization analysis to construct a model for the specific orientation of PR subunits within the hexameric complex. We found intersubunit distances to average 16 Å between neighboring 55 residues and that residues 177 are >20 Å apart from each other. These distance constraints show that PR has a defined and radial orientation within a hexamer, with the 55-site of the A-B loop facing the hexamer core and the 177-site of the E-F loop facing the hexamer exterior. Dynamic nuclear polarization measurements of the local solvent dynamics complement the electron spin-resonance-based distance analysis, by resolving whether protein surfaces at positions 55, 58, and 177 are exposed to solvent, or covered by protein-protein or protein-detergent contacts.