Martin A. Schroer

Intercalation in Layered Metal–Organic Frameworks: Reversible Inclusion of an Extended π-System

We report the synthesis of layered [Zn(2)(bdc)(2)(H(2)O)(2)] and [Cu(2)(bdc)(2)(H(2)O)(2)] (bdc = benzdicarboxylate) metal-organic frameworks (MOF) carried out using the liquid-phase epitaxy approach employing self-assembled monolayer (SAM) modified Au-substrates. We obtain Cu and Zn MOF-2 structures, which have not yet been obtained using conventional, solvothermal synthesis methods. The 2D Cu(2+) dimer paddle wheel planes characteristic for the MOF are found to be strictly planar, with the planes oriented perpendicular to the substrate. Intercalation of an organic dye, DXP, leads to a reversible tilting of the planes, demonstrating the huge potential of these surface-anchored MOFs for the intercalation of large, planar molecules.

Exploring the Piezophilic Behavior of Natural Cosolvent Mixtures

Proteins are only marginally stable and are hence very sensitive to environmental conditions, such as high and low temperatures or high hydrostatic pressures. In nature, living organisms are able to compensate for extreme environmental conditions and hence rescue proteins from denaturation by using osmolytes. Organic osmolytes are accumulated under anhydrobiotic, thermal, and pressure stresses. Among those osmolytes are amino acids, polyols and sugars (e.g., glycerol and trehalose), methylamines such as trimethylamine-Noxide (TMAO), and urea. TMAO has been found to enhance protein folding and ligand binding most efficiently. On the other hand, urea, a highly concentrated waste product in mammalian kidneys, is a perturbant. It is also a major organic osmolyte in marine elasmobranch fishes. Interestingly, TMAO has been found to counteract perturbations imposed by urea and hydrostatic pressure in deep-sea animals, most effectively at a 2:1 urea:TMAO ratio. In the deep sea, hydrostatic pressures up to the 1 kbar (100 MPa) range prevail, and living organisms have to cope with such extreme environmental conditions. High hydrostatic pressure generally destabilizes the protein structure, inhibits polymerization of proteins and ligand binding. Interestingly, TMAO has been shown to largely offset these pressure effects. In fact, it was found that the amount of TMAO in the cells of a series of marine organisms increases linearly with the depth of the ocean. For that reason, TMAO is thought to serve as pressure counteractant. The term “piezolyte” has been coined for such kind of cosolute. About the underlying mechanism of stabilization by TMAO at ambient pressure conditions several experimental and theoretical (molecular dynamics simulations) articles have been published in recent years. TMAO is largely excluded from the protein surface and enhances the water structure causing greater organization through more and stronger hydrogen bonding among water molecules. However, the mechanism of this “chemical chaperon” at high hydrostatic pressure (HHP) conditions is still unclear. To yield a deeper understanding of this phenomenon, we determined the intermolecular interaction of dense protein solutions in the absence and presence of cosolvent mixtures of TMAO and urea also under HHP conditions. Small-angle Xray scattering (SAXS) experiments on dense lysozyme solutions have been carried out in the pressure range from 1 bar up to 4 kbar. The SAXS technique accurately monitors structural alterations of the protein solution and yields quantitative information on the state-dependent protein– protein interaction potential. As lysozyme is a highly stable protein, pressure-induced effects will only be attributed to changes in the protein–protein interaction of the native protein and how this is influenced by osmolytes. No pressureinduced unfolding of the protein occurs in the pressure range covered. Complementary thermodynamic data, that is, the temperature of unfolding and the volume change upon unfolding of the protein, were obtained by differential scanning (DSC) and pressure perturbation calorimetry (PPC), respectively. To verify that the protein is folded at all solution conditions studied, SAXS measurements on diluted lysozyme solutions (cP= 10 mgmL ) were carried out in the whole pressure range covered. For diluted protein solutions, the scattering intensity I(q) is proportional to the form factor P(q) (q= (4p/l)sin(V/2) is the wave vector transfer, l the wavelength of the X-rays, and V the scattering angle), which depends on the structure and size of the protein. For dilute lysozyme solutions, the radius of gyration of the particle, Rg, could be determined. We found a constant Rg value of (15.1 0.4) up to 4 kbar, indicating the absence of unfolding even at the highest pressure applied. In the case of concentrated protein solutions, the interaction between the particles gives rise to an additional scattering contribution. This SAXS signal can be described as the product of the form factor and an effective structure factor, which is related to the intermolecular structure factor S(q). To relate the structure factor to the protein–protein interaction potential, statistical mechanical model approaches have to be employed. Here, the mean-spherical approximation (MSA) in combination with the DLVO (Derjaguin– Landau–Verwey–Overbeek) potential V(r) has been used. The pair potential V(r) is given as the sum of a hard sphere potential VHS(r), a repulsive screened Coulomb-like potential VSC(r) and an attractive Yukawian potential VY(r), which is frequently used to describe protein–protein interactions (for details, see the Supporting Information). [*] Y. Zhai, Prof. Dr. R. Winter Fakult t Chemie, TU Dortmund Physikalische Chemie—Biophysikalische Chemie Otto-Hahn Str. 6, 44227 Dortmund (Germany) E-mail: roland.winter@tu-dortmund.de

Unique Features of the Folding Landscape of a Repeat Protein Revealed by Pressure Perturbation

The volumetric properties of proteins yield information about the changes in packing and hydration between various states along the folding reaction coordinate and are also intimately linked to the energetics and dynamics of these conformations. These volumetric characteristics can be accessed via pressure perturbation methods. In this work, we report high-pressure unfolding studies of the ankyrin domain of the Notch receptor (Nank1-7) using fluorescence, small-angle x-ray scattering, and Fourier transform infrared spectroscopy. Both equilibrium and pressure-jump kinetic fluorescence experiments were consistent with a simple two-state folding/unfolding transition under pressure, with a rather small volume change for unfolding compared to proteins of similar molecular weight. High-pressure fluorescence, Fourier transform infrared spectroscopy, and small-angle x-ray scattering measurements revealed that increasing urea over a very small range leads to a more expanded pressure unfolded state with a significant decrease in helical content. These observations underscore the conformational diversity of the unfolded-state basin. The temperature dependence of pressure-jump fluorescence relaxation measurements demonstrated that at low temperatures, the folding transition state ensemble (TSE) lies close in volume to the folded state, consistent with significant dehydration at the barrier. In contrast, the thermal expansivity of the TSE was found to be equivalent to that of the unfolded state, indicating that the interactions that constrain the folded-state thermal expansivity have not been established at the folding barrier. This behavior reveals a high degree of plasticity of the TSE of Nank1-7.

High-Pressure SAXS Study of Folded and Unfolded Ensembles of Proteins

A structural interpretation of the thermodynamic stability of proteins requires an understanding of the structural properties of the unfolded state. High-pressure small-angle x-ray scattering was used to measure the effects of temperature, pressure, denaturants, and stabilizing osmolytes on the radii of gyration of folded and unfolded state ensembles of staphylococcal nuclease. A set of variants with the internal Val-66 replaced with Ala, Tyr, or Arg was used to examine how changes in the volume and polarity of an internal microcavity affect the dimensions of the native state and the pressure sensitivity of the ensemble. The unfolded state ensembles achieved for these proteins with high pressure were more compact than those achieved at high temperature, and were all very sensitive to the presence of urea and glycerol. Substitutions at the hydrophobic core detectably altered the conformation of the protein, even in the folded state. The introduction of a charged residue, such as Arg, inside the hydrophobic interior of a protein could dramatically alter the structural properties, even those of the unfolded state. The data suggest that a charge at an internal position can interfere with the formation of transient hydrophobic clusters in the unfolded state, and ensure that the pressure-unfolded form of a protein occupies the maximum volume possible. Only at high temperatures does the radius of gyration of the unfolded state ensemble approach the value for a statistical random coil.

The Effect of Ionic Strength, Temperature, and Pressure on the Interaction Potential of Dense Protein Solutions: From Nonlinear Pressure Response to Protein Crystallization

Understanding the intermolecular interaction potential, V(r), of proteins under the influence of temperature, pressure, and salt concentration is essential for understanding protein aggregation, crystallization, and protein phase behavior in general. Here, we report small-angle x-ray scattering studies on dense lysozyme solutions of high ionic strength as a function of temperature and pressure. We show that the interaction potential changes in a nonlinear fashion over a wide range of temperatures, salt, and protein concentrations. Neither temperature nor protein and salt concentration lead to marked changes in the pressure dependence of V(r), indicating that changes of the water structure dominate the pressure dependence of the intermolecular forces. Furthermore, by analysis of the temperature, pressure, and ionic strength dependence of the normalized second virial coefficient, b2, we show that the interaction can be fine-tuned by pressure, which can be used to optimize b2 values for controlled protein crystallization.

Concentration dependent effects of urea binding to poly(N-isopropylacrylamide) brushes: a combined experimental and numerical study

The binding effects of osmolytes on the conformational behavior of grafted polymers are studied in this work. In particular, we focus on the interactions between urea and poly(N-isopropylacrylamide) (PNIPAM) brushes by monitoring the ellipsometric brush thickness for varying urea concentrations over a broad temperature range. The interpretation of the obtained data is supported by atomistic molecular dynamics simulations, which provide detailed insights into the experimentally observed concentration-dependent effects on PNIPAM-urea interaction. In particular, in the low concentration regime (cu ≤ 0.5 mol L(-1)) a preferential exclusion of urea from PNIPAM chains is observed, while in the high concentration regime (2 ≤ cu ≤ 7 mol L(-1)) a preferential binding of the osmolyte to the polymer surface is found. In both regimes, the volume phase transition temperature (Ttr) decreases with increasing urea concentration. This phenomenon derives from two different effects depending on urea concentration: (i) for cu ≤ 0.5 mol L(-1), the decrease of Ttr is explained by a decrease of the chemical potential of bulk water in the surrounding aqueous phase; (ii) for cu ≥ 2 mol L(-1), the lower Ttr is explained by the favorable replacement of water molecules by urea, which can be regarded as a cross-linker between adjacent PNIPAM chains. Significant effects of the concentration-dependent urea binding on the brush conformation are noticed: at cu = 0.5 mol L(-1), although urea is loosely embedded between the hydrated polymer chains, it enhances the brush swelling by excluded volume effects. Beyond 0.5 mol L(-1), the stronger interaction between PNIPAM and urea reduces the chain hydration, which in combination with cross-linking of monomer units induces the shrinkage of the polymer brush.

Structural Plasticity of Staphylococcal Nuclease Probed by Perturbation with Pressure and pH

The ionization of internal groups in proteins can trigger conformational change. Despite this being the structural basis of most biological energy transduction, these processes are poorly understood. Small angle X‐ray scattering (SAXS) and nuclear magnetic resonance (NMR) spectroscopy experiments at ambient and high hydrostatic pressure were used to examine how the presence and ionization of Lys‐66, buried in the hydrophobic core of a stabilized variant of staphylococcal nuclease, affect conformation and dynamics. NMR spectroscopy at atmospheric pressure showed previously that the neutral Lys‐66 affects slow conformational fluctuations globally, whereas the effects of the charged form are localized to the region immediately surrounding position 66. Ab initio models from SAXS data suggest that when Lys‐66 is charged the protein expands, which is consistent with results from NMR spectroscopy. The application of moderate pressure (<2 kbar) at pH values where Lys‐66 is normally neutral at ambient pressure left most of the structure unperturbed but produced significant nonlinear changes in chemical shifts in the helix where Lys‐66 is located. Above 2 kbar pressure at these pH values the protein with Lys‐66 unfolded cooperatively adopting a relatively compact, albeit random structure according to Kratky analysis of the SAXS data. In contrast, at low pH and high pressure the unfolded state of the variant with Lys‐66 is more expanded than that of the reference protein. The combined global and local view of the structural reorganization triggered by ionization of the internal Lys‐66 reveals more detectable changes than were previously suggested by NMR spectroscopy at ambient pressure. Proteins 2011.

Sequential single shot X-ray photon correlation spectroscopy at the SACLA free electron laser

Hard X-ray free electron lasers allow for the first time to access dynamics of condensed matter samples ranging from femtoseconds to several hundred seconds. In particular, the exceptional large transverse coherence of the X-ray pulses and the high time-averaged flux promises to reach time and length scales that have not been accessible up to now with storage ring based sources. However, due to the fluctuations originating from the stochastic nature of the self-amplified spontaneous emission (SASE) process the application of well established techniques such as X-ray photon correlation spectroscopy (XPCS) is challenging. Here we demonstrate a single-shot based sequential XPCS study on a colloidal suspension with a relaxation time comparable to the SACLA free-electron laser pulse repetition rate. High quality correlation functions could be extracted without any indications for sample damage. This opens the way for systematic sequential XPCS experiments at FEL sources.

Colloidal crystallite suspensions studied by high pressure small angle x-ray scattering

We report on high pressure small angle x-ray scattering on suspensions of colloidal crystallites in water. The crystallites made out of charge-stabilized poly-acrylate particles exhibit a complex pressure dependence which is based on the specific pressure properties of the suspending medium water. The dominant effect is a compression of the crystallites caused by the compression of the water. In addition, we find indications that also the electrostatic properties of the system, i.e. the particle charge and the dissociation of ions, might play a role for the pressure dependence of the samples. The data further suggest that crystallites in a metastable state induced by shear-induced melting can relax to a similar structural state upon the application of pressure and dilution with water. X-ray cross correlation analysis of the two-dimensional scattering patterns indicates a pressure-dependent increase of the orientational order of the crystallites correlated with growth of these in the suspension. This study underlines the potential of pressure as a very relevant parameter to understand colloidal crystallite systems in aqueous suspension.

Adsorption of nanoparticles at the solid-liquid interface.

The adsorption of differently charged nanoparticles at liquid-solid interfaces was investigated by in situ X-ray reflectivity measurements. The layer formation of positively charged maghemite (γ-Fe(2)O(3)) nanoparticles at the aqueous solution-SiO(2) interface was observed while negatively charged gold nanoparticles show no adsorption at this interface. Thus, the electrostatic interaction between the particles and the charged surface was determined as the driving force for the adsorption process. The data analysis shows that a logarithmic particle size distribution describes the density profile of the thin adsorbed maghemite layer. The size distribution in the nanoparticle solution determined by small angle X-ray scattering shows an average particle size which is similar to that found for the adsorbed film. The formed magehemite film exhibits a rather high stability.