Roland Winter

Synchrotron X-ray and neutron small-angle scattering of lyotropic lipid mesophases, model biomembranes and proteins in solution at high pressure.

In this review we discuss the use of X-ray and neutron diffraction methods for investigating the temperature- and pressure-dependent structure and phase behaviour of lipid and model biomembrane systems. Hydrostatic pressure has been used as a physical parameter for studying the stability and energetics of lipid mesophases, but also because high pressure is an important feature of certain natural membrane environments and because the high pressure phase behaviour of biomolecules is of importance for several biotechnological processes. Using the pressure jump relaxation technique in combination with time-resolved synchrotron X-ray diffraction, the kinetics of different lipid phase transformations was investigated. The techniques can also be applied to the study of other soft matter and biomolecular phase transformations, such as surfactant phase transitions and protein un/refolding reactions. Several examples are given. In particular, we present data on the pressure-induced unfolding and refolding of small proteins, such as Snase. The data are compared with the corresponding results obtained using other trigger mechanisms and are discussed in the light of recent theoretical approaches.

Origins of life and biochemistry under high-pressure conditions

Life on Earth can be traced back to as far as 3.8 billion years (Ga) ago. The catastrophic meteoritic bombardment ended between 4.2 and 3.9 Ga ago. Therefore, if life emerged, and we know it did, it must have emerged from nothingness in less than 400 million years. The most recent scenarios of Earth accretion predict some very unstable physico-chemical conditions at the surface of Earth, which, in such a short time period, would impede the emergence of life from a proto-biotic soup. A possible alternative would be that life originated in the depth of the proto-ocean of the Hadean Earth, under high hydrostatic pressure. The large body of water would filter harmful radiation and buffer physico-chemical variations, and therefore would provide a more stable radiation-free environment for pre-biotic chemistry. After a short introduction to Earth history, the current tutorial review presents biological and physico-chemical arguments in support of high-pressure origin for life on Earth.

Effect of pressure on membranes

Besides temperature, hydrostatic pressure has been used as a physical-chemical parameter for studying the energetics and phase behavior of membrane systems. First we review some theoretical aspects of lipid self-assembly. Then, the temperature and pressure dependent structure and phase behavior of lipid bilayers, differing in chain configuration, headgroup structure and composition as revealed by using thermodynamic, spectroscopic and scattering experiments is discussed. We also report on the lateral organization of phase-separated lipid membranes and model raft mixtures as well as the influence of peptide and protein incorporation on membrane structure and dynamics upon pressurization. Also the effect of other additives, such as ions, cholesterol, and anaesthetics is discussed. Furthermore, we introduce pressure as a kinetic variable. Applying the pressure-jump relaxation technique in combination with time-resolved synchrotron X-ray diffraction, the kinetics of various lipid phase transformations was investigated. Finally, also new data on pressure effects on membrane mimetics, such as surfactants and microemulsions, are presented.

Effects of pressure-induced membrane phase transitions on inactivation of HorA, an ATP-dependent multidrug resistance transporter, in Lactobacillus plantarum.

ABSTRACT The effects of pressure on cultures of Lactobacillus plantarum were characterized by determination of the viability and activity of HorA, an ATP-binding cassette multidrug resistance transporter. Changes in the membrane composition of L. plantarum induced by different growth temperatures were determined. Furthermore, the effect of the growth temperature of a culture on pressure inactivation at 200 MPa was determined. Cells were characterized by plate counts on selective and nonselective agar after pressure treatment, and HorA activity was measured by ethidium bromide efflux. Fourier transform-infrared spectroscopy and Laurdan fluorescence spectroscopy provided information about the thermodynamic phase state of the cytoplasmic membrane during pressure treatment. A pressure-temperature diagram for cell membranes was established. Cells grown at 37°C and pressure treated at 15°C lost >99% of HorA activity and viable cell counts within 36 and 120 min, respectively. The membranes of these cells were in the gel phase region at ambient pressure. In contrast, cells grown at 15°C and pressure treated at 37°C lost >99% of HorA activity and viable cell counts within 4 and 8 min, respectively. The membranes of these cells were in the liquid crystalline phase region at ambient pressure. The kinetic analysis of inactivation of L. plantarum provided further evidence that inactivation of HorA is a crucial step during pressure-induced cell death. Comparison of the biological findings and the membrane state during pressure treatment led to the conclusion that the inactivation of cells and membrane enzymes strongly depends on the thermodynamic properties of the membrane. Pressure treatment of cells with a liquid crystalline membrane at 0.1 MPa resulted in HorA inactivation and cell death more rapid than those of cells with a gel phase membrane at 0.1 MPa.

Temperature- and pressure-dependent phase behavior of monoacylglycerides monoolein and monoelaidin.

We used x-ray and neutron diffraction to study the temperature- and pressure-dependent structure and phase behavior of the monoacylglycerides 1-monoelaidin (ME) and 1-monoolein (MO) in excess water. The monoacylglycerides were chosen for investigation of their phase behavior because they exhibit mesomorphic phases with one-, two-, and three-dimensional periodicity, such as lamellar, an inverted hexagonal and bicontinuous cubic phases, in a rather easily accessible temperature and pressure range. We studied the structure, stability, and transformations of the different phases over a wide temperature and pressure range, explored the epitaxial relations that exist between different phases, and established a relationship between the chemical structure of the lipid molecules and their phase behavior. For both systems, a temperature-pressure phase diagram has been determined in the temperature range from 0 to 100 degrees C at pressures from ambient up to 1400 bar, and drastic differences in phase behavior are found for the two systems. In MO-water dispersions, the cubic phase Pn3m extends over a large phase field in the T,p-plane. At temperatures above 95 degrees C, the inverted hexagonal phase is found. In the lower temperature region, a crystalline lamellar phase is induced at higher pressures. The phases found in ME-water include the lamellar crystalline Lc phase, the L beta gel phase, the L alpha liquid-crystalline phase, and two cubic phases belonging to the crystallographic space groups Im3m and Pn3m. In addition, the existence of metastable phases has been exploited. Between coexisting metastable cubic structures, a metric relationship has been found that is predicted theoretically on the basis of the curvature elastic energy approximation only.

Effect of hydrostatic pressure on water penetration and rotational dynamics in phospholipid-cholesterol bilayers.

The effect of high hydrostatic pressure on the lipid bilayer hydration, the mean order parameter, and rotational dynamics of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) cholesterol vesicles has been studied by time-resolved fluorescence spectroscopy up to 1500 bar. Whereas the degree of hydration in the lipid headgroup and interfacial region was assessed from fluorescence lifetime data using the probe 1-(4-trimethylammonium-phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), the corresponding information in the upper acyl chain region was estimated from its effect on the fluorescence lifetime of and 3-(diphenylhexatrienyl)propyl-trimethylammonium (TMAP-DPH). The lifetime data indicate a greater level of interfacial hydration for DPPC bilayers than for POPC bilayers, but there is no marked difference in interchain hydration of the two bilayer systems. The addition of cholesterol at levels from 30 to 50 mol% to DPPC has a greater effect on the increase of hydrophobicity in the interfacial region of the bilayer than the application of hydrostatic pressure of several hundred to 1000 bar. Although the same trend is observed in the corresponding system, POPC/30 mol% cholesterol, the observed effects are markedly less pronounced. Whereas the rotational correlation times of the fluorophores decrease in passing the pressure-induced liquid-crystalline to gel phase transition of DPPC, the wobbling diffusion coefficient remains essentially unchanged. The wobbling diffusion constant of the two fluorophores changes markedly upon incorporation of 30 mol% cholesterol, and increases at higher pressures, also in the case of POPC/30 mol% cholesterol. The observed effects are discussed in terms of changes in the rotational characteristics of the fluorophores and the phase-state of the lipid mixture. The results demonstrate the ability of cholesterol to adjust the structural and dynamic properties of membranes composed of different phospholipid components, and to efficiently regulate the motional freedom and hydrophobicity of membranes, so that they can withstand even drastic changes in environmental conditions, such as high external hydrostatic pressure.

Pressure perturbation calorimetric studies of the solvation properties and the thermal unfolding of proteins in solution—experiments and theoretical interpretation

We used pressure perturbation calorimetry (PPC), a relatively new and efficient technique, to study the solvation and volumetric properties of amino acids and peptides as well as of proteins in their native and unfolded state. In PPC, the coefficient of thermal expansion of the partial volume of the protein is deduced from the heat consumed or produced after small isothermal pressure jumps, which strongly depends on the interaction of the protein with the solvent or cosolvent at the protein-solvent interface. Furthermore, the effects of various chaotropic and kosmotropic cosolvents on the volume and expansivity changes of proteins were measured over a wide concentration range with high precision. Depending on the type of cosolvent and its concentration, specific differences were found for the solvation properties and unfolding behaviour of the proteins, and the volume change upon unfolding may even change sign. To yield a molecular interpretation of the different terms contributing to the partial protein volume and its temperature dependence, and hence a better understanding of the PPC data, molecular dynamics computer simulations on SNase were also carried out and compared with the experimental data. The PPC studies introduced aim to obtain more insight into the basic thermodynamic properties of protein solvation and volume effects accompanying structural transformations of proteins in various cosolvents on one hand, as these form the basis for understanding their physiological functions and their use in drug designing and formulations, but also to initiate further valuable applications in studies of other biomolecular and chemical systems.

Inhibiting islet amyloid polypeptide fibril formation by the red wine compound resveratrol.

Grapes for amyloids: The red wine compound resveratrol can effectively inhibit the formation of IAPP amyloid that is found in type II diabetes. Our in vitro inhibition results do not depend on the antioxidant activity of resveratrol. Further, the markedly enhanced cell survival in the presence of resveratrol also indicates that the small oligomeric structures that are observed during β‐sheet formation are not toxic and could be off‐pathway assembly products.

Small‐Molecule Inhibitors of Islet Amyloid Polypeptide Fibril Formation

Newly synthesized proteins in the cell adopt a functional folded state resulting from a highly regulated process. Failure to form this functional state leads to the degradation of proteins inside the cell. However, under certain conditions, some proteins can also adopt an alternative state by the assembly of unfolded or partially folded monomers or protein fragments into a b-sheet structure called amyloid fibril. In spite of arising from diverse amino acid sequences, they have a similar fibrillar structure that binds the dye Congo red. These amyloids are involved in a number of devastating diseases including Alzheimer&s disease, prion diseases, and type-II diabetes mellitus. In the type-II diabetes, deposition of extracellular amyloid plaques in pancreatic beta cells has been observed in humans. Biochemical analysis of the plaques revealed the presence of a 37-residue peptide called islet amyloid polypeptide (IAPP) or amylin, which is co-secreted with insulin. It has a disulfide bond between residues 2–7 and the C-terminus is amidated. IAPP is also known to interact with lipid membranes which are able to induce and foster the fibril formation. Recently, a possible mechanism of IAPP fibril formation at anionic lipid interfaces has been proposed in which it has been shown that IAPP forms b-sheet-rich amyloid fibrils via an intermediate a-helical state. The presence of IAPP amyloid finally leads to the apoptosis of pancreatic beta cells. However, it is still not clear whether the fibrils themselves or their intermediate states are responsible for the cell death. In nature, IAPP amyloid fibril formation can be prevented by altering the primary amino acid sequence, such as in rat IAPP where three proline residues, which are absent in the human IAPP, are thought to prevent the amyloid fibril formation. Recently, Kapurniotu&s group succeeded in the synthesis of conformationally constrained analogues of IAPP, which are methylated at amide bonds and do not fibrillize. Inhibition of amyloid fibril formation is considered to be a potentially key therapeutic approach towards diabetes and other amyloid-related diseases. Surprisingly, very little attempt has been made to inhibit IAPP fibril formation by small-molecule inhibitors. Small-molecule inhibitors have advantages over peptide inhibitors because they could more easily cross the blood brain barrier, avoid immunological response, and are more stable in biological fluids and tissues. In addition, the high flexibility of peptide inhibitors may, for entropic reasons, prevent efficient binding. This problem may be overcome by synthesis of conformationally restricted peptides. The bottleneck in the discovery of small-molecule inhibitors of amyloid fibril formation is the lack of structural information about amyloids. However, this did not prevent the discovery of small-molecule inhibitors for other amyloid fibrils such as, Ab and tau, which are involved in Alzheimer&s disease. In a recent study on a cellular model of tau aggregate inhibition, two rhodanine-scaffold (2-thioxothiazolidin-4one) based inhibitors have been identified which have very low cell toxicity. These compounds were chosen because of the presence of a rhodanine heterocyclic core, which is biocompatible, non-mutagenic, and has a drug-like profile. Inspired by these results, we wanted to explore whether these compounds will also inhibit amyloid fibril formation of IAPP which has a completely different amino acid sequence but shares a similar fibrillar morphology with the tau aggregate. To our knowledge, this is the first study on such smallmolecule inhibitors of IAPP amyloid formation. The compounds 1 and 2 (Figure 1) were synthesized as described earlier. Amyloid fibril formation was carried out in 10 mm sodium phosphate buffer at pH 7.5 for 96 h. To reveal the effect of the two potential inhibitors, different concentrations of the compounds were added to the buffer solution. Fibril formation was quantified by measuring the fluorescence intensity of the amyloid-specific dye thioflavin T (ThT) at a wavelength of 480 nm. The fluorescence intensity of amyloid fibrils increases upon binding to ThT. The efficiency of inhibition was monitored by measuring the ThT fluorescence intensity with respect to that of pure IAPP aggregate without inhibitor (100%). It is evident from Figure 1b that both compounds have a marked inhibitory effect. The concentration at which half of the fibril formation is inhibited (IC50) is 1.23 mm for compound 1, and 0.45 mm for compound 2. A similar trend has been observed for the aggregation of tau, with IC50 values of 0.67 and 0.26 mm for compounds 1 and 2, respectively. From the results on tau and IAPP aggregation it is clear that compound 2 is a more [*] Dr. R. Mishra, D. Sellin, S. Jha, Prof. Dr. R. Winter Faculty of Chemistry Physical Chemistry I—Biophysical Chemistry Technical University Dortmund Otto-Hahn-Strasse 6, 44227 Dortmund (Germany) Fax. (+49)231-755-3901 E-mail: roland.winter@uni-dortmund.de

Pressure-Jump Small-Angle X-Ray Scattering Detected Kinetics of Staphylococcal Nuclease Folding

The kinetics of chain disruption and collapse of staphylococcal nuclease after positive or negative pressure jumps was monitored by real-time small-angle x-ray scattering under pressure. We used this method to probe the overall conformation of the protein by measuring its radius of gyration and pair-distance-distribution function p(r) which are sensitive to the spatial extent and shape of the particle. At all pressures and temperatures tested, the relaxation profiles were well described by a single exponential function. No fast collapse was observed, indicating that the rate limiting step for chain collapse is the same as that for secondary and tertiary structure formation. Whereas refolding at low pressures occurred in a few seconds, at high pressures the relaxation was quite slow, approximately 1 h, due to a large positive activation volume for the rate-limiting step for chain collapse. A large increase in the system volume upon folding implies significant dehydration of the transition state and a high degree of similarity in terms of the packing density between the native and transition states in this system. This study of the time-dependence of the tertiary structure in pressure-induced folding/unfolding reactions demonstrates that novel information about the nature of protein folding transitions and transition states can be obtained from a combination of small-angle x-ray scattering using high intensity synchrotron radiation with the high pressure perturbation technique.