Low Hydration Levels Research Articles

Helical structure and dynamics in membrane polypeptides.

Direct determination of the conformational properties of polypeptides and proteins reconstituted in phospholipid bilayer membranes is not easily achieved, since the main high-resolution structural methods (X-ray crystallography and high-resolution NMR spectroscopy) are rarely applicable to membrane systems. It is expected that solid-state NMR methods will make increasing contributions in this area [l], although some methods of sample preparation (e.g. oriented bilayers obtainable only at low hydration levels) can perturb the structure and orientation of the membrane-bound polypeptides. This is particularly the case for ion channel and membranelytic peptides whose amino acid composition and structural properties result in membrane-bound forms ‘poised’ between surface and membraneinserted states [2-41. In the light of these difficulties, extraction of the (normally helical) polypeptide into an ‘isotropic’ environment (a non-aqueous solution or detergent micelle) suitable for high-resolution NMR spectroscopy is often used to obtain structural and dynamic information [5]. Such studies are undertaken with the implicit assumption that the polypeptide has ‘intrinsic structural properties’ that can be assessed in the environment used to substitute for the membrane. Several questions follow that are the subject of this brief review. First, to what extent can intrinsic conformational properties really be defined and second, to what extent are they relevant for the membrane-bound polypeptide? Third, if a polypeptide has intrinsic (helical) properties, can these be predicted either by simulation or by comparison with semi-empirical methods for defining sequence-dependent helical propensities. The latter point is of interest in relation to the ‘two-stage model’ for membrane protein folding proposed by Popot and Engelman [6], in which the transmembrane helices of membrane proteind are proposed to be autonomously stable in a bilayer environment. If the limits of stable hydrogen bonding in transmembrane helices can be defined accurately, either by prediction from their sequences, by definition of ‘intrinsic’ helical

Synthesis of cross-linked polymers by reaction of sucrose and diepoxide monomers: Characterization and nuclear magnetic resonance study

The synthesis of a new cross-linked structure, with sucrose and diepoxide monomers, possessing the properties of a highly swellable hydrogel polymer is reported. Characterization studies of its structure and water effects were carried out in dried and hydrated samples by CP-MAS 13C n.m.r., by wide-band 1H and 2H n.m.r. and by differential thermal analysis. The results indicate that the sample is completely amorphous and disordered, that methyl groups are present and that there is strong water structuring due to hydrogen bonding, with an absence of free water at low levels of hydration. Two processes are manifest in the temperature dependence of the 2H line width.

The hydration structure studied by TSDC. Myoglobin and haemoglobin

The coordination scheme of water in myoglobin and haemoglobin, the two oxygen carrier proteins in vertebrates, is still far from being fully understood. The TSDC (thermally stimulated depolarization currents) technique is a sensitive tool to study water molecules bound to biological macromolecules and characterized by mobility and order degree, quite different from those of bulk water. TSDC spectra for both proteins were recorded in the temperature range 100 to 300 K, at very low hydration levels (h=0.01 to 0.62) obtained with different procedures. Different classes of water molecules were found: the preferred hydration sites and the water dipole activation energies are discussed. In particular, the existence of a critical hydration level which induces a partially irreversible structural transition was monitored in myoglobin. The technique allowed estimation of the average number of hydrogen bonds established by the water molecules in the inner cages of haemoglobin at different hydration levels.

Dextran hydrogels for the controlled release of proteins

The release of proteins from non-degrading and enzymatically degrading dextran hydrogels was investigated. These dextran hydrogels were obtained by radical polymerization of aqueous solutions of glycidyl methacrylate derivatized dextran (dex-GMA). It was shown by FTIR analysis that by a proper selection of the polymerization conditions, hydrogels could be obtained in which more than 95% of the methacrylate groups had reacted within 20 min. The release of three model proteins (lysozyme, albumin, IgG) from non-degrading dextran gels with a varying initial water content and crosslink density was studied. For gels with a high equilibrium water content, the amount of protein released was proportional to the square root of time. The diffusion of proteins in these highly hydrated gels could be effectively described by the free volume theory. On the other hand, the release of the proteins from hydrogels with a low hydration level was marginal and did not follow the free volume theory, indicating that in these gels screening occurred. Dextran hydrogels can be made degradable by coencapsulation of dextranase. The degradation rate could be established by determination of reducing oligosaccharides and was dependent on the amount of dextranase in the gel. The release of a model protein (IgG) from degrading dextran hydrogels was investigated as a function of the amount of dextranase present in the gel and the gel characteristics. In the absence of dextranase no significant release of IgG from the hydrogels occurred, indicating that the protein diameter is larger than the hydrogel mesh size. However, in the presence of dextranase the release of IgG from the gels abruptly increased after a certain time when the enzyme had degraded the hydrogel to a certain extent.

The phase morphology and crystal growth habits of DNA fractions. Optical microscopy and light scattering

Polydisperse DNA of reasonable molecular weights was prepared from a mammalian source via sonication and fractionation. A method for characterizing the molecular weight using gel electrophoresis is described. Quiescent crystallization was studied in thin films of one of the fractions induced by rapidly changing the hydration state isothermally. We report the occurrence of the semicrystalline nature of DNA. The crystal growth occurring via aggregates is best described as sheaves and spherulites from DNA gels in the relative humidity range (RH) corresponding to A-DNA. These habits exhibit primary nucleation and secondary growth, which closely resemble those of melt-crystallized, synthetic macromolecules and, in a follow-up report, will be shown to be lamellar in nature. Small, needle-like crystals are observed for B-DNA hydration levels, and are unstable at lower hydration levels. A transformation from needle to lamellar crystals can occur, even when the primary nucleation of lamellar forms is otherwise absent at that hydration level, through a cylindrical phase exhibiting selective reflection of colored bands. The hydration level plays, in part, the role of the supercooling in this system and the long-known hysteresis in the formation and dissolution of the A-DNA (crystals) can now be viewed in light of those factors known to operate in semicrystalline systems. A morphological phase diagram is developed and is in accord with the known physical evidence. Because this preparation and these morphological observations are without precedence, substantial detail into methodology is included for this first article in the series.

Molecular dynamics simulation evidence of anomalous diffusion of protein hydration water.

The mean square displacement of protein hydration water has been found to increase nonlinearly in time, as observed for long times, by molecular dynamics simulations at low hydration levels and in proximity of the protein surface at full hydration. While such an anomalous diffusion is traced back to the more general properties of disordered media, some caution in the use of the self-diffusion coefficient $D$ to characterize the water dynamics in these systems is suggested.

Gas Phase Reactions of Hydrated Halides with Chlorine

A selected ion flow tube (SIFT) equipped with a supersonic expansion ion source was used to study the reactions of hydrated halides X-(D2O)n (X = F, n ≤ 6; X = Cl, n ≤ 8; X = Br, n ≤ 16; X = I, n ≤ 13) with Cl2 at temperatures ranging from 140 to 400 K. The bare halide reactions were observed to produce Cl- with rate constants ranging from one-third to two-thirds the collision rate. At low hydration levels the reactions proceeded at nearly the collision rate via a ligand switching mechanism, displacing one or more water molecules to produce a trihalide (i.e., XCl2-(D2O)m + (n − m)D2O where m < n). The rate constants decreased for the fluoride and chloride clusters with each increase in hydration level at n ≥ 4 and n ≥ 6, respectively. The bromide and iodide clusters continued to react at nearly the collision rate over the entire range of hydration levels studied. The implications these results have on the plausibility of halide surface reactions occurring on atmospheric aerosols and the structures of hali...

A Solid-State 2H NMR Investigation of Purine Motion in a 12-Base-Pair RNA Duplex

Solid-state 2H NMR spectroscopy has been used to investigate the base dynamics of a RNA oligonucleotide with a defined sequence, [r(CGCGAAUUCGCG)] 2, which contains the RNA analogue of the EcoRI binding site. The C8 protons of all purines in the self-complementary dodecamer were exchanged for deuterons. The quadrupole-echo lineshapes and spin-lattice relaxation times as a function of hydration for the sample in the form of the Na salt have previously been reported. In that study the 2H NMR lineshapes and T 1 values of [r(CG*CG*A*A*UUCG*CG*)] 2 were compared with those of the analogously labeled DNA sequence, [d(CG*CG*A*A*TTCG*CG*)](2) (Wang et al., J.Am. Chem. Soc. 114, 6583, 1992). It was concluded that the amplitudes of purine motion for DNA and RNA are similar at all hydration levels; however, the rate difference observed at low-hydration levels may or may not persist at high hydration. Here the internal motions of the purine bases in the RNA oligomer have been thoroughly investigated. Three models were used to simulate the motion: (1) two-site jump, (2) diffusion in a cone, and (3) restricted diffusion on the surface of a cone. The purine motion is best simulated by the restricted-diffusion on a cone model with an amplitude of ±9.5° and a rate between 8.0 × 10 6 rad/s at 90% RH and 8.4 × 10 8 rad/s at 0% RH. This small amplitude and fast rate of purine motion for RNA are similar to previous results obtained for DNA purines.

Thermotropic phase properties of 1,2-di-O-tetradecyl-3-O-(3-O-methyl- beta-D-glucopyranosyl)-sn-glycerol

The hydration properties and the phase structure of 1,2-di-O-tetradecyl-3-O(3-O-methyl-beta-D-glucopyranosyl)-sn-glycerol (3-O-Me-beta-D-GlcDAIG) in water have been studied via differential scanning calorimetry, 1H-NMR and 2H-NMR spectroscopy, and x-ray diffraction. Results indicate that this lipid forms a crystalline (Lc) phase up to temperatures of 60-70 degrees C, where a transition through a metastable reversed hexagonal (Hll) phase to a reversed micellar solution (L2) phase occurs. Experiments were carried out at water concentrations in a range from 0 to 35 wt%, which indicate that all phases are poorly hydrated, taking up < 5 mol water/mol lipid. The absence of a lamellar liquid crystalline (L alpha) phase and the low levels of hydration measured in the discernible phases suggest that the methylation of the saccharide moiety alters the hydrogen bonding properties of the headgroup in such a way that the 3-O-Me-beta-D-GlcDAIG headgroup cannot achieve the same level of hydration as the unmethylated form. Thus, in spite of the small increase in steric bulk resulting from methylation, there is an increase in the tendency of 3-O-Me-beta-D-GlcDAIG to form nonlamellar structures. A similar phase behavior has previously been observed for the Acholeplasma laidlawii A membrane lipid 1,2-diacyl-3-O-(6-O-acyl-alpha-D-glucopyranosyl)-sn-glycerol in water (Lindblom et al. 1993. J. Biol. Chem. 268:16198-16207). The phase behavior of the two lipids suggests that hydrophobic substitution of a hydroxyl group in the sugar ring of the glucopyranosylglycerols has a very strong effect on their physicochemical properties, i.e., headgroup hydration and the formation of different lipid aggregate structures.

The amplitude of local angular motion of purines in DNA in solution.

Nuclear magnetic resonance and optical experiments are combined to determine the rms amplitude of local angular motion of purines in DNA in solution. A 12 base-pair duplex DNA with the sequence d(CGCGAATTCGCG)2 is deuterated at the H8 positions of adenine and guanine by exchange with solvent at 55 degrees C. The deuterium nmr spectrum of this DNA is measured at 30 mg/mL at 30 degrees C in an 11.76 Tesla magnet (76.75 MHz). The time-resolved fluorescence polarization anisotropies (FPA) of this same sample and also a greatly diluted sample (0.215 mg/mL) were measured after addition of ethidium. FPA measurements of the dilute sample yield the hydrodynamic radius, RH = 9.94 +/- 0.2 A, while those at the nmr concentration are employed to characterize the collective motions in terms of either an enhanced viscosity or dimer formation. The rms amplitude of local angular motion was determined by analyzing the 2H-nmr spectrum, in particular the line width, using recently developed theory for the transverse relaxation rate (RQ2) together with essential information about the collective motions from these and other optical studies. When the principal-axis frame of the electric field gradient tensor is assumed to undergo overdamped libration around each of its three body-fixed axes in an isotropic deflection potential, then the rms amplitude of local angular motion around any single axis is found to lie in the range 10 degrees-11 degrees, provided the high DNA concentration acts to enhance the viscosity, and is about 9 degrees-11 degrees, if it acts to produce end-to-end dimers. The proton nmr relaxation data of Eimer et al. are reanalyzed and shown to yield an rms amplitude of angular motion of the cytosine H5-H6 internuclear vector of 9 degrees-10 degrees, depending upon its orientation with respect to the helix axis. In all of these analyses, full account is taken of the collective twisting and bending deformations, which have a small but significant effect on the results. It is shown that the rms amplitudes of local angular motion do not depend strongly on the model (potential), provided that isotropic rotation around the same number of axes is allowed and that one compares rms angles of the same dimensionality. The rms amplitudes of local angular motion in solution are comparable to those observed for the same sequence at low levels of hydration in the solid state.

The hydration of proteins in nearly anhydrous organic solvent suspensions.

Water sorption isotherms at 27 degrees C have been measured for lysozyme and chymotrypsin in suspensions of toluene, di(n-butyl) ether, n-propanol, and a solution of 1M n-propanol in benzene. Sorption isotherms for the different suspensions are compared by converting solvent water content to the thermodynamic activity of water in each solvent. The sorption behavior is also compared to that for the two proteins hydrated from the vapor phase. At low water activities, all sorption isotherms are similar when compared on the basis of water activity. However, at higher activities, water sorption by the proteins in the organic suspensions is suppressed relative to the sorption of water vapor. The greatest suppression is observed for n-propanol, which suggests that the suppression may be due to a competition for water-binding sites on the protein by the organic solvent. Sorption isotherms at low water activities have also been predicted using a thermodynamic model in which it is assumed that water binds selectively to the ionizable residues on the surface of the protein. A comparison of predicted and measured sorption isotherms shows that the model can provide reasonable estimates of water sorption in nonpolar or moderately polar organic solvent suspensions at low levels of hydration.

Hydration and protein dynamics: an ESR and ST-ESR spin labelling study of human serum albumin

Human serum albumin has been studied at low hydration level by the ESR spin labelling technique, under the assumption that a covalently bound spin-label is a reporter of the protein internal dynamics. At room temperature, the presence of a double component signal allowed us to monitor the influence of increasing hydration level on internal protein dynamics as well as on the superficial water dynamics. The ESR results have shown that the first 20 g of water per 100 g of protein activate the internal protein dynamics and that superficial water dynamics starts at higher hydration values. ESR experiments at low temperature have shown that at −160°C ≲T≲−80°C, the label experiences an increasing environmental polarity with increasing temperature in the samples with hydration values higher than about 20 g of water per 100 g of protein. The results are discussed in connection with both conformational substates of the protein and hydration water dynamics.

13C-CP/MAS NMR studies of the cyclomalto-oligosaccharide (cyclodextrin) hydrates

The 13C-CP/MAS NMR spectra of cyclomaltohexaose (α-cyclodextrin) hexahydrate, cyclomalto-heptaose (β-cyclodextrin) “undecahydrate”, cyclomalto-octaose (ψ-cyclodextrin) “octadecahydrate”, and of the same materials at lower levels of hydration are compared with solution NMR data, structures obtained from single crystal diffraction studies, and with previous reports of the 13C-CP/MAS NMR spectra. The chemical shifts of the C-1 and C-4 resonances can be correlated with the conformation about the (1 → 4) linkage. The chemical shifts of the C-6 resonances are also sensitive to hydrogen-bonding interactions, as shown by the spectral changes on loss of water from the structures. The results suggest that, for resonances of carbon atoms close to a centre of significant conformational change, chemical shifts may be predicted on the basis of conformation alone, but for the resonances of more distant atoms, changes in chemical shift due to conformational change may be masked by the effects of alterations in the local environment.

Structural Investigations of Human Stratum Corneum by Small-Angle X-Ray Scattering

The structure of human stratum corneum was investigated with small-angle X-ray scattering (SAXS). At room temperature the scattering curve was characterized by a strong intensity at low scattering vector (Q less than 0.8 nm-1) and two complicated diffraction peaks originating from a lamellar structure of the lipids. The lamellar lipid structure in the stratum corneum transformed to a disordered structure between 65 degrees C and 75 degrees C, the same temperature region at which a thermal lipid transition occurred. After cooling down to room temperature a recrystallization of at least a part of the lipids took place, after which only one unit cell with a repeat distance of 13.4 nm could be detected. Comparison of the scattering curve of the stratum corneum after crystallization with the scattering curve of the stratum corneum before recrystallization leads to the conclusion that in the original curve the lipids are arranged in two unit cells with repeat distances of 6.4 nm and 13.4 nm. From model calculations it appears that the latter unit cell consists of more than one bilayer. The scattering curves of stratum corneum hydrated to various levels were measured. A change in the water content of stratum corneum between 6% w/w and 60% w/w (fully hydrated) did not result in swelling of the bilayers, but the scattering curve obtained with stratum corneum hydrated to 60% w/w differed from those at lower hydration levels: the scattering curve at 60% w/w showed only the diffraction peaks corresponding to a unit cell with a repeat distance of 6.4 nm. This observation implies that the ordering of a part of the lipids is reduced at very high water contents, which may explain the strong penetration-enhancing effects of water in the stratum corneum.

Structure and thermotropic properties of hydrated N-stearoyl sphingomyelin bilayer membranes

Hydrated multibilayers of N-stearoyl sphingomyelin were investigated as a function of hydration using differential scanning calorimetry (DSC) and X-ray diffraction. Anhydrous N-stearoyl sphingomyelin exhibits an endothermic transition at 75°C (Δ H=3.8kcal/mol) are increasing hydration proessively lowers the transition temperature and increases the transition enthalpy, until limiting values ( T m = 45°C, Δ H = 6.7kcal/mol) are observed for hydration values > 21.4% H 2O. At low hydration levels, < 20% H 2O, an additional transition is observed at approx. 20°C. X-ray diffraction studies at temperatures below (22°C) and above (55°C) the main endothermic transition confirm that the bilayer gel (sharp 4.2Åreflection) → bilayer liquid crystal (diffuse 4.5Åreflection) transition occurs at all hydration levels with limiting bilayer hydration occurring at approx. 31.5% H 2O in the gel phase and at approx. 35% H 2O in the liquid crystal phase. The thermotropic properties and metastability of this partial synthetic N-stearoyl sphingomyelin differ in some respects from that of the previously studied synthetic dl-erythro- N-stearoyl sphingomyelin (Estep, T.N., Calhoun, W.I., Barenholz, Y., Biltonen, R.L., Shipley, G.G. and Thompson, T.E. (1980) Biochemistry 19, 20–24), suggesting an influential role of the interfacial molecular conformation.

Intracellular water in Artemia cysts (brine shrimp): Investigations by deuterium and oxygen-17 nuclear magnetic resonance

The dormant cysts of Artemia undergo cycles of hydration-dehydration without losing viability. Therefore, Artemia cysts serve as an excellent intact cellular system for studying the dynamics of water-protein interactions as a function of hydration. Deuterium spin-lattice (T(1)) and spin-spin (T(2)) relaxation times of water in cysts hydrated with D(2)O have been measured for hydrations between 1.5 and 0.1 g of D(2)O per gram of dry solids. When the relaxation rates (I/T(1), I/T(2)) of (2)H and (17)O are plotted as a function of the reciprocal of hydration (1/H), an abrupt change in slope is observed near 0.6 g of D(2)O (or H(2) (17)O)/gram of dry solids, the hydration at which conventional metabolism is activated in this system. The results have been discussed in terms of the two-site and multisite exchange models for the water-protein interaction as well as protein dynamics models. The (2)H and (17)O relaxation rates as a function of hydration show striking similarities to those observed for anisotropic motion of water molecules in protein crystals.It is suggested here that although the simple two-site exchange model or n-site exchange model could be used to explain our data at high hydration levels, such models are not adequate at low hydration levels (<0.6 g H(2)O/g) where several complex interactions between water and proteins play a predominant role in the relaxation of water nuclei. We further suggest that the abrupt change in the slope of I/T(1) as a function of hydration in the vicinity of 0.6 g H(2)O/g is due to a change in water-protein interactions resulting from a variation in the dynamics of protein motion.

Regulation of trehalose metabolism by Streptomyces griseus spores

Spores of Streptomyces griseus contain trehalose and trehalase, but trehalose is not readily hydrolyzed until spore germination is initiated. Trehalase in crude extracts of spores, germinated spores, and mycelia of S. griseus had a pH optimum of approximately 6.2, had a Km value for trehalose of approximately 11 mM, and was most active in buffers having ionic strengths of 50 to 200 mM. Inhibitors or activators or trehalase activity were not detected in extracts of spores or mycelia. Several lines of evidence indicated that trehalose and trehalase are both located in the spore cytoplasm. Spores retained their trehalose and most of their trehalase activity following brief exposure to dilute acid. Protoplasts formed by enzymatic removal of the spore walls in buffer containing high concentrations of solutes also retained their trehalose and trehalase activity. Protoplasts formed in buffer containing lower levels of solutes contained low levels of trehalose. The mechanism by which trehalose metabolism is regulated in S. griseus spores is unresolved. A low level of hydration of the cytoplasm of the dormant spores and an increased level of hydration during germination may account for the apparent inactivity of trehalase in dormant spores and the rapid hydrolysis of trehalose upon initiation of germination.

The influence of cold acclimation on the lipid composition and cryobehaviour of the plasma membrane of isolated rye protoplasts

Destabilization of the plasma membrane, which is a primary cause of freezing injury, is a consequence of freeze-induced osmotic stresses and cell dehydration. However, the mechanism of injury depends on the magnitude of the osmotic stress and the extent of cell dehydration. Over the range of 0 to — 5 °C, destabilization of the plasma membrane in protoplasts isolated from non-acclimated rye leaves is a result of osmotic excursions, because freeze-induced osmotic contraction results in endocytotic vesiculation of the plasma membrane and sufficiently large area reductions are irreversible. At lower temperatures, the protoplasts are subjected to extremely large osmotic pressures ( — 12 MPa at —10 °C), and there are several changes in the ultrastructure of the plasma membrane, including the formation of aparticulate domains and lamellar- to hexagonaln-phase transitions. These changes, which are manifestations of demixing of the membrane components, are predicted by a theory of bilayer interactions at low levels of hydration. During cold acclimation, the cryobehaviour of the plasma membrane is altered; osmotic contraction results in the reversible formation of exocytotic extrusions and the propensity for dehydration-induced demixing and lamellar- to hexagonaln-phase transitions is decreased. In both cases, the differential behaviour is also observed in liposomes prepared from plasma membrane lipids isolated from non-acclimated and cold-acclimated leaves. However, as no lipid species are unique to the plasma membrane of either non-acclimated or cold-acclimated leaves, the differential behaviour is caused by altered lipid-lipid interactions because of different proportions of the lipid species. Hence the behaviour of the plasma membrane can be altered by using a protoplast-liposome fusion procedure to selectively modify the lipid composition of the plasma membrane. These studies provide direct evidence that the increased cryostability of the plasma membrane is a consequence of alterations in its lipid composition.

Interaction of water with oriented DNA in the A- and B-form conformations

High resolution 2H nuclear magnetic resonance (NMR) was used to investigate the interaction of D2O with solid samples of uniaxially oriented Li-DNA (B-form DNA) and Na-DNA (A- and B-form DNA). At low levels of hydration, 0 approximately 4 D2O/nucleotide, the 2H spectra shows a very weak (due to short T2) broad single resonance, suggestive of unrestricted rotational diffusion of the water. At approximately 5 or more D2O/nucleotide, the Li-DNA (B-form) spectra suddenly exhibit a large doublet splitting, characteristic of partially ordered water. With increasing hydration, the general trend is a decrease of this splitting. From our analysis we show that the DNA water structure reorganizes as the DNA is progressively hydrated. The D2O interaction with Na-DNA is rather different than with Li-DNA. Below 10 D2O/nucleotide Na-DNA is normally expected to be in the A-form, and a small, or negligible splitting is observed. In the range 9-19 D2O/nucleotide, the splitting increases with increasing hydration. Above approximately 20 D2O/nucleotide Na-DNA converts entirely to the B-form and the D2O splittings are then similar to those found in Li-DNA. We show that the complex Na-DNA results obtained in the range 0-20 D2O/nucleotide are caused by a mixture of A- and B-DNA in those samples.

Static disorder and librational motions of the purine bases in films of oriented Li-DNA

Solid-state 2H nuclear magnetic resonance line shapes have been obtained from folded films of oriented Li-DNA molecules with the purine bases selectively labeled with deuterium at the 8-position. From line shape simulations, the static base tilts as well as the anisotropic motional amplitudes were determined as a function of hydration level and temperature. It was found that the average tilt angle of the bases is close to 0 ° and at a hydration of ten water molecules per nucleotide the distribution width of tilt angles about this average cannot be larger than 9 ° (standard deviation). A slightly increased distribution width is observed at low hydration levels. The motional amplitudes are hydration dependent, with the tilting motion ranging from 4 ° for the driest, up to 15 ° for the wettest sample, and slightly larger amplitudes are observed for the twisting motion. The amplitude of the twisting motion is unaffected by a temperature decrease down to −60 °C, in contrast to the tilting motion that is suppressed at low temperatures.