Gert Rapp

Time-resolved X-ray diffraction study of structural changes associated with the photocycle of bacteriorhodopsin.

The time course of structural changes accompanying the transition from the M412 intermediate to the BR568 ground state in the photocycle of bacteriorhodopsin (BR) from Halobacterium halobium was studied at room temperature with a time resolution of 15 ms using synchrotron radiation X‐ray diffraction. The M412 decay rate was slowed down by employing mutated BR Asp96Asn in purple membranes at two different pH‐values. The observed light‐induced intensity changes of in‐plane X‐ray reflections were fully reversible. For the mutated BR at neutral pH the kinetics of the structural alterations (tau 1/2 = 125 ms) were very similar to those of the optical changes characterizing the M412 decay, whereas at pH 9.6 the structural relaxation (tau 1/2 = 3 s) slightly lagged behind the absorbance changes at 410 nm. The overall X‐ray intensity change between the M412 intermediate and the ground state was about 9% for the different samples investigated and is associated with electron density changes close to helix G, B and E. Similar changes (tau 1/2 = 1.3–3.6 s), which also confirm earlier neutron scattering results on the BR568 and M412 intermediates trapped at ‐180 degrees C, were observed with wild type BR retarded by 2 M guanidine hydrochloride (pH 9.4). The results unequivocally prove that the tertiary structure of BR changes during the photocycle.

The tertiary structural changes in bacteriorhodopsin occur between M states: X-ray diffraction and Fourier transform infrared spectroscopy.

The tertiary structural changes occurring during the photocycle of bacteriorhodopsin (BR) are assigned by X‐ray diffraction to distinct M states, M1 and M2. Purple membranes (PM) of the mutant Asp96Asn at 15, 57, 75 and 100% relative humidity (r.h.) were studied in a parallel X‐ray diffraction and Fourier transform infrared (FTIR) spectroscopic investigation. Light‐dependent conformational changes of BR‐Asp96Asn are observed at high hydration levels (100 and 75% r.h.) but not in partially dehydrated samples (57 and 15% r.h.). The FTIR spectra of continuously illuminated samples at low and high hydration, despite some differences, are characteristic of the M intermediate. The changes in diffraction patterns of samples in the M2 state are of the same magnitude as those of wild‐type samples trapped with GuaHCl in the MG state. Additional large changes in the amide bands of the FTIR spectra occur between M2 and MG. This suggests, that the tertiary structural changes between M1 and M2 are responsible for the switch opening the cytoplasmic half‐channel of BR for reprotonation to complete the catalytic cycle. These tertiary structural changes seem to be triggered by a charge redistribution which might be a common feature of retinal proteins also in signal transduction.

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.

Accelerated Formation of Cubic Phases in Phosphatidylethanolamine Dispersions

By means of x-ray diffraction we show that several sodium salts and the disaccharides sucrose and trehalose strongly accelerate the formation of cubic phases in phosphatidylethanolamine (PE) dispersions upon temperature cycling through the lamellar liquid crystalline-inverted hexagonal (Lalpha-HII) phase transition. Ethylene glycol does not have such an effect. The degree of acceleration increases with the solute concentration. Such an acceleration has been observed for dielaidoyl PE (DEPE), dihexadecyl PE, and dipalmitoyl PE. It was investigated in detail for DEPE dispersions. For DEPE (10 wt% of lipid) aqueous dispersions at 1 M solute concentration, 10-50 temperature cycles typically result in complete conversion of the Lalpha phase into cubic phase. Most efficient is temperature cycling executed by laser flash T-jumps. In that case the conversion completes within 10-15 cycles. However, the cubic phases produced by laser T-jumps are less ordered in comparison to the rather regular cubic structures produced by linear, uniform temperature cycling at 10 degrees C/min. Temperature cycles at scan rates of 1-3 degrees C/min also induce the rapid formation of cubic phases. All solutes used induce the formation of Im3m (Q229) cubic phase in 10 wt% DEPE dispersions. The initial Im3m phases appearing during the first temperature cycles have larger lattice parameters that relax to smaller values with continuation of the cycling after the disappearance of the Lalpha phase. A cooperative Im3m --> Pn3m transition takes place at approximately 85 degrees C and transforms the Im3m phase into a mixture of coexisting Pn3m (Q224) and Im3m phases. The Im3m/Pn3m lattice parameter ratio is 1. 28, as could be expected from a representation of the Im3m and Pn3m phases with the primitive and diamond infinite periodic minimal surfaces, respectively. At higher DEPE contents ( approximately 30 wt%), cubic phase formation is hindered after 20-30 temperature cycles. The conversion does not go through, but reaches a stage with coexisting Ia3d (Q230) and Lalpha phases. Upon heating, the Ia3d phase cooperatively transforms into a mixture of, presumably, Im3m and Pn3m phases at about the temperature of the Lalpha-HII transition. This transformation is readily reversible with the temperature. The lattice parameters of the DEPE cubic phases are temperature-insensitive in the Lalpha temperature range and decrease with the temperature in the range of the HII phase.

A dual detector single readout system for simultaneous small (SAXS) and wide-angle X-ray (WAXS) scattering

Abstract A data acquisition system is described which allows to simultaneously record X-ray scattering patterns in different angular regimes both with high spatial and temporal resolution. It consists of two linear detectors with delay-line readout connected in series. A few examples illustrate its application in the study of the polymorphism of lipid systems and their phase transitions as well as of synthetic polymers.

Structure of the stable and metastable ripple phase of dipalmitoylphosphatidylcholine

Dipalmitoylphosphatidylcholine (DPPC) dispersed in excess water forms a stable ripple phase upon heating from the gel phase and a metastable ripple phase Pβ′ (mst) upon cooling from the liquid crystalline phase. The X-ray diffraction pattern of Pβ′ (mst) displays several reflections in the range from 1/25 to 1/2.8 nm−1, which can all be indexed on a two-dimensional monoclinic lattice (space group p2) with a=26.2, b=8.63 nm and γ=107°. In contrast to the stable ripple phase, which shows a sawtooth like surface profile and an almost constant bilayer thickness, the electron density map of the metastable ripple phase shows an almost symmetric surface profile with a modulation length of 26.2 nm. The lipid bilayer thickness varies from 3.9 to 4.4 nm, which most likely arises from a continuous periodic change of the tilt of the chains to the surface normal of between 30 and 40 degrees. A further important feature of the structure is the staggered stacking of the bilayers with water pockets enclosed.

Relaxation of chemically skinned guinea pig taenia coli smooth muscle from rigor by photolytic release of adenosine-5′-triphosphate

SummaryThe mechanical events following release of ATP from P3-1-(2-nitro)phenylethyladenosine-5′-triphosphate (caged-ATP) in skinned guinea pig taenia coli smooth muscle in rigor were investigated. A rigor force of about 25–35% of the maximal active force was obtained by removing ATP at the plateau of a maximal active contraction. In the rigor solution free-Mg2+ was 2mm, ionic strength 90mm and pH 7.0. When caged-ATP (12.5mm) was diffused into the preparation there was no change in the rigor force. Photolytic production of about 2mm ATP was achieved with a xenon flash lamp. Following illumination, force decreased with an approximate initial rate constant of 0.7 s−1. The rate of relaxation was increased in the presence of inorganic phosphate (at 3mm: 1.3 s−1; 10mm: 2.2 s−1). At higher Mg2+ concentrations the rate of relaxation was slower (5mm: 0.2 s−1) and at lower concentrations the rate was faster (0.5mm: 1.2 s−1). An increased rate of relaxation was observed when ionic strength was increased to 150mm (2.2 s−1). Phosphate increased the rate of relaxation at the different levels of Mg2+ (0.5–10mm) and ionic strength (90, 150mm). In preparations shortened (by 1–3%) to give reduced rigor force, a small transient increase in tension was recorded after ATP release. In comparison to the rates of ATP-induced dissociation of actomyosin in solution, reported in the literature, the rate of relaxation from rigor is slower. This may reflect a slow rigor cross-bridge dissociation or mechanical interactions not associated with cross-bridges in the muscle fibre. However, the results may also be interpreted on the basis of a model proposed for striated muscle by Goldmanet al. (1984) where the relaxation from rigor in the absence of Ca2+ involves a phase of reattaching cross-bridges whose lifetime in a tension-producing state is influenced by phosphate.

New evidence for gel-liquid crystalline phase coexistence in the ripple phase of phosphatidylcholines

Abstract Experimental evidence supporting the hypothesis of gel-liquid crystalline phase coexistence in the stable ripple phase of diacylphosphatidylcholines has been obtained from time-resolved X-ray small- (SAXS) and wide-angle diffraction (WAXS) in the millisecond to second time domain. The pretransition of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) exhibits a thin lamellar liquid crystalline intermediate phase (designated Lα*) if driven far away from equilibrium by an infrared temperature jump (T-jump) technique. The findings can be described by a two-step model. (1) Instantaneously with the T-jump, an anomalously thin lamellar liquid crystalline intermediate phase (d = 5.6–5.8 nm) forms, coexisting with the original gel-phase Lβ′. Within the first seconds, the lamellar repeat distance of the intermediate increases to a value of about 6.7 nm. A closer examination of these kinetics reveals two relaxation components: a fast process, proceeding within tenths of a second, and a slow process, on the time scale of a few seconds. (2) Finally, both the liquid crystalline and the gel-phase relax into the stable ripple phase Pβ′. The total process time of the transition is nearly independent of the addition of NaCl, but varies strongly with the chain length of the lecithin species.

Mixing behavior of saturated short-chain phosphatidylcholines and fatty acids: Eutectic points, liquid and solid phase immiscibility, non-lamellar phases

Phase diagrams of the hydrated pseudo-binary mixtures dilauroylphosphatidylcholine (DLPC)/lauric acid (LA) and dimyristoylphosphatidylcholine (DMPC)/myristic acid (MA) have been constructed using high-sensitivity DSC and time-resolved X-ray diffraction. They are of a different type compared to those of the longer chain plhosphatidylcholine (PC)/fatty acid (FA) mixtures, the latter being of maximum azeotropic point type. Eutectic points were distinguished in the phase diagrams, at 75 mol’% MA and 49°C for the DMPCjMA mixture, and at ca. 67 mol% LA and 29°C for the DLPC/LA mixture. Regions of liquid&liquid and solid-solid phase separation have been located according to the shape of the phase diagrams and observed by X-ray diffraction. Limited regions (2-4°C) of liquid-liquid phase immiscibility exist at compositions with slightly prevailing fatty acid molar content. For instance, at 67 mol’K MA, a phase separation between L, phase enriched in DMPC and H,, phase enriched in MA takes place in the temperature range 51-55°C. Solid phase immiscibility is detected between 60 and 90 mol% fatty acid. The studied PC/FA mixtures form compound subgel polymorphic phases (one in the DMPCjMA mixture, with DMPCIMA 1:2 molar stoichiometry, and two in the DLPC/LA mixture, with about 40 and 60 mol’% LA, respectively) upon low-temperature equilibration. In the liquid crystalline phase region, non-lamellar phases dominate the phase diagrams, especially in their fatty acid-rich part. With increasing FA content, the nonlamellar phases arrange in the sequence: bicontinuous cubic phases (Ia3d, Pn3m, Im3m) + hexagonal phase (H,,) + micellar cubic

Effect of high pressure on the structure of dipalmitoylphosphatidylcholine bilayer membranes: a synchrotron-X-ray diffraction and FT-IR spectroscopy study using the diamond anvil technique

Abstract The effect of high pressure, up to 20 kbar, on multilamellar vesicles of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) was examined by using FT-IR spectroscopy and Synchrotron-X-ray diffraction in the small- and wide-angle scattering region. The temperature and pressure range studied permitted us to explore the phase behaviour of DPPC from the liquid–crystalline phase through various gel phases up to the pressure region where bulk water freezes and freeze-induced dehydration of the membrane occurs. To our knowledge this is the first study where the structure and phase behaviour of an amphiphilic lipid system has been studied up to these high pressures. The use of pressure to study the lipid gel state has revealed several new phases which may not exist at very low temperatures at ambient pressure.