Werner Kreutz

Evidence for the protonation of two internal carboxylic groups during the photocycle of bacteriorhodopsin: Investigation of kinetic infrared spectroscopy

The amino acid sequence of bacteriorhodopsin (BR) [ 1,2], its three-dimensional structure at low resolution [3] and projected structure at 3.7 A resolution [4] have been determined. Attempts have been made to fit the amino acid sequence into the threedimensional structure [5,6] and the retinal binding site has been established [7-91. To understand the proton pumping mechanism, molecular events in the chromophore and protein should be known as well as the structural information. Resonance Raman spectroscopy provides information on the chromophore (review [lo]), whereas kinetic infrared spectroscopy [ 11 ,121 detects molecular changes in the chromophore as well as the protein. It has been applied successfully in investigations of rhodopsin [ 131 and bacteriorhodopsin. For the latter, the BR570-M412 difference spectrum has been measured over 17001500 cm-’ and 1300-l 100 cm-’ [ 121; a preliminary study on the BR570-L550 difference spectrum in the same spectral range has been published [ 141. Having extended the spectral range beyond 1700 cm-‘, we now report evidence for the presence of two carboxylic acid residues being protonated and re-deprotonated during the photocycle. While the protonation of one group occurs simultaneously with the formation of M412, the time course for the second group is slower and does not reflect kinetics observed for chromophoric changes. In addition, the two groups can be identified by their different absorption maxima. The time constant for re-deprotonation is similar for both groups coinciding with that for the reformation of BR570 from M412. These results will be discussed with respect to a possible involvement of carboxylic groups in the proton pumping mechanism [ 15,171 and with respect to their role regarding the spectroscopic properties of BR [15,16,18-211.

Structure of human low-density lipoprotein subfractions determined by X-ray small-angle scattering

The structure of low-density lipoprotein (LDL) particles from three different density ranges (LDL-1: d = 1.006-1.031 g/ml; LDL-3: d = 1.034-1.037 g/ml; LDL-6: d = 1.044-1.063 g/ml) was determined by X-ray small-angle scattering. By using a theoretical particle model, which accounted for the polydispersity of the samples, we were able to obtain fits of the scattering intensity that were inside the noise interval of the measured intensity. The assumption of deviations from radial symmetry is not supported by our data. This implies a spread-out conformation of the apolipoprotein B (apoB) molecule, which appears to be localized in the outer surface shell. A globular structure is not consistent with our data. Furthermore, different models exist concerning the structure of the cholesterol ester core below the phase transition temperature. The electron density data suggest an arrangement in which the steroid moieties are localized at average radii of 3.2 and 6.4 nm. Model calculations show that packing problems can only be avoided if approximately half of the acyl chains of each shell are pointing towards the center of the particle, the other half towards the surface. This arrangement of the acyl chains has never been proposed before. The LDL particles of different density classes differ mainly with respect to the size of the core but also with respect to the width of the surface shells. Model calculations show that the size of different LDL particles can be accurately predicted from the compositional data.

Measurements of fast light-induced light-scattering and -absorption changes in outer segments of vertebrate light sensitive rod cells

AbstractFlash-induced changes of light-absorption and of light-scattering of vertebrate rod outer segments (ROS) from frog and cattle in suspension were measured at 380 and 800 nm. The photometer used allows the observation of light intensity changes under well defined angles.We studied the successive decrease of the signal amplitude in series of flashes. One flash bleaches about 1% rhodopsin.The following results are discussed:1.The signal at 380 nm is a superposition of the absorption change caused by formation of metarhodopsin II and of a biphasic additional signal. The latter exists only for the initial range of bleaching (15 to 25% rhodopsin).2.At 800 nm three scattering signals are observed which are characterized by their successive amplitude decrease and time course:N: A small signal with time course and successive amplitude decrease comparable to the metarhodopsin II absorption change, probably arising from a structural change within the disc membrane. Ni: A slow signal, disappearing with the first flash, which may be understood as an outer membrane effect. P: A biphasic signal with a successive decrease rate, by a factor of 10 to 20 higher than that of the metarhodopsin II signal. The two kinetically different components are separated by variation of the observation angle. Two regions of different extension appear to change structurally with different time course. “P” may reflect an influence of the light-induced transmitter release on disc shape and/or mass.

Light-induced Fourier transform infrared (FTIR) spectroscopic investigations of the primary donor oxidation in bacterial photosynthesis

Fourier transform infrared (FTIR) difference spectroscopy of the primary electron donor (P) photo–oxidation has been performed for reaction centers (RCs) and chromatophores of purple photosynthetic bacteria. In the 1800–650 cm−1 spectral region highly reproducible absorbance changes were obtained that can be related to specific changes of individual bond absorption. Several bands in the difference spectra are tentatively assigned to changes of intensity and position of the keto and ester C−O vibrations of the P bacteriochlorophylls, and a possible interpretation in terms of changes of their environment or type of bonding to the protein is given. Small difference bands in the amide I and II region allow only minor protein conformational changes.

Time-resolved Infrared Spectroscopy of the Ca2+-ATPase THE ENZYME AT WORK

Changes in the vibrational spectrum of the sarcoplasmic reticulum Ca2+-ATPase in the course of its catalytic cycle were followed in real time using rapid scan Fourier transform infrared spectroscopy. In the presence of Ca2+, the cycle was induced by the photochemical release of ATP from a biologically inactive precursor (caged ATP, P3-1-(2-nitro)phenylethyladenosine 5′-triphosphate). Absorbance changes arising from ATP binding to the ATPase were observed within the first 65 ms after initiation of ATP release. After ATP binding, up to two subsequent partial reactions of the ATPase reaction cycle were observed depending on the buffer composition (10 mM CaCl2 + 330 mM KCl or 1 mM CaCl2 + 20% Me2SO): (i) formation of the ADP-sensitive phosphoenzyme (kapp = 0.79 s−1 ± 15% at 1°C, pH 7.0, 10 mM CaCl2, 330 mM KCl) and (ii) phosphoenzyme conversion to the ADP-insensitive phosphoenzyme concomitant with Ca2+ release (kapp = 0.092 s−1 ± 7% at 1°C, pH 7.0, 1 mM CaCl2, 20% Me2SO). Each reaction step could well be described by a single time constant for all associated changes in the vibrational spectrum, and no intermediates other than those mentioned above were found. In particular, there is no evidence for a delay between the transition from ADP-sensitive to ADP-insensitive phosphoenzyme and Ca2+ release. In 2H2O a kinetic isotope effect was observed: both the phosphorylation reaction and phosphoenzyme conversion were slowed down by factors of 1.5 and 3.0, respectively. The small amplitudes of the observed changes in the infrared spectrum indicate that the net change of secondary structure is very small and of the same order of magnitude for ATP binding, phosphorylation, and phosphoenzyme conversion. Therefore, our results do not support a distinction between minor and major secondary structure changes in the catalytic cycle of the ATPase, which might be expected according to the classical E1-E2 model.

Molecular changes in the sarcoplasmic reticulum calcium ATPase during catalytic activity: A Fourier transform infrared (FTIR) study using photolysis of caged ATP to trigger the reaction cycle

Fourier transform infrared spectroscopy was used to study ligand binding and conformational changes in the Ca2+‐ATPase of sarcoplasmic reticulum. Novel in infrared difference spectroscopy, the catalytic cycle in the IR sample was started by photolytic release of ATP from an inactive, photolabile ATP‐derivative (caged ATP). Small, but characteristic infrared absorbance changes were observed upon ATP release. On the basis of model spectra, the absorbance changes corresponding to the trigger and substrate reactions, i.e. to photolysis of caged ATP and hydrolysis of ATP, were separated from the absorbance changes due to the active ATPase reflecting formation of the phosphorylated Ca2E1P enzyme form. A major rearrangement of ATPase conformation as the result of catalysis can be excluded.

Crystallization of the photosynthetic light‐harvesting pigment‐protein complex B800‐850 of Rhodopseudomonas capsulata

The B800‐850 light‐harvesting complex of Rhodopseudomonas capsulata was crystallized in the presence of detergents. The crystals were obtained by a vapour diffusion technique, using polyethylene glycol as a precipitant. Crystals grew to a size of 0.5 × 0.5 × 0.3 mm within two weeks. Two different crystal forms were obtained; one is supposed to be triclinic, space group PI, the other orthorhombic, space group C2221. Both crystal forms diffract to approximately 1.0 nm. Absorption spectra and polyacrylamide gel electrophoresis demonstrate that all expected components, i.e. three polypeptides of apparent M r,8000, 10000 and 14000, bacteriochlorophyll a and carotenoids, are present and in native conformation.

Flash-induced kinetic infrared spectroscopy applied to biochemical systems.

A flash photolysis apparatus with monitoring infrared beam is described allowing measurements of relative transmission changes of 10−3 in times of a few milliseconds. The investigation of the photodissociation of CO-myoglobin confirms the results obtained by static infrared difference spectroscopy. The application of our method to the rhodopsin/Meta II transition reveals signals which can tentatively be ascribed to the disappearance of the C=C-band of the protonated N-retinylidene Schiff base in rhodopsin. The developed method will be compared with other existing methods of kinetic vibronic spectroscopy such as kinetic resonance Raman spectroscopy and kinetic Fourier infrared spectroscopy.

pH-Dependent LAK Cell Cytotoxicity

In the microenvironment of many solid tumors the pH is considerably lower (mean pH between 6.6 to 7.2) than the pH in normal tissue (pH 7.0-7.5). Therefore, the influence of acidic pH on the cytotoxic activity of lymphokine-activated killer cells (LAK cells) after different culture periods was tested. K-562 human erythroleukemia cells were selected as target cells. Cell killing was measured using a two-color flow cytometric method. At physiological pH of 7.4, LAK cell-mediated cytotoxicity ranged from 15 to 48% (E:T ratio = 50:1). The specific lysis of target cells was considerably reduced (up to 70% inhibition of specific lysis) under acidic conditions (pH 6.8, 6.3, 5.8). This effect was independent of donors, duration of the culture period, and the E:T ratio in the cytotoxic assay. As pH gradients surrounding tumor cells may reach values below pH 6.0 at the cell surface, the pH-dependence of LAK cell cytotoxicity could at least partially explain the inhibition of the natural immune response in solid tumors. Therapeutic immunological strategies concerning the enhancement of the natural immune response like LAK cell and IL-2 immunotherapy including IL-2 gene therapy may only be successful if a simultaneous inhibition of the acidification process and an elevation of tumor pH is achieved.

Changes of protein structure, nucleotide microenvironment, and Ca2+-binding states in the catalytic cycle of sarcoplasmic reticulum Ca2+-ATPase: investigation of nucleotide binding, phosphorylation and phosphoenzyme conversion by FTIR difference spectroscopy

Changes of infrared absorbance of sarcoplasmic reticulum Ca(2+)-ATPase (EC 3.6.1.38) associated with partial reactions of its catalytic cycle were investigated in the region from 1800 to 950 cm-1 in H2O and 2H2O. Starting from Ca2E1, 3 reaction steps were induced in the infrared cuvette via photolytic release of ATP and ADP: (a) nucleotide binding, (b) formation of the ADP-sensitive phosphoenzyme (Ca2E1P) and (c) formation of the ADP-insensitive phosphoenzyme (E2P). All reaction steps caused distinct changes of the infrared spectrum which were characteristic for each reaction step but comparable for all steps in the number and magnitude of the changes. Most pronounced were absorbance changes in the amide I spectral region sensitive to protein secondary structure. However, they were small--less than 1% of the total protein absorbance--indicating that the reaction steps are associated with small and local conformational changes of the polypeptide backbone instead of a large conformational rearrangement. Especially, there is no outstanding conformational change associated with the phosphoenzyme conversion Ca2E1P-->E2P. ADP-binding induces conformational changes in the ATPase polypeptide backbone with alpha-helical structures and presumably beta-sheet or beta-turn structures involved. Phosphorylation is accompanied by the appearance of a keto group vibration that can tentatively be assigned to the phosphorylated residue Asp351. Phosphoenzyme conversion and Ca(2+)-release produce difference signals which can be explained by the release of Ca2+ from carboxylate groups and a change of hydrogen bonding or protonation state of carboxyl groups.