Membrane Pharmacy Structure Dynamics  Research group : Priv.Doz. Dr. Thomas Nawroth  Technology

Fig.1: The strategy of structure and membrane research leads from heterogenous cells over isolated homogenous systems (proteins, lipids, membranes, nucleic acids) to reconstituted model membranes and protein-detergent complexes  (after T20). Molecular genetics supplies cells with specific proteins, chemical synthesis gives us lipids, polymers and compounds for optical system manipulation, e.g. caged-compounds. The molecular homogenous systems are suitable for structure investigation (SAXS, SANS, ASAXS, X-ray diffraction, EXAFS / XANES, especially with time resolution).

The supply of the biological systems require the growth of cells from a genetically defined strain and subsequently the isolation and purification of proteins in the native state. For investigation of the structure-function relation and molecular biochemistry, this means isolation of milligram-amounts of pure proteins (e.g. 99%) in a functional competent and homogenous state. While working with proteins of the bioenergetic system, this includes the preparation and purification of native membranes, e.g. protoplast membrane vesicles (PMV) from Micrococcus luteus, or membranes from cell organelles (mitochondria). During the study artificial model membranes have to be prepared, e.g. liposomes, which are sufficient for function and structure investigation, e.g. with neutron scattering. The technology for supplying the biological system is presented in the protein preparation page.
Some scientific investigations require the chemical synthesis of several small molecules for the manipulation of the system or the generation of a specific signal during structure investigation. In the MPSD group those synthesis work is done at two compound classes: i) the synthesis of caged compounds (caged-ATP, caged-nucleotides, caged-tritium, caged acids / proton; ref. T7, T9, T14, T15 , T20) and ii) the synthesis of structure labels, i.e. artificial metal labels for proteins, lipids and polymers, e.g. Europium-chelates, and fluorescent or coloured photoaffinity labels (e.g. the hydrophobic labels in ref. F15). An overview and some receipts are given in the Chemical synthesis chapter.
 

Fig.2: A 4-beam dual-wavelength flash illumination device  for the manipulation of proteins and membranes by light. The sample (e.g. reconstituted Bacteriorhodopsin/ ATP-synthase liposomes) is illuminated homogenously by two opposite beams of visible light (green, 550 +- 40 nm; 1000 mW/cm2). After a reaction delay the sample is flash-irradiated by two lateral UV sources (300-380 nm; 80 J) for photolabeling or activation of caged compounds. Analogous devices are used for time resolved X-ray and neutron scattering with caged compounds (caged-acid/ proton) (after T20).

A variety of experiments about enzymatic analysis, surface labeling, structure research with X-rays and neutrons, and generation of functional states in time resolved studies requires a sofisticated optical setup. Especially experiments with photogeneration of compounds, e.g. for light-driven ATP-synthesis, cleavage of caged compounds and photoaffinity labeling or crosslinkig, yield unambigous results only if the optical system is setup properly, i.e. if the sample is homogenously illuminated at any point. Similar precautions have to be observed, if the status of the system is examined by optics, e.g. fi probing the protein conformation by a fluorescence measurement. For triggering of a time resolved experiments by light, e.g. photolysis of a caged acid or caged ATP, the light has to be generated in a short time (< 1 ms) and large amount (several J/ml sample). For this purpose the MPSD groups uses currently mainly (UV) flash illumination. The technologies are presented in the optics page.
If the molecular reaction (motion) is slower than a few milliseconds and if the system is insensitive to shearing forces, it can be investigated in a more simple way after triggering by a concentration jump during rapid mixing of the compounds with a stopped flow device. The MPSD group has constructed for this purpose several devices and sample environments since 1987 (ref. F14, T14, T20) and improved the technology for the use at high flux synchrotrons (ref , S##, F##) and time resolved neutron scattering (R##). A result is shown in the structural film of working F₁ATPase. The mixing technology is demonstarted in the rapid mixing page.
 

Fig.3: A flow-through quartz capillary for structure investigation of protein solutions as used at DESY-HASYLAB, Hamburg, at the JUSIFA beamline (B1). Analogous, but more complicated sample environments with helium-jet cooling and stopped-flow mixing devices or flash units are used for time resolved studies at the high flux synchrotrons (ESRF, ELETTRA) and for time resolved neutron scattering at the ILL, Grenoble.

X-ray and neutron scattering experiments of biological systems require special sample environments, if examined with high flux, high precision and / or time resolution. Thus the MPSD group has in cooperation with industry constructed in the last 20 years several sample environments for structure research of biomolecules in solution with X-ray synchrotron radiation, neutrons and spectroscopy. A sketch of an recent helium cooled sample environment SBox4 for high flux synchrotron light scattering is shown in Fig.4 and ref. S##. The principal setup of a typical X-ray setup is shown in Fig.5. This actual environment, which is suitable also for temperature jump (cold trap) experiments, is presented in the sample environment page.
The control of time resolved experiments and data aquisition is done at the MPSD group by a combination of computer- and digital eletronics (TTL). During this several novel digital and electronics divices were constructed in cooperation with industry. The first experiments in the early 80's where done with timer-chains. Since 1988 we switched over to microcomputer systems (with directly coded binary code for hardware controller CPU's) and TTL-electronics for the fast process steps (microseconds to milliseconds) and PC-computer programs for the slower steps (> 100 ms), providing the parameter and data aquisition / handshake (e.g. F14, T14, T20). An actual example is given in the WEB for time resolved neutron scattering of liposomes (http://www.ill.fr ; see experiment reports ... database-settings: instrument = D22 ; name = Nawroth ; experiment = Test-251 and further). Some technological aspects are presented in the computer control and electronics page.
 

Fig.4: The modular sample environment SBox4 is constructed for low background high-precission experiments with spectroscopy, X-ray and neutron scattering. The sample cell is cooled by two helium jets. It can be observed with an internal CCD camera. The three thermostats at the right hand side allow temperature jump experiments with the stopped-flow / cold trap method. This enviroment has been used at ESRF Grenoble, beamlines ID1, ID2A and ID24, and at DESY-HASYLAB Hamburg, beamline BW4 (USAXS); a smaller version without temperature jump equipment (SBox2) also at ELETTRA Trieste, beamline 5.2 (SAXS).

Fig.5: Principal setup for a time resolved X-ray scattering and absorption experiment at a high flux synchrotron. The active working sample is produced from stock solutions by rapid mixing with a stopped-flow device. The helium atmosphere reduces air scattering and beam heating. Radiation demage can be suppressed by slow continous sample displacement by drag buffer with a second drive (from R6, Fig.1).