Membrane Pharmacy Structure Dynamics 

Research group : Priv.Doz. Dr. Thomas Nawroth 


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Science - Investigation of the structure-function relation of proteins and membranes

The scientific work of the MPSD group follows an interdisciplinary concept : The investigation of molecular motions in biological systems requires the parallel application of biophysics, biochemistry, molecular biology and physics, especially structure investigations. This is the concept of "biophysical chemistry", which is in detail explained at the MPSD_concept page. The investigations are in part done locally in the MPSD group, a part is done in several collaborations (open group concept). Thus the work is done in parallel with the following complementary methods:
Structural biology studies
Biological activity studies
Biophysical studies
Biochemical preparations
Chemical synthesis

Structural biology studies

- structure investigation of proteins in solution, e.g. by neutron- and X-ray small angle scattering, EXAFS / XANES:
 .. F1ATPase from Micrococcus luteus
 .. ATP-synthase from Rhodospirillum rubrum and Micrococcus luteus in detergent solution (TDOC)
 .. monomeric Bacteriorhodopsin from Halobacterium salinarium (halobium) in detergent solution (TDOC)
 .. Cytochrome oxidase from Micrococcus luteus in detergent solution (TDOC)
- structure investigation of lipid membranes (liposomes) during energization (pH-jump) by time resolved neutron scattering
- structure investigation of working F1ATPase during ATP hydrolysis by time resolved X-ray small angle scattering (TR-SAXS after ATP addition ().
- time resolved XANES / EXAFS of metal proteins for detection of fast motions
- freeze fracture electron microscopy of liposomes (with and without protein) and native membrane preparations.

Biological activity studies

.- estimation of the three coupled activities of ATP-synthase: proton-transport, ATP-synthesis (F16) and ATP-hydrolysis. Only the estimation of the functional coupling of these three activities can exclude artefacts, e.g. by structural demage during protein isolation. Those studuies have been done with ATP-synthases from beef heart mitochondria (F17, F22), Micrococcus luteus (F23, F18) and Rhodospirillum rubrum (F3).
 - estimation of ATP hydrolysis of the F1-fragment (F1ATPase) of ATP-synthase, e.g. from Micrococcus luteus (F8, F18, F20)
 - estimation of proton related redox activities of cytochrome oxidoreductases, e.g. Ubiquinol-oxidase and Cytochrome-c oxidase from Micrococcus luteus (F19) or Cytochrome-o complex from Rhodospirillum rubrum (F12).
 - estimation of proton transport across membranes under stress, e.g. after energization by a pH-jump using a stopped flow rapid mixing device or flash photolysis of caged acids (T15, T20).
- Photolabeling of proteins and membranes, e.g. with fluorescent photoaffinity labels (F15) or metal labels

Biophysical studies

- spectroscopy of metal proteins (terminal oxidases in respiration), time resolved spectroscopy
- redox titration of oxiddo-reductases
- time resolved estimation of proton fluxes by light spectroscopy of dye entrapped liposomes after a pH-jump (stopped flow addition of acid, acid generation by caged acids).
- BLM (black lipid membrane, planar lipid membrane) electrophysiological investigations, especially with membrane proteins (ATP-synthase) and lipid analogs / pharmaceutical compounds
- EPR study of terminal oxidase

Biochemical preparations

The biochemical preparations are the prerequisite of any other studies. Especially the time resolved structure investigation of working proteins  require a very high quality of the isolated proteins: highly pure (>98%), native (fully functional) and kinetically homogenous preparations, even with the difficult integral membrane proteins. As presented in T20 the required knowledge was obtained by the MPSD group in more than 50 "man-years" work (see thesis list). In some cases the precedures were recently developed to the biotechnical level for structural biology investigations (up to 200 mg F1ATPase, ATP-synthase and terminal oxidases from Micrococcus luteus, monomeric Bacteriorhodopsin from Halobacterium salinarium).
Due to the complex properties and molecular interactions, the scientific work with membrane proteins requires six steps for the deliviry of the scientific objects:
- growth of cells, if required after genetic manipulation: In the MPSD group we grow the bacteria: Micrococcus luteus, Rhodospirillum rubrum, Halobacterium salinarium (S9 from D. Oesterheldt).
- isolation of membranes, if required from organelles (eucariotes, e.g. beef heart mitochondria)
- isolation and purification of proteins, in case of membrane proteins with detergents
- isolation and purification of lipids, if required chemical (semi)synthesis
- preparation of model membranes from lipids (liposomes, BLM)
- reconstitution of membrane proteins into model membranes (liposomes)

Chemical synthesis

- synthesis of caged acids (proton) for the generation of a membrane proton potential (energization) by a pH-jump after flash illumination. This enables us to experiments with membrane energization without any "helper protein" (e.g. Bacteriorhodopsin) or shear stress (stopped-flow), as required for time resolved structure and function investigations of membranes and membrane proteins after energization by a pH-jump. A recent application is the time resolved neutron scattering of liposomes, by which a proton potential dependence of the lipid membrane structure was detected.
- synthesis of caged nucleotides, caged-ATP, caged-ADP, and HPLC purification for time resolved structure and function investigations with flash-photolysis activation of the biological system.
- synthesis of fluorescent and coloured hydrophobic photoaffinity labels (F15, T20)
- synthesis of metal labels for proteins , lipids and polymers: quasicovalent chelate labels bearing heavy metals, e.g.Europium (Eu), Terbium (Tb), Gadolinium (Gd), Samarium (Sm), Iron (Fe), Uranium (U). These are use for X-ray structure investigations (ASAXS, MAD phasing, EXAFS / XANES) and fluorescence studies (in case of Eu the fluorescence is a strong phosphorescence of 0.5 ms decay !).

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email to:   update : 15.10.2012