Membrane Pharmacy Structure Dynamics(MPSD)
Research group : Priv.Doz. Dr. Thomas Nawroth
[ Impressum-Disclaimer | Membrane-Proteins
|Membrane proteins investigated by the MPSD group in the last ten years. Currently the work is focussed on bacterial ATP-synthase, its catalytic head fragment F1ATPase and Cytochrome-c oxidase COX (from [T20]).|
Membranes are hetrogenous complexes of lipid bilayers, membrane proteins
and other macromolecules (carbohydrates, cell sceleton elements). The biological
function depends strongly on structure and
the moleclar action between lipids and proteins. The investigation of structure
and function requires a concept of isolation,
purification and reconstitution yielding model membrane systems. This can
be most easily done with bacterial systems, which can be cultured in large
amounts and manipulated, e.g. by moleculare genetics. Because of the complex
dependence between structure and function the investigation of membrane
proteins requires the parallel application of methods of structural biology,
biophysics and biochemistry (functional analysis). This is the interdisciplinary
of biophysical chemistry.
The energy metabolism of most cells (bioenergetics) depends on ion pumping membrane proteins, which are proton pumps in most recent organisms but sodium or other ion translocating proteins in archeae (archaebacteria) and extremophiles. The reason for this is the intrinsic flexibilty of membrane protein structure, which is a consequence of the hydrophobic effect. This results in a binding energy by stripping of hydration water during membrane insertion of hydrophobic molecules but not in a directed force between the membrane components. Thus membrane components can move in the membrane plane with minimal energy input (lateral translation or rotation).
The isolation and purification of the membrane proteins requires the solubilization of the protein by replacement of the lipids by detergents (both are amphiphiles). As shown in the below figure, the native structure and thus the biological function is only retained if weak, non-denaturating detergents of highest chemical quality are used (no peroxides or long chain impurities). The methods and technology of membrane protein purification is subject of the lecture "Methods of membrane biochemistry". The methods for structure research of membranes and membrane proteins in solution are subjects of the lecture "Biophysical chemistry of membranes and membrane proteins". You can learn the technology of membrane protein purification and some techniques for biophysical chararacterization by participation in our practical course "Advanced practicum in membrane biochemistry".
|The purification and investigation of membrane proteins requires the retainment of the function competent structure (native), which is obtained by solubilzation with weak, non-denaturating detergents [T20].|
ATP-synthase : The enzyme (M=500,000) converts the energy of the ion transmembrane transport to that stored in the energy rich compound ATP during molecular rearrangements. F1ATPase is the catalytic head fragment of ATP-synthase (M=400.000). The pumped ions are mostly protons, but as found by Prof. P. Dimroth, in some cases sodium ions are transported. The static structure of an ADP-inhibited modification of F1ATPase was resolved with atomar resolution by X-ray crystallography (Nobel avard: J.E: Walker 1997). Nevertheless the reaction mechanism is essentially unknown. The reson is the lack of time resolution in the very most studies. We investigate molecular motion inside ATP-synthase and F1ATPase by time resolved X-ray small angle scattering after activation by rapid mixing methods (stopped-flow) or flash photochemistry (caged ATP, caged acids - proton). This yields a structural film of working ATP-synthase. The structural dynamics upon molecular regulation of ATP-synthase are investigated by comparative static X-ray small angle scattering. The structure of reconstituted ATP-synthase in situ is studied by Neutron small angle scattering of contrast matched liposomes.
Cytochrome-oxidase COX, Quinol-oxidaseQOX (Cytochrome-o) and Cytochrome-c reductase (Cytochrome-bc1 complex) are Cytochrome-group containing oxido-reductases, which combine an oxidation of external coenzymes (ubiquinol, cytochrome-c) with the proton membrane transport in oxidative phosphorylation or photosynthesis (bacteria). We investigate molecular motions of Cytochrome-c oxidase COX and Quinol-oxidase QOX [F19] from Micrococcus luteus during regulation by comparative X-ray small angle scattering of enzyme modifications (oxidized, reduced, inhibited ...). Structural dynamics have been shown in cooperation with PD. Dr. W. Doster by time resolved optical spectroscopy after flash irradiation of carbon monoxide (CO) inhibited Cytochrome-c oxidase COX from Micrococcus luteus. In cooperation with PD. Dr. T. A. Link this was compared to Cytochrome-c oxidase from beef heart mitochondria. The Cytochrom-o complex [F12] and the Cytochrome-bc1 complex (Cytochrome-c reductase) from Rhodospirillum rubrum have been investigated by biochemical methods.
Bacteriorhodopsin is a light
driven proton pump and the parent of the 7-helix receptor protein family.
The protein of 27,000 mass burries a retinal chromophor in the center of
a hollow protein cylinder, which consists of seven amphipatc helices (the
outer side is hydrophobic). In Halobactium salinarium (formerly
called Halobacterium halobium) the protein is arranged in natural
2D-crystals, which consist of a hexagonal arrangement of Bacteriorhodopsin
trimers and 15% membrane spanning lipids. These specialized membrane domains
are called "purple membrane" PM, which is
the operative unit of halobacterial photosynthesis. Bacteriorhodopsin is
extremely stable: during our ERA sub-experiment [F21],
purple membrane samples survived a 6 month flight in space (extreme vacuum
an cold, dark (no sun light) samples). For biochemical and structural investigations
we solubilize the purple membrane by detergent and purify the otained monomeric
Bacteriorhodopsin mBR bei HPLC [F5].
The stzucture of mBR in detergent solution is invetigated by neutron small
angle scattering, X-ray small angle scattering and time resolved flash-photolabeling
using our 3-domain photolabels (photoreactive, selective, detectable).
Those "lantern-labels" are available with metal-heads, detectable with
anomalous X-ray scattering (Eu, Tb), Mössbauer spectroscopy (Fe) or
NMR (Gd), and with fluorescent or colored head groups (dansyl, dabsyl ...)
[F15]. The labels can be introduced in
polymers for the detection of molecular motions in "motile polymers".
|The reconstitution of purified membrane proteins into liposomes as model membranes is done by our "detergent incubation of preformed liposomes" procedure [F3]. Proteoliposomes for functional analysis [F16] may contain several membrane proteins [F17,F18,F22,F23], whereas those for structue investigation contain only one protein molecule in small unilamellar vesicles (SUV) [F3,F9].|