Membrane Pharmacy Structure Dynamics

Research group : Priv.Doz. Dr. Thomas Nawroth

Structural Biology

preliminary [ Impressum-Disclaimer ] Topics: Structure function relation, Overwiew, X-ray diffraction, Small angle scattering of solutions, EXAFS and XANES, Electron microscopy, Electron diffraction, Special techniques

Structure function relation

Fig.1: The description and manipulation of biological function requires three regimes of life sciences: Structural biology, Molecular genetics and Biochemistry or Biophysics (i.e. function analysis / enzymatics, isolation and purification of systems and protein chemistry)

The biological function of proteins, membranes, carbohydrates and nucleic acids depends on the molecular structure at several levels. The chemical structure (sequence of components) and the fold is determined by the genetic code. Nevertheless it is until now impossible to predict the structure of the biopolymers in a satisfying way, nor is it possible to describe the structure dynamics, e.g. molecular motion of flexible systems, e.g. bioenergetic proteins or molecular motors (muscle). Thus the structure of biological systems has to be examined experimentally with respect to the functional state. This is the subject of "Structural Biology", i.e. structure reseach at biological systems. As depicted in figure1, this is one one the three scientific regimes required for the description and manipulation of biological function, preferably in an interdisciplinary concept. As shown in the overwiew table, the techniques of structural biology differ in spatial resolution, restrictions of the status of the sample and in the acceptable heterogeneity of the specimen.

Overview of structural biology methods

method	imaging property	resolution	size of visual domain	homogenous sample required (isolation and purification)	restrictions of sample / specific preparation	time resolution for dynamics estimation possible
X-ray diffraction with monochromatic beam	indirect	0.2 - 0.5 nm	single molecule (20 nm)	yes, 3D- crystals of highly purified, structural homogenous protein, 100 mg material	well ordered 3D- crystals and metal derivatives or anomaouls scatterers incorporated (MAD phasing)	restricted (flash photolysis), at high flux synchrotorns, e.g. ESRF - ID14 beamlines; ELETTRA - 5.1 beamline
X-ray Laue-diffraction with polychromatic beam	indirect	0.2 - 0.5 nm	single molecule (10 nm)	yes, 3D- crystals of highly purified, structural homogenous protein, 100 mg material	well ordered 3D- crystals and derivatives required; restricted to small proteins of high scattering power	yes, e.g. ESRF - ID beamline
SAXS, small amgle X-ray scattering; ASAXS, anomalous small amgle X-ray scattering	no, only by molecular modelling	1 nm	single particle or cluster (500 nm)	yes, solution of highly purified, structural homogenous protein, 1-10 mg material	no, but purified, structural homogenous protein required; domain differentiation in complexes by metal labeling possible	yes, TR-SAXS by rapid mixing (stopped flow technique) or flash photolysis (caged compounds, chromoproteins) at high flux synchrotrons, e.g. ESRF - ID2A, ID1, ID13 beamlines; ELETTRA - SAXS (5.2)
SANS, small amgle neutron scattering	no, only by molecular modelling	1 nm	single particle or cluster, membranes (2000 nm)	yes, solution of highly purified, structural homogenous protein or membranes, 5-50 mg material	no, but purified, structural homogenous protein required; domain differentiation in complexes by specific deuteration or spin labeling possible	yes, TR-SANS at high flux neutron sources, e.g. ILL - D22 , D11 beamlines
inelastic and quasielastic neutron scattering, TOF	no, only by molecular modelling	(1 nm)	single domains in proteins or clusters, membranes	yes, solution or crystal of highly purified, structural homogenous protein or membranes, 10-100 mg material	no, but purified, structural homogenous material required; domain differentiation in complexes by specific deuteration or spin labeling possible	yes, TOF experiments at high flux neutron sources, e.g. ILL - D22 and several other beamlines
Neutron diffraction	indirect	0.3 - 0.5 nm	single molecule (20 nm)	yes, 3D- crystals of highly purified, structural homogenous protein, some 100 mg material	well ordered 3D- crystals and deuterium derivative(contrast phasing)	no; method available only at high flux neutron sources, e.g. ILL
EXAFS and XANES	indirect, only by molecular modelling	0.001 nm	0.5 nm, environment of metal center	restricted, some 100 mg material	sample should be homogenous in metal centers	yes, at high flux synchrotorns, e.g. at ESRF - beamlines ID26 (scanning), ID24 (energy dispersive)
2D/3D- NMR	indirect from NOE's and angles	0.4 nm	single molecule (10 nm)	yes, solution of highly purified, structural homogenous protein, 100 mg material	¹³C- and ¹⁵N-derivatives required; restricted to small proteins (< 40,000 mass with 800 MHz-NMR)	no, until now only by simulation of protein flexibility
FT-IR	no, only by molecular modelling	only in labeled domains	single molecule (20 nm)	yes, solution of highly purified, structural homogenous protein, 100 mg material	¹³C- derivatives required; site specific isotope labeling	yes
microscopy (light)	yes	500 nm ( = light wavelength)	micrometer	no	thin solid sample / cut	yes, video microscopy
fluorescence microscopy (light)	yes	< 500 nm (light wavelength)	micrometer	no	fluorescence labeling, thin solid sample / cut	yes, video microscopy
laser scanning microscopy	yes	150 nm ( = 1/4 wavelength)	micrometer	no	thin solid sample / cut	restricted
confocal laser microscopy	indirect, modelling	1-2 nm (chromophore)	single molecule (20 nm)	yes	single molecule is held in the focus of a laser, radiation demage risk	yes
electron microscopy, negative stain technique	yes	2 - 3 nm	0,1 - 10 micrometer	no	stain with heavy element, may produce artefacts with membranes and flexible proteins	no
electron microscopy, freeze fracture technique	yes, indirect	2 - 3 nm	0,1 - 10 micrometer	no	rapid freezing (vitrification) and subsequent cut and metal sputtering in vacuuum	restricted, by time resolved freezing of the samples
electron diffraction (2D-crystallography)	indirect	0.3 - 1 nm	single molecule (20 nm)	yes, 2D- membrane protein crystals	2D- crystals of vaccum stable sample derivative required	no
AFM , atomic force microscopy	indirect	1-2 nm	0,1 - 10 micrometer	no	solid sample with rigid surface required	no
Photoaffinity labeling, Photoaffinity crosslinking	no	0,5 nm group distance	1-5 nm in single particle or cluster, membranes	recommended for unambigous results	molecule must have a few reactive groups at surface	yes, but not usual; by flash photochemistry
MD , molecular dynamics structure simulation	by theory	< 0.1 nm	single molecule (10 nm)	- (theory)	restricted to small moleules and short times (picoseconds)	yes, may be improved, if more experimental data on molecular motion become available

X-ray diffraction

Small angle scattering of solutions

EXAFS and XANES

Electron microscopy

Electron diffraction

Special techniques

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email to: nawroth@MPSD.de update : 15.10.2013