Expertise in Structural Biology
Liquid nuclear magnetic resonance spectroscopy
Liquid nuclear magnetic resonance (liquid NMR) spectroscopy is a powerful spectroscopic technique that allows a three-dimensional structure elucidation of a given molecule at atomic resolution. During NMR studies the solubilised molecules under investigation are applied to a constant and high magnetic field. NMR spectroscopy relies on the absorptiometry of electromagnetic radiation in the radio frequency region of magnetically active nuclei, like 15N, 1H and 13C. The absorbed radio frequency depends strongly on the chemical environment of the magnetically active nucleus which is influenced among others by its chemical bonds as well as molecular conformations. Thus, absorption frequencies provide important spectral information about the three-dimensional structure of the molecule under investigation.
Extraordinary advances occurred during the past decade in structural biology by liquid NMR spectroscopy. The precision and accuracy of solution structures by NMR improved dramatically and the upper range of molecular weights accessible to NMR structure determination is still increasing. In structural biology NMR is applied to study high resolution, three-dimensional structures of biological macromolecules like proteins as well as the structural basis of affinity and specificity of protein-protein or protein-ligand interactions. In addition, NMR can provide a realistic dynamic view of interfaces of these interactions or the transient intramolecular interfaces seen during protein folding which are not available by other methods. The determination of three dimensional structures as well as the investigation of interacting proteins of biologically and medically relevant proteins with NMR allow the prediction of hitherto unknown functions of these proteins, e.g. of viral proteins of HIV and SARS coronavirus. A detailed insight into the mechanism of interaction of viral proteins with host cell factors by liquid NMR allows the identification and optimization of artificial ligands of the host cell proteins. This may result in new drugs that block specific host virus interactions.
A - Preparation of a regulatory protein 7a of SARS-CoV in thin walled glass tube for NMR measurements
B – Insertion of the NMR tube into the probe of our high field (800 MHz) NMR spectrometer
C – 1H- 15N-HSQC NMR spectrum of SARS-CoV 7a
D – Three-dimensional solution structure of the according protein
For further information see:
K. Hänel et al. (2006), J. Biomed. Sci. 13, 281-293; K. Hänel & D. Willbold (2007), Biol. Chem. 388, 1325-1332
The technique of analytical ultracentrifugation (AUC) was developed by Svedberg and Lysholm in 1927. With AUC one is able to determine molecular weight, shape and stoichiometry of macromolecules and macromolecular complexes. AUC is an absolute method not depending on standards for comparison.
The method offers the opportunity to retrieve structural information about biological macromolecules in solution for a broad concentration range and under a wide variety of solvent conditions. The hydrodynamic behaviour of macromolecules under the influence of centrifugal forces is determined by their molecular size and molecular shape and the physical and chemical properties of the solvent.
Two different experimental protocols are used:
- Sedimentation equilibrium centrifugation (SEC) is utilized to determine the molecular weight of a molecule or a molecular complex.
- Sedimentation velocity centrifugation (SVC) or moving boundary sedimentation is utilized to determine sedimentation coefficients or sedimentation coefficient distributions by observation of the particle behaviour during the sedimentation process.
Figure: XL-A from Beckman-Coulter (www.beckmancoulter.com). On the left side a number of radial cell scans are shown as obtained during a sedimentation velocity run. On the right side the setup within the rotor chamber with rotor and optics arm is shown.
Fluorescence correlation spectroscopy
Fluorescence correlation spectroscopy (FCS) is a method to detect molecular parameters like e.g. diffusion time, diffusion coefficent. Threrefore FCS can be used to analyse interaction of molecules (Elson & Magde, 1974; Magde et al., 1974; overview: Schwille & Haustein, 2001). In Fluorescence Correlation Spectroscopy the fluctuation of the fluorescence intensity is recorded in a very small volume, i.e. in femtoliter range via a confocal optic system. The photon statistic method FIDA (Fluorescence Intensity Distribution Analysis) was developed in 1999 by Kask (Kask, P., Palo, K., Ullman, D., and Gall, K. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 13756-13761) and in parallel by Chen et al. (1999) Biophys. J. 77, 553-567, where it was named PCH (Photon Count Histogram). This method deliver detailed information about a fluorescent tracer diffusing through the open 3D volume of the size of one bacteria (roughly one femto-liter). As long as enough fluorescent molecules are available (nano-molar range) this methods are well suited for diagnostic or drug discovery.
Surface plasmon resonance spectroscopy
The Surface plasmon resonance (SPR) method is based on the physical phenomenon of surface plasmon resonance (SPR) and detects binding of molecules to a surface.
In the case of total reflection a so called evanescent field with limited depth of penetration (~ 300 nm) is generated. If the resonance conditions are met this evanescent field is able to interact with the surface plasmon of a thin metallfilm. Thereby the resonance conditions depent on the following parameters: (i) angle of incidence, (ii) refractive indices, (ii) wavelength. In the SPR instrument the wavelength is constant and therefore changes in the refractive index near the surface can be followed by measuring changes in the resonance angle. Because mass-adsorption to the surface leads to changes in the refractive index the Biacore instrument can be used to measure time-resolved binding of biomolecules to a surface.
The resulting sensorgramm is a plot of the SPR-signal in relative units (RU) against time. A relative unit is equivalent to 1 pg of molecule bound to the surface of the flow-cell which has an area of 1 mm
Figure:Total reflection: I = I0, attentuated total reflection: I < I0 (SPR)