Techniques

Understanding the interplay between structure and function of kinases is essential for the analysis of complex cellular signal transduction events. The main focus of our group is research on the molecular basis to fine tune kinase activity employing biochemical, biophysical, cell biological and bioinformatics tools all summarized under the term Biomolecular Interaction Analysis (BIA). In Kassel BIA encompasses surface plasmon resonance (SPR, Biacore technology), fluorescence polarisation (FP), isothermal titration calorimetry (ITC), bioluminescence resonance energy transfer (BRET), FRET and microfluidic electrophoretic mobility shift assay (MEMSA). Those BIA–techniques are applied in several research grants funded by the Deutsche Forschungsgemeinschaft (DFG), the German Federal Ministry of Education and Research (BMBF), the European Union, the Michael J. Fox Foundation for Parkinson’s Research as well as the University of Kassel.
The group has a long standing expertise in particular in surface plasmon resonance, a technique that is exquisitely sensitive and capable of monitoring the association and dissociation rate constants of biomolecular interactions. We run several Biacore instruments as well as SPR-devices from Sierra Sensors and are in mutual exchange with the Biaffin GmBH & CoKG, a spin off from the Biochemistry department. In addition, the group is part of the Center of Interdisciplinary Nanostructure Science and Technology (CINSaT), generating miniaturized devices for advanced biosensing at increased sensitivity and throughput. This includes devices combining SPR with magnetooptical effects (MO-SPR) as well as nanocrytalline diamond surfaces in biotechnological applications.

• Merker, D, Kesper, M, Kailing, ML, Herberg, F, Reithmaier, JP, Pavlidis, IV, Popov, C Nanostructured modified ultrananocrystalline diamond surfaces as immobilization support for lipases Diamond & Related Materials 90 (2018) 32–39)

We recently developed a Bioluminescence Resonance Energy Transfer (BRET)-based assay to investigate protein-protein interactions of PKA regulatory (RI/RII) and catalytic (Cα/PrKX) subunits in living mammalian cells (Diskar et al., 2005; 2006; 2007; Prinz et al., 2004; 2006a; 2006b). It is based on the physical principle of resonance energy transfer, which was first described by Förster in 1948 (Förster, 1948).

BRET is a proximity assay based on the detection of energy transfer between Renilla luciferase (RLuc) used as the donor protein and a variant of Green Fluorescent Protein (GFP, here: GFP²) as the acceptor. Upon oxidation of the substrate Deep Blue C, a coelenterazine derivative, RLuc emitts light at 410 nm. If GFP² is in close proximity to the luciferase, part of the light will be transferred by resonance to GFP², and, in turn, GFP² will emitt light at 515 nm. Both proteins have been genetically modified and optimized for the use this assay and for high expression results in eukaryote cell culture.

We have designed and cloned various vectors expressing recombinant proteins using the BRET system, by fusing the coding sequences of mainly human RI-, RII and C- subunits of PKA to the N- and C- terminus of either GFP² or Rluc.
When RLuc and GFP² are expressed as fusion proteins in a cell, the interaction event of the target proteins (here: RI/RII- and C- subunits of PKA) can be measured by the signal ratio of green over blue light (515/410nm) using an α-Fusion™ multilabel reader.

An advantage of BRET over the earlier developed FRET (Fluorescence Resonance Energy Transfer) is that BRET does not require excitation by an external light source, thus eliminating photo bleaching and high fluorescent background problems in the cell-based assay (Prinz et al., 2008).

We successfully established a medium throughput assay system in 96-well microplate format using the BRET-technology suitable for probing cAMP analogs affecting the activity of PKA by enhancing or attenuating the dissociation of PKA holoenzyme (Gesellchen et al., 2006; Moll et al., 2006; Prinz et al., 2006a; 2006b). The assay was also utilized for detecting anchoring dependent signalling propagation mediated by A-kinase anchoring proteins (Hundsrucker et al., 2006; Prinz et al., 2006a) and isoform specific regulation of PKA (Diskar et al., 2007).

More literature:
- Förster, T. (1948) Zwischenmolekulare Energiewanderung und Fluoreszenz. Ann. Physik 2, 55-75.

In biological and medical research the essential aspect of fluorescence polarisation (FP) is, that FP provides information on the rotational mobility of a fluorescently labelled molecule. Since the mobility of the fluorescent molecule changes upon binding to a large macromolecule, one can utilize FP to determine and quantify biomolecular interactions. Consequently, fluorescence polarization has been used now for many years to study biochemical systems, for example protein-protein and protein-ligand interactions (Jameson et al., 2003; Moll et al., 2006c). The advantages of this method are that the assay is inexpensive (low sample amount needed) and rapid with a sensitivity close to classical radioligand binding assays in a homogenous high throughput format. The theoretical principles of FP (or fluorescence anisotropy, which are both describing the same physical phenomenon) have been extensively described (Lakowicz, 1999; Moll et al., 2006a, 2006b; 2006c).

Since 2006 we establish in our group direct and indirect FP assay formats to basically determine the binding of labelled and unlabeld cyclic nucleotides (e.g. cAMP and derivatives) to cyclic nucleotide binding proteins and domains thereof (Moll et al., 2006a).

So far we therefore measure the affinity of hundreds of cyclic nucleotides to cAMP dependent protein kinase (Moll et al., 2006a; 2008; Schweinsberg et al., 2008), Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels (Lolicato et al., 2011) and cGMP-dependent protein kinase.

More literature:
- Jameson, D.M. and Croney, J.C. (2003). "Fluorescence polarization: past, present and future." Comb Chem High Throughput Screen 6(3): 167-173.
- Lakowicz, J.R. (1999). "Fluorescence Anisotropy. Principles of fluorescence Spectroscopy." New York, Kluwer academic/Plenum Publishers: 291-319.
 

Isothermal Titration Calorimetry (ITC) is a label free in vitro method to characterize an interaction, e.g. between a receptor and a ligand. The method based on monitoring the released (or absorbed) heat upon ligand binding. Therefore, a solution of ligand molecules is titrated into a fixed concentration of receptor molecules (Figure left). A thermodynamic profile of a given interaction can be calculated derived from the produced heat per ligand injection (Figure right side top). In detail, the change in free Gibbs free energy (ΔG, proportional to equilibrium binding constant), the enthalpy change (ΔH), the stoichiometry (n) and the resultant entropic content of the interaction ( TΔS) can be calculated (Figure right bottom). These values allow insight into the driving forces of an interaction, and, combined with structural information provides evidence of the formation and breaking of individual noncovalent-bonds as well as the dynamic and flexibility of the system adding crucial pieces to a full understanding of the chemical basis of an interaction in a structure function approach.

Kinase assays are essential to analyze enzymatic activities and kinetics in vitro. Using a microfluidic electrophoretic mobility shift assay (MEMSA) the phosphorylation of substrate peptides can be monitored in real-time with high sensitivity. In an ‘off-chip’ mode, reaction components are prepared and subsequently sampled by capillary ‘sipper’ tubes in a vacuum-driven flow. By applying two different voltages, an electric field is generated. The additional negative charge of phosphorylated peptides leads to a mobility shift in the microfluidic system. Therefore, the non-phosphorylated substrate peptides can be separated from the phosphorylated product peptides and subsequently quantified. To allow for sensitive detection, a certain amount of the applied substrate peptide is fluorescently labeled.
In the Biochemistry department this technique is primarily used to the analysis of protein kinases with relatively low specific activity i.e. LRRK2, MAST kinase and PKG.

In the Biochemistry department several Biosensors based on the principle of Surface Plasmon Resonance (SPR) are employed. We are running a Biacore 3000, a T 200 System and a Sierra SPR sensor device.
SPR arises when light illuminates thin conducting films (gold is used in the case of Biacore instruments) under specific conditions. The resonance is a result of the interaction between electromagnetic vectors in the incident light and free electron clouds, called plasmons, in the conductor. SPR can arise because of a resonant coupling between the incident light energy and surface plasmons in the conducting film at a specific angle of incident light. Absorption of the light energy causes a characteristic drop in the reflected light intensity at that specific angle (Figure 1).
The resonance angle θ is sensitive to a number of factors, including the wavelength of the incident light, the nature and thickness of the conducting film, and the temperature. Most important for this technology, the angle depends on the refractive index of the medium opposite to the incident light. When other factors are kept constant, the resonance angle is a direct measure of the refractive index of the medium. Only the angle on which SPR occurs is altered and detected with the diode array detector; the intensity of the “shadow” in the reflected light is unchanged. One thousand RU correspond to a 0.1° arc in the SPR angle.

Interaction analyses are performed between a “ligand”, covalently or non-covalently coupled to a sensor chip and the “analyte”, molecules in a flow phase. Figure 2 describes the principle of the interaction for a standard application.

While the interactions of protein to proteins or Protein DNA interactions are readily to be determined the use of SPR for small molecules is more difficult since in principle SPR is amass sensing device. However competition experiments and more sensitive devices have overcome this limitations (Moll, D, 2006).

Besides those classical applications more specialized and advance setups are possible (see Role of metals and nucleotides in the function and control of Protein kinases) SPR is also used to kincetically characterize and optimize cyclic nucleotides analogues as tools for biochemical and pharmaceutical research (see Development of cyclic nucleotide analogs for EPAC, HCN and cdiGMP-binding molecules).

Novel developments combine SPR with magnetooptical phenomena and such approaches are co-developed in collaboration with the Physics department at Kassel University (Kämpf, K, 2012).