Inhibitor solubility assay
Blebbistatin has low solubility in aqueous solutions, which hinders its use in several cellular and in vivo assays. We optimize the structure of our new compounds to achieve significantly higher solubility, enhancing their applicability. Solubility of the new derivatives is determined by measuring the absorbance spectra of the soluble fraction under different solvent conditions. Analysis of the absorbance maximum of each of the derivatives shows a linear dependency on the applied concentration until their solubility limit. Above the solubility limit maximum absorbance values become constant or even show an inverse dependency on concentration at higher concentrations due to aggregation of the precipitating molecules.
Motor protein expression
We express recombinant human non-muscle myosin 2 (NM2) isoforms NM2A, NM2B and NM2C in Sf9-baculovirus expression system. NM2 constructs consist of the myosin motor domain supplemented with an artificial lever arm of alpha-actinin, which mimics the fully activated form of NM2s in the absence of light chains. We also express recombinant Dictyostelium discoideum myosin 2 motor domain in Dictyostelium expression system because most of the structural insights on the working mechanism of myosin 2 and the structural background of blebbistatin inhibition have been studied with the various mutant forms of this protein.
We purify NM2A, NM2B and NM2C human myosin isoforms with FLAG-tag affinity chromatography. The FLAG-tag has been cloned to the C-terminus of the proteins in order to purify intact, full-length myosins. Dictyostelium myosin 2 motor domain is purified with Ni-NTA affinity chromatography after serial isolation steps including a final selective detachment of actin and myosin. Beside the recombinant myosin 2 isoforms we purify fast skeletal muscle myosin 2 from rabbit psoas muscle, smooth muscle myosin 2 from chicken gizzard, and cardiac myosin 2 from porcine left ventricle using the well established purification protocols. We perform limited proteolysis on skeletal and cardiac muscle myosin 2s with α-chymotripsin and on smooth muscle myosin 2 with papain to achieve S1 fraction (containing the motor domain, the lever arm, the regulatory- and the essential light chains). This step is followed by anionic exchange chromatography to obtain high purity S1 fractions. We also collaborate with other laboratories to test our compounds on human skeletal and cardiac myosin 2 isoforms.
The basic biochemical test for all new compounds is the measurement of their effect on the actin activated ATPase rate of seven different myosin isoforms (expressed human NM2A, NM2B, NM2C and Dictyostelium myosin 2, and tissue purified rabbit fast skeletal muscle myosin 2, chicken gizzard smooth muscle myosin 2 and porcine left ventricle cardiac myosin 2). We perform the measurements in a 384-well plate format using a PK/LHD coupled ATPase assay. The inhibitory effect of all molecules is determined at 50 μM inhibitor concentration and the ones that show more than 20% inhibition are characterized in more detail.
We utilize versatile intrinsic and extrinsic fluorescence signals during the ATPase cycle of myosin in the absence and in the presence of actin. Time resolved fluorescence, steady-state and equilibrium fluorescence measurements are performed in our state-of-the-art fluorimeters. We are especially interested in whether the differences in the structure of our new inhibitors (compared to blebbistatin) change the molecular mechanism of inhibition (e.g. whether the blocked state remains the same). We are also keen on elucidating the effect of our molecules on the distribution of myosin heads between their open and super relaxed states (SRX), most importantly in cardiac and skeletal myosins.
Rapid enzyme kinetics
In order to characterize the detailed kinetic parameters of the actomyosin ATPase cycle – especially in the presence of the different new inhibitors – we follow pre-steady-state reactions using an array of rapid kinetic instruments. We use our stopped-flow apparatuses to measure fast reaction steps by following intrinsic and extrinsic fluorescence signals. We can also perform fast kinetic experiments at temperatures that are higher than the denaturation temperature of the proteins in our temperature-jump-stopped apparatus of which principles had been developed and published by our group. This allows us to more precisely characterize the thermodynamics of enzymatic reactions and also enables us to measure enzyme reactions at their native temperatures that may be higher than the optimal measuring temperature of the isolated myosins. Furthermore, we can also perform sophisticated quench-flow experiments detecting either fluorescence signals or radioactivity form isotope-labeled nucleotides.