One of the big challenges in Drug Discovery today is that all current life science tools working at the molecular level either measure static structure or average binding kinetics; the crucial and often very complex mechanical details of the underlying dynamic process are often not revealed. Dynamic Single-Molecule (DSM) analysis reveals these details. This reduces the risk of failure of drugs in costly late-stage clinical trials, thereby decreasing the overall cost of drug development dramatically.
DSM analysis has the unique ability to combine live imaging, manipulation and force-displacement measurement with Ångström precision for the study of DNA-protein interactions and protein conformational changes. This combination gives the crucial dynamic and functional mechanistic information that is complementary to structural data (X-ray crystallography) and ensemble average kinetics (SPR or calorimetry). In pharma, DSM can play a significant role in two stages of the Drug Discovery Process: Target Validation, and Hit-to-Lead.
Probe and film DNA-protein interactions in real-time at the single-molecule level
Scientists can use optical tweezers to trap beads and catch a biomolecule, such as DNA, in between. This biomolecule can then be manipulated by moving the beads, while the force and extension are measured. Fluorescently labeled proteins can be visualized with confocal or STED fluorescence microscopy. The combination of optical tweezers with simultaneous multicolor fluorescence measurements allows correlating the mechanical properties of the DNA with the protein activity.
The C-Trap provides the ability to apply and measure force and extension of the tethered DNA while simultaneously visualizing proteins as they interact with the DNA. In this way, it is possible to film proteins and directly characterize their effect. When DNA-protein interactions are measured at the single-molecule level the exact mechanisms of DNA organization, replication, translation, and repair can be studied in high detail, including conformational changes.
The film in Figure 1 shows the position of bound XRCC4 and XLF on DNA over time at protein concentrations of 5 nM. These are two repair proteins involved in non-homologous end joining which can associate with each other to form complexes capable of bridging DNA. The film shows XLF-XRCC4 protein tandems binding to double-stranded DNA and rapidly diffusing as they are searching for damage sites.
Expore the posibilities:
- Achieve better target validation and understanding of the druggable mechanism of action early in the process by investigating the complex dynamic molecular mechanisms of DNA-protein interactions.
- Visualize and measure the dynamics of DNA-protein interactions under the influence of pharmaceuticals to gain insight into the drug’s mechanism of action.
Study protein conformational changes in real-time at the single-molecule level
Scientists can use optical tweezers to trap beads and catch a protein in between. The folding and unfolding of the protein can then be monitored by moving the beads while measuring the force and extension. The combination of optical tweezers with simultaneous multicolor fluorescence measurements allows correlation of the global mechanical properties of the protein with the local structural properties.
The C-Trap system provides the ability to apply and measure force and extension of a protein target while simultaneously obtaining the fluorescence signals from e.g. FRET fluorophores. This allows one to identify the (in)active and intermediate states and resolve the interaction energies of proteins. All these together provide important insights into the protein’s functional mechanism. Because of the C-Trap’s unique microfluidics system, this can be done under different experimental conditions: in the presence or absence of (ant)agonists, co-factors and/or pharmaceuticals, within the same experiment.
Figure 1 shows the obtained equilibrium dynamics trace of calmodulin – a calcium-binding protein. The graph reveals that calmodulin switches between two major states, an open and a closed one, without a clear preference. We can resolve intermediate steps as calmodulin occasionally jumps to a third state for short periods of time.
1 Full-length CaM protein at 10 mM Ca2+ showing equilibrium dynamics between multiple states, represented by the dashed grey lines. Data is recorded at 50 kHz (grey line) and averaged at 200 Hz (red line). The histogram quantifies the most populated states in the inset (right panel) showing two peaks at 6.5 ± 0.1 pN and 7.8 ± 0.09 pN (mean ± standard deviation).
Explore the possibilities:
- Study the mechanism behind the biological function of proteins by looking at the conformational changes of protein targets, in real-time and with Ångström resolution.
- Observe conformational changes of protein targets in the presence of different pharmaceuticals. Find out whether the desired mechanism(s) of action is fulfilled by the drug in a quick, easy and effective manner.