Wyatt Technology Corporation has announced that its instruments have been selected by scientists at the Department of Biochemistry, University of Texas Health and Science Center, San Antonio to study the dynamics of macromolecular complex assembly and disassembly.
A new application note details how, with the aid of Wyatt Technology’s DynaPro Dynamic Light Scattering instrument, the researchers studied the underlying mechanisms by reconstituting clathrin coat assembly and disassembly from purified components. They found that the new method gave comparable results to the traditional sedimentation method, but in a fraction of the time and using a fraction of the material.
The aggregation and bonding together of sets of macromolecules to form a complex (known as macromolecular complexes) provides the backbone of most cellular processes, including gene regulation and signal transduction. The intrinsic complexity of the assembly of macromolecular structures makes it difficult for biomedical engineers to ascertain the physical interactions between the components of the cell.
In order to understand the process better, modern biology needs to advance from the reductionist approach into the systemic properties of biological systems in order to understand how the dynamics of cellular processes emerge from the interactions among different molecular components.
For this particular application, Wyatt’s Dynapro Dynamic Light Scattering (DLS) instrument measured clathrin coat assembly, since it was able to distinguish individual clathrin triskelia from assembled clathrin coats. Clathrin coat assembly reactions were run with different concentrations of AP180 clathrin assembly protein, and subsequently analyzed by the Dynapro or by the conventional sedimentation assay. Both approaches gave very similar results.
Further tests were run to see if DynaPro could be used to study the kinetics of clathrin coat disassembly. The kinetics were monitored in real time in the DynaPro instrument and the data fit compared against single exponential decays.
Unlike the sedimentation method, which only permits endpoint measurements, the DynaPro made it possible to measure the effects of the different mutations on the rate at which Hsc70 disassembles clathrin coats quantitatively. This approach should be adaptable to real time monitoring of any macromolecular complex assembly/disassembly reactions.