In the last year we have developed a technique for measuring macromolecular size-distributions in concentrated solutions. The goal was to study proteins at concentrations closer to the intracellular environment, where weak interactions can govern a wide spectrum of behaviour, including dynamic multi-protein complex formation and liquid-liquid phase transition. A major difficulty for studying macromolecules at high concentration are thermodynamic non-ideality and long-range hydrodynamic interactions, which couple the motion of one molecule to the motion of all others. This coupling invalidates linearity assumptions underlying size-distribution analyses with any previous biophysical method. We have discovered a computational strategy to extract the necessary information from the evolution of sedimentation velocity boundary profiles, using a mean-field approximation in order to quantify nonideality parameters and simultaneously determine particle size distributions. This presents the first method that can report on both polydispersity and macromolecular interactions in the nonideal regime. The new approach can exploit the strongly size-dependent hydrodynamic resolution of sedimentation velocity, and extends the concentration range of the method by more than an order of magnitude. The development of improved 3D printed sample holders was necessary to enable sedimentation experiments at high concentrations, avoiding optical abberations from ensuing refractive index gradients. In other work, we have continued our collaboration with the National Institutes of Standards and Technology (Dr. Jeffrey Fagan and Dr. Thomas LeBrun) to develop a lithographic mask on sapphire substrate as a standard reference material for radial calibration in AUC. This radial calibration completes the set of calibration measurements that is indispensable to accurately measure macromolecular sizes and hydrodynamic shapes. To disseminate knowledge of analytical ultracentrifugation we have made this technique a major focus in our workshops at NIH and the FEBS Practical Course in Prague, distributed data analysis software SEDFIT with new models, maintained an email user-list and discussion group of 800 colleagues, and shared cell designs at the NIH 3D Print Exchange.
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