Dynamics of Protein Assemblies by Analytical Ultracentrifugation

National Institute Of Biomedical Imaging And Bioengineering, Bethesda

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Abstract

In the last year we have further improved nonideal sedimentation velocity (nonideal SV) as a technique for measuring macromolecular size-distributions in highly concentrated solutions. The goal was to study proteins at concentrations closer to the intracellular environment, where weak interactions can govern a wide spectrum of behavior, 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 each molecule to the motion of all others. This coupling invalidates linearity assumptions underlying size-distribution analyses with any previous biophysical method. Recently we have developed a mean-field approximation as a computational strategy to tackle the nonideality. This can simultaneously quantify nonideality parameters of particles in solution, and simultaneously determine particle size distributions, and presents the first method that can report on both polydispersity and macromolecular interactions in the nonideal regime. We have now been able to demonstrate that this approach offers an opportunity to the study of weak protein interactions. This was accomplished in sedimentation studies with model proteins with tunable attractive and repulsive electrostatic interactions, and in a study of gamma crystallin. However, we have also discovered a protein size-dependence of the highest concentration that we can study by nonideal SV, which poses a limit of approximately half the serum concentration for antibodies. While this is already a tenfold increase from previous techniques, and opens windows for new applications that have been exploited in different projects, we have pursued strategies to further increase the concentration to reach physiological levels. An experimental problem is presented by optical aberrations in the strong refractive index gradients of the sedimentation boundary. This can be minimized by short optical pathlengths, and therefore we have further improved our custom 3D printed sample holders. We have characterized the stability and precision of these sample holders to ensure they do not interfere with molecular migration in the centrifugal field. Furthermore, we have developed a technique for measuring the magnitude of any optical distortions. Increasing the concentration limit also requires further improvements in computational data analysis, because linear approximations of hydrodynamic interactions fail in suspensions of particles with greater than 5% volume occupancy. For this reason, we have refined the description of hydrodynamic and thermodynamic interactions incorporating higher-order terms derived from statistical fluid dynamics. In other work, we have continued our collaboration with the National Institutes of Standards and Technology to produce lithographic masks 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 hands-on workshop at NIH, started planning an upcoming FEBS Practical Course in Grenoble, distributed data analysis software SEDFIT with the new models, maintained an email user-list and discussion group of >800 colleagues, and shared cell designs at the NIH 3D Print Exchange.