Development of Fluorescence Detection In Analytical UltraCentrifugation
Analytical ultracentrifugation, though a classical biophysical discipline, has undergone a renaissance in the last decade due to new computational capabilities and new instrumentation, with increasing applications in structural biology and immunology, for example, for the study of protein interactions and multi-protein complexes, and in the biotechnology industry for the characterization of protein pharmaceuticals and nanoparticles for drug delivery.
Very recently, the Schuck lab has embarked on the use of fluorescence detection in analytical ultracentrifugation (fdAUC). Using a commercial fluorescence detector, in combination with newly developed computational tools, we have achieved unprecedented sensitivity, which allows the characterization of protein assemblies in the low picomolar concentration range, and from unpurified cell extracts. This has great potential to serve as a complementary tool to single-molecule fluorescence microscopy, by measuring the size of macromolecular complexes in fdAUC after their observed co-localization in cellular imaging. Unfortunately, the commercial fluorescence detector is limited to a single excitation wavelength and allows only very constrained sedimentation and detection modes.
We are developing a novel, flexible ultracentrifugal fluorescence detection platform capable of accommodating high-speed multi-channel excitation and emission detection, controlling switchable polarizers and filters, and detecting time-resolved fluorescence. The long-term goal is to integrate this detection platform into a commercial analytical ultracentrifuge system. In the meantime, we are in the process of building a mock-up centrifuge to optimize the optics design, and test data acquisition and signal processing modes.
The design of real-time signal processing requires measurement and synchronization with sample rotation in the microsecond time-scale, sample pre-processing across the nanosecond time scale, control of optical elements for radial scanning in the time-scale of seconds, and storage of data for further analysis across a time-scale of hours. The methods, instrumentation and algorithms accomplishing these tasks will form the backbone for the implementation of different fdAUC modes envisioned above.
We anticipate multiple milestones in this project, each expanding our capability for macromolecular characterization. The first – likely achievable even with the mock-up – will be a stationary AUC detector for rapidly-sedimenting and non-diffusing particles, which will greatly extend the size-range for AUC analysis. After installation in the ultracentrifuge, we anticipate multi-spectral detection in standard sedimentation velocity modes will increase the complexity of interacting protein systems that can be studied. The Schuck lab has several collaborative projects studying molecules from different immunological pathways where this could be tested and immediately exploited, while ongoing work could optimize sensitivity and explore different strategies.
The BESIP student working on this project would have interest in analytical ultracentrifugation, signal processing, optics, and prototyping. The intern will work closely with the interdisciplinary team to further advance the prototype system and conduct initial experiments. Possible tasks include the development of real-time signal processing algorithms to determine rotational speed to a very high accuracy, development of data analysis algorithms, optimization of the optical system, and implementation of new sample centerpieces using 3D printing technologies. Based on the state of the prototype, the intern will also conduct testing using real-world samples and help conduct initial experiments demonstrating the effectiveness of the prototype.
Schuck lab: The Section of the Dynamics of Macromolecular Assembly develops biophysical methods to study protein interactions and the assembly of multi-protein complexes. Hallmarks of multi-protein complexes are multi-valent interactions and cooperativity. In the molecular machinery of cellular processes these constitute ubiquitous mechanisms for the integration and transfer of information. Therefore, our focus is on the development of approaches for multi-component systems where several different macromolecular components interact to allow association and dissociation of different co-existing complexes in different states. We are interested in the characterization of the number of assembly states, and their size, shape, and the interaction energetics. Complementary to crystallographic techniques, such solution interaction studies can provide information on the assembly principles of structurally polymorph multi-protein complexes.
Our group has been particularly active in the development of quantitative hydrodynamic methods using sedimentation velocity analytical ultracentrifugation in conjunction with mathematical modeling of reaction/diffusion/sedimentation processes. We have recently embarked on the development of novel fluorescence detection approaches, which allow unprecedented sensitivity and resolution. We are also pursuing other techniques, including optical biosensing, isothermal titration calorimetry and global multi-method modeling approaches. We are dedicated to the dissemination of methodology and have implemented our computational analysis methods in widely used software.
We are involved in several collaborative applications in various fields including immunological protein complexes, viral proteins, membrane receptor complexes, and eye lens crystallins.
Pohida group: Provides electrical, electronic, electro-optical, mechanical, computer, and software engineering expertise to NIH projects that require in-house technology development. Collaborations involve advanced signal transduction and data acquisition; real-time signal and image processing; control and monitoring systems (e.g. robotics, process automation); and rapid prototype development. Collaborations result in the design of first-of-a-kind biomedical/clinical research systems, instrumentation, and methodologies.