TR&D Project 1. The Sample Stage: Tools for Isolating and Preserving Macromolecular Hierarchies Any given macromolecule may make stable, dynamic or transient interactions with other macromolecules. These interactions form a hierarchical network, comprising the cellular interactome; and this whole network is surrounded by a macromolecular milieu of other complexes that jostle with it, and can form vicinal interactions. TR&D1 represents the first module of our pipeline, where the challenge is to preserve with high fidelity and isolate the local native intracellular macromolecular environment of any macromolecule of interest. To access and then isolate a macromolecule and its interactors in their native hierarchical assemblies, we must break open the cell. However, this disrupts the cell?s large-scale organization; and factors such as dilution, change in milieu, the intermingling of normally segregated components, loss of energy regeneration, and exposure to degradative enzymes cause disassembly, scrambling and aggregation. All these factors worsen as the time lengthens between cell disruption and analytic isolation of the assemblies. Our solution is both to capture as rapidly and efficiently as possible these assemblies, and preserve their native state as much as possible. To accomplish this, we will produce optimized reagents for the ultra-efficient detection and capture of assemblies. We will continue to develop baits for both widely-used existing tags and targets for which the addition of tags are not practical. As well as other affinity capture reagents, our Center provides offers a unique BTRR service in its platform to generate nanobodies, outstanding baits for affinity capture approaches. We will also continue to develop methods for faithfully preserving native cellular interactions for subsequent isolation of endogenous complexes. We accomplish this by freezing complexes in place inside the cell in a state of ?suspended animation?, then cryomilling to access the cell?s complexes while they are still frozen; these complexes must be isolated quickly, cleanly and conveniently from its surrounding cellular lysate in such a way as to preserve the desired characteristics of the complex while minimizing the adherence of additional contaminating materials. Here, we will develop means for rapidly optimizing these affinity captures, which are both portable to (i) bench scale for any other researcher and (ii) readily accessible, affordable robotic platforms to maximize throughput and reproducibility. Because these technologies must be readily accessible to any laboratory, we are focusing on establishing simple, robust and widely used reagents and approaches for the efficient and rapid isolation of macromolecular complexes. We wish to extend this progress and develop enabling new technologies to allow any researcher to readily and comprehensively access the information in the dynamic interactomes of both normal and diseased living systems.
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