Kinetic dissection of macromolecular complex formation with minimally perturbing fluorescent probes.

Michael B Cory, Zachary M Hostetler, Rahul M Kohli

Methods in enzymology 2022

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The formation of macromolecular complexes containing multiple protein binding partners is at the core of many biochemical pathways. Studying the kinetics of complex formation can offer significant biological insights and complement static structural snapshots or approaches that reveal thermodynamic affinities. However, determining the kinetics of macromolecular complex formation can be difficult without significant manipulations to the system. Fluorescence anisotropy using a fluorophore-labeled constituent of the biologic complex offers potential advantages in obtaining time-resolved signals tracking complex assembly. However, an inherent challenge of traditional post-translational protein labeling is the orthogonality of labeling chemistry with regards to protein target and the potential disruption of complex formation. In this chapter, we will discuss the application of unnatural amino acid labeling as a means for generating a minimally perturbing reporter. We then describe the use of fluorescence anisotropy to define the kinetics of complex formation, using the key protein-protein-nucleic acid complex governing the bacterial DNA damage response-RecA nucleoprotein filaments binding to LexA-as a model system. We will also show how this assay can be expanded to ask questions about the kinetics of complex formation for unlabeled variants, thus assessing assembly kinetics in more native contexts and broadening its utility. We discuss the optimization process for our model system and offer guidelines for applying the same principles to other macromolecular systems. Copyright © 2022 Elsevier Inc. All rights reserved.

Citation

Michael B Cory, Zachary M Hostetler, Rahul M Kohli. Kinetic dissection of macromolecular complex formation with minimally perturbing fluorescent probes. Methods in enzymology. 2022;664:151-171