Approaches to quantitatively analyze protein complex assembly and regulation

Abstract

Biological macromolecular complexes occupy high-dimensional conformational landscapes corresponding to their assembly and regulation. For many protein complexes, these conformational landscapes encode diverse functional states of the complex, and indeed, there is a growing appreciation of the critical significance of protein dynamics in driving protein function. As such, there is a need for approaches to experimentally and quantitatively resolve these conformational landscapes to better understand: 1) the diverse structural states these complexes sample; 2) how these states and their dynamics are linked to biological function; and 3) how these landscapes are modulated by binding partners or environmental signals, or over the course of complex assembly. State-of-the-art structural approaches including heterogeneous cryogenic electron microscopy (cryo-EM) offer one potentially powerful mechanism for resolving these landscapes, and we present here approaches to resolve large numbers (100s-1,000s) of volumes from a single dataset. Moreover, I explore methods to analyze the resulting large volume ensembles using supervised and unsupervised approaches, and demonstrate the power of these approaches by applying them to identify a proofreading role for the universally-conserved methyltransferase KsgA in biogenesis of the bacterial small ribosomal subunit. However, many of the most highly dynamic protein complexes – involved in critical cellular processes like environmental sensing and signal transduction – remain inaccessible to structural techniques like heterogeneous cryo-EM. I suggest that combining structural approaches with high-throughput techniques, including proteomic and library-based approaches, has substantial power to shed light on the function and regulation of such complexes. As an example, I couple a high-throughput reporter assay with deep mutational scanning, finding highly distributed phosphorylation regulated assembly of the Atg1 complex in Saccharomyces cerevisiae in response to diverse environmental cues. Taken together, my work generates a toolbox of complementary approaches for quantitatively characterizing the assembly and regulation of protein complexes, and I anticipate substantial utility in applying these approaches to broadly characterize the functional and regulatory landscapes of protein complexes.