Towards in situ membrane protein structural biology using a multimodal integrative approach
Creators: name: Jiang, Jennifer
Publisher: name: No Publisher Supplied
Year: 2024

description: Membrane proteins mediate many key physiological functions and are highly acclaimed targets for therapeutic intervention. In fact, more than half of clinically approved drugs target human membrane proteins. The structure and functional dynamics of membrane proteins are intricately coupled to their surrounding membrane environment, making it critical to study them within the cellular context. Within biological membranes, lipid-lipid and lipid-protein interactions play major roles in the molecular organization of the membrane into distinct membrane microdomains featuring unique lipid and protein composition. These membrane microdomains are proposed to be involved in the spatiotemporal coordination of signal transduction and other essential cellular processes. However, the structural characteristics and functional implications of these membrane domains and their associated membrane protein complexes remain poorly understood. Understanding cellular processes at the molecular and structural level represents the next frontier of biomedical research. In recent years, an integrative spatial and multimodal approach shows great promise in synergizing various experimental and computational techniques to yield significant structural and mechanistic insights into complex biological processes. Cryo-electron tomography (cryo-ET) is gradually becoming a key cornerstone of in situ cell biology research. This imaging modality rapidly emerged as a mainstream structural biology technique due to its unique capability to preserve and directly visualize the structural architecture of subcellular structures and macromolecular assemblies in their native cellular landscape. Cryo-ET provides unique opportunities to bridge the gap between light microscopy of whole cells and in vitro structure determination methods. Recent advances in the field have revolutionized cellular structural biology and have opened new avenues for understanding the structure and functional relationships of biomolecular complexes in their unperturbed state. Despite its potential, structural characterization by cryo-ET suffers from many inherent challenges. To overcome these limitations, cryo-ET is commonly integrated with complementary methods such as mass spectrometry (MS), structure prediction, biochemical and biophysical methods, to obtain a comprehensive glimpse into the subcellular universe at molecular resolution. In this thesis, a multidisciplinary approach is used to investigate the structural characteristics of membrane microdomains and associated membrane proteins in two distinct biological membrane systems: thylakoid membrane from a marine diatom and plasma membrane from a human fungal pathogen. In Part II, we examine the heterogenous distribution of photosynthetic protein complexes in thylakoid membranes of Phaeodactylum tricornutum. To aid in the identification and localization of photosynthetic protein complexes embedded in isolated thylakoid membranes, we developed an integrative workflow that combines on-grid immunogold labeling and in situ cryo-electron tomography. By applying this methods development to isolated thylakoid membranes, we discovered two subpopulations of Photosystem II (PSII) within native thylakoid membranes, specifically semicrystalline arrays composed of dimeric PSII cores and PSII holocomplexes. This experimental workflow can be further applied to a range of biological specimens and facilitate structural studies of other protein complexes, especially those with no known 3D structures. In Part III, we investigate the structural architecture of plasma membrane microdomains in fungi and their functional relevance in the context of antifungal drug treatment. By combining cryo-ET with MS and biochemical and biophysical analyses, we provide structural and spatial insights into fungal plasma membrane proteins and confirm the existence of membrane compartments. In addition, our data suggest that echinocandin antifungals may interact with the local membrane environment to perturb the spatial distribution of membrane protein complexes. Future work towards unraveling the structure and dynamics of membrane-embedded protein complexes and their organization into higher-order structures within biological membranes will provide crucial information on their role in regulating various cellular processes.