Protein-mediated membrane fusion is a highly regulated biological process essential for cellular and organismal functions and infection by enveloped viruses. During viral entry, the membrane fusion reaction is catalyzed by specialized protein machinery on the viral surface. These viral fusion proteins undergo a series of dramatic structural changes during membrane fusion where they engage, remodel, and ultimately fuse with the host membrane. The structural and dynamic nature of these conformational changes and their impact on the membranes have long-eluded characterization. Furthermore, the native pre-fusion structural and conformational dynamics of these fusion machines remains unclear as the conventional structural approaches employed by structural biologists are not well suited for studying these dynamic protein machines on the viral surface. The objective of this dissertation is to characterize the complete mechanism of Influenza virus hemagglutinin (HA) fusion activation and membrane fusion, and to profile and characterize the structural and conformational dynamics of the HIV-1 Env fusion glycoprotein on the viral surface. In chapter 2 I use continuous labeling HDX-MS to characterize the structural dynamics and conformational homogeneity of the HIV-1 Env fusion glycoprotein on the surface of two distinct engineered and authentic viral vaccine platforms. By HDX-MS we observed significant amounts of non-native Env present in one vaccine platform, whereas all Env present in the other resembled trimeric Env in the closed conformation. In chapter 3, I use pulse labeling HDX-MS to characterize the mechanism of HA fusion activation and HA mediated membrane fusion in situ using whole infectious virions. Our data reveal how concurrent reorganizations at the HA1 receptor binding domain interface and HA2 fusion subunit produce a dynamic fusion intermediate ensemble in full-length HA. In contrast, the soluble HA ectodomain transitions directly to the post-fusion state with no observable intermediate. These data provide unprecedented insight into the structural mechanics of HA which has served as the prototypical class 1 viral fusion protein and informed our understanding about how all class 1 viral fusion proteins function. In chapter 4 I present developments and improvements on the HDX-MS workflows that will enable more complete characterizations of HA's mechanism and the structural and conformational dynamics of other class 1 viral fusion proteins. Together these works have dramatically furthered our understanding of the structural mechanics of class 1 fusion proteins and lay the foundation for future studies on influenza virus and other enveloped viruses and their membrane fusion machinery.

Membrane fusion is an essential step in various developmental, physiological, and pathological processes in eukaryotes. Specific fusion proteins catalyze the merger of two lipid bilayers. The molecular mechanisms of many membrane fusion reactions still remain unclear. Here, we characterized the molecular details of various adhesion and fusion proteins involved in viral-cell, placental trophoblast, and sperm-egg fusion events to better understand their mechanisms. In total, nine crystal structures of these human and viral glycoproteins are presented. Furthermore, structural comparisons combined with mutagenesis, biochemical, biophysical, and cell culture experiments allowed us to investigate the implications of specific residues of these proteins in mediating membrane fusion. Structures of class I viral fusion proteins revealed that electrostatic interactions are critical for post-fusion stability in viruses that fuse at neutral pH, whereas the salt bridges do not play a stabilizing role in fusion proteins that proceed through low pH. Instead, hydrophobic residues stabilize the fusion core, and histidine/arginine residues in the chain reversal region stabilize a helix-dipole moment. Moreover, the structure of human syncytin-1 fusion protein unveiled a striking overall structural similarity to class I viral fusion proteins, and the presence of salt bridges within the fusion subunit zipper the inner and outer helices. These electrostatic interactions stabilize the post-fusion structure and provide the energetics for trophoblast cell fusion in placentation. While class I fusion proteins share similar structural and functional features, the mechanism of sperm-egg fusion requires the involvement of sperm Izumo1 and egg Juno proteins. Atomic resolution structures of Izumo1 and Juno display an interface stabilized through extensive interactions and a major conformational change within Izumo1 upon Juno binding. Moreover, mutational studies at the Izumo1-Juno interface revealed the structural determinants required for binding. We provide a comprehensive analysis that now identifies general trends and signatures important in viral-cell and cell-cell fusion events.

In Viral Membrane Proteins: Structure, Function, and Drug Design, Wolfgang Fischer summarizes the current structural and functional knowledge of membrane proteins encoded by viruses. In addition, contributors to the book address questions about proteins as potential drug targets. The range of information covered includes signal proteins, ion channels, and fusion proteins. This book has a place in the libraries of researchers and scientists in a wide array of fields, including protein chemistry, molecular biophysics, pharmaceutical science and research, bioanotechnology, molecular biology, and biochemistry.

Viruses are major pathogens in humans, and in the organisms with which we share this planet. The massive health and economic burden these agents impose has spurred a huge research effort to understand their most intimate details. One outcome of this effort has been the production, in many but certainly not all cases, of effective vaccines and therapies. - other consequence has been the realization that we can exploit viruses and put them to work on our behalf. Viruses are still seen to have the most - tential as vehicles for gene delivery and other therapeutic applications. However, their ability to exploit cellular functions to their own ends makes viruses not only highly effective pathogens but also exquisite experimental tools. Work with viruses underpins much of our current understanding of molecular cell biology and related fields. For membrane traffic in parti- lar, viruses have been crucial in providing insights into key cellular fu- tions and the molecular mechanisms underlying these events.

Viral Fusion Mechanisms presents the first comprehensive review on this exciting topic. The book focuses on molecular mechanisms rather than phenomonology and examines a wide range of viruses, including influenza, HIV, Sendai, SFV, Vaccinia, VSV, and RSV. Recent theoretical work on dissecting protein-mediated membrane fusion is discussed, and the most promising new technologies for elucidating mechanisms are highlighted. Viral Fusion Mechanisms is an essential reference for biophysicists, cell biologists, colloid chemists, immunologists, microbiologists, molecular biologists, and virologists.

This balanced volume provides a broad and coherent overview of recent progress in membrane fusion research—highlighting an interdisciplinary treatment of the subject from the fields of biophysics, biochemistry, cell biology, virology, and biotechnology—in a single volume., Featuring easy-access sections on the general properties of membranes and applications of membrane fusion techniques, this valuable sourcebook outlines membrane structure, lipid polymorphism, and intermembrane forces ... covers membrane fusion in model systems ... presents the fusogenic properties of enveloped viruses ... discusses the fusion and flow of intracellular membranes and cell-cell fusion occurring during fertilization and myogenesis ... offers applications of membrane fusion techniques in cell-biological research and biotechnology ... and more. Supplying a comprehensive view of this exciting topic, Membrane Fusion is a working resource for molecular, cell, and membrane biologists; biophysicists; biochemists; virologists; biotechnologists; microbiologists; immunologists; physiologists; and graduate and medical school students in biophysics, biochemistry, physiology, virology, cell biology, and biotechnology.