Browsing by Subject "Nuclear Magnetic Resonance, Biomolecular"
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Item Analysis of Synaptotagmin-SNARE Complex Interactions by One-Dimensional NMR Spectroscopy(2013-06-05) Zhou, Amy 1986-; Rice, Luke M.; Rizo-Rey, José; Brautigam, Chad A.; Yu, HongtaoThe mechanism of calcium-triggered neurotransmitter release is mediated by numerous proteins at the neuronal synapse. The SNARE proteins form a complex that mediates fusion between the synaptic vesicle and plasma membrane. The protein synaptotagmin-1 is the major sensor for the calcium concentration. Synaptotagmin-1 and the SNARE complex are therefore believed to interact in order to couple the calcium concentration to membrane fusion. The detailed mechanism of the interaction is still unclear, because the technical difficulties in probing the system have rendered it intractable with respect to traditional biochemical and biophysical methods. I will present an analytical method based on one-dimensional NMR spectroscopy that overcomes these limitations. The experiments are based on the current working model that synaptotagmin-1 facilitates the SNAREs’ role in membrane fusion in a calcium-dependent manner. My data suggest that the synaptotagmin-1/SNARE complex interaction is calcium-dependent and mediated primarily by the synaptotagmin C2B domain. Further, the polybasic region of C2B constitutes the primary binding site, while the two arginine residues at the bottom of the domain mediate additional interactions that lead to aggregation and precipitation. These results help clarify the complex mechanism of synaptotagmin-1/SNARE coupling, as well as to illustrate the usefulness of 1D NMR to study such protein-protein interactions. Alternative methods to probe such interactions are explored. The advantage of a related competition assay lies in its sampling of the primary binding site with little interference of the other binding mode(s). However, experimental artifacts hindered the application of the assay to my system. A diffusion-based method is another route for studying protein-protein interactions, provided there is sufficient dynamic range to allow for meaningful interpretations. The SNARE-, calcium-, and lipid-binding profiles of an extended synaptotagmin-like protein (E-Syt) were also characterized. E-Syts are of interest because they can shed light on the evolution of proteins in the synaptotagmin family. In addition, they can reveal general governing principles of tandem C2-domain proteins which often function in signal transduction and membrane trafficking.Item A Fragile Native State Structure: An Aryl Hydrocarbon Receptor Nuclear Translocator (ARNT) Variant Exhibits Slow Interconversion Kinetics Between two Different Folds(2009-09-04) Evans, Matthew Ryan; Gardner, Kevin H.The aryl hydrocarbon receptor nuclear translocator (ARNT) is a promiscuous basic helix-loop-helix Period/ARNT/Single-minded (bHLH-PAS) protein that controls various biological pathways by forming heterodimeric transcriptional regulator complexes with several other bHLH-PAS proteins via the beta-sheet surfaces of its two PAS domains. The beta-sheets of PAS domains are involved in many intermolecular interactions with other proteins and natural cofactors in order to detect environmental changes in sensor PAS proteins. As part of a study of the HIF-2 alpha and ARNT PAS-B heterodimer, site-directed mutagenesis was performed on the ARNT PAS-B domain. Interestingly, one point mutation on the ARNT beta-sheet surface (Y456T) resulted in a new conformation of the domain that existed in equimolar concentrations with the wild-type conformation. Subsequent studies demonstrated that the two conformations are in equilibrium and that relative populations of the two conformations can be perturbed by additional mutations. Using solution NMR spectroscopy, we solved the high resolution solution structure of a mutant ARNT PAS-B domain in the new conformation, demonstrating that it contains a three-residue slip in register and accompanying inversion of the central beta-strand. In addition, this new conformer has a greater than hundred-fold reduction in affinity for HIF-2 alpha PAS-B, disrupting the hypoxia response pathway. Solution NMR measurements of the interconversion kinetics have let us establish that these two conformations interconvert slowly (40 min at RT) with a linear Arrhenius temperature-dependence of the interconversion rate. Stopped-flow unfolding experiments using GdmHCl on Y456T, revealed a similarly slow unfolding rate (25 min at RT) and an energy barrier to unfold of approximately 13 kcal/mol, which is nearly identical to that for the interconversion process. These data indicate that the protein must undergo a global unfolding process in order to interconvert between conformations. Lastly, these relative populations of Y456T can be affected by compound preferentially binding into the core of one of these conformations. This discovery highlights the malleability of PAS beta-sheets and suggests ARNT may act as a regulatory switch to control different biological pathways. Furthermore, this system presents a great opportunity to further understand the structural and kinetic impact of beta-strand slips observed in nature.Item Physical Studies of Actin Nucleation and Conformational Dynamics(2017-09-06) Zahm, Jacob Aaron; Tomchick, Diana R.; Rosen, Michael K.; Rice, Luke M.; Yu, HongtaoActin is a 42 kilodalton ATPase that exists ubiquitously in eukaryotic cells. Unlike other ATPases, however, actin, under suitable conditions, can polymerize, forming helical filaments. Cells, in orchestrating their myriad cellular processes, utilize actin's intrinsic capacity to polymerize, but do so in a tightly controlled fashion, such that new filaments only appear when and where the cell needs them to suit specific purposes. Such control exists at two different levels. Firstly, the stability of actin filaments is subject to "intrinsic" control arising from the state of bound nucleotide. ATP binding favors incorporation of actin monomers into filaments. This incorporation augments actin's ATP hydrolysis activity, and the conversion of ATP to ADP in the nucleotide binding cleft considerably destabilizes filaments, facilitating the return of filament subunits to free monomers. The structural mechanism through which nucleotide conveys information throughout the actin monomer to influence polymerization behavior remains poorly understood and represents a persistent fundamental biological question. In this work I, for the first time, apply modern muti-resonance NMR methods to begin to answer these questions. In addition to the aforementioned intrinsic control, cellular actin is subject to "extrinsic" control via the action of nucleation factors. In order to form a growing filament, actin must proceed through a nucleation step in which monomers must assemble into a thermodynamically and kinetically disfavored nucleus, which ultimately proceeds to a growing filament. Nucleation factors accelerate the rate of filament formation by binding to actin monomers and arranging them into the prerequisite nucleus. In this work, I reveal the crystal structure of actin monomers in complex with the bacterially derived nucleation factor, VopL. The structure represents the first high resolution snapshot of a filament-like nucleation intermediate, and reveals general principles underlying the action of nucleation factors.Item Structural and Functional Studies of C2-Domain Proteins Involved In Neurotransmitter Release(2007-05-22) Dai, Han; Rizo-Rey, JoséNeurotransmitter release is mediated by synaptic vesicle exocytosis at presynaptic nerve terminals. This process is extremely fast and strictly regulated by the intracellular Ca2+ concentration. To achieve this exquisite regulation, many proteins are involved. The central fusion machinery includes the SNARE proteins synaptobrevin, syntaxin and SNAP-25, as well as Munc18. Besides these universal components, many other neuronal specific proteins are also involved in regulating Ca2+-triggered release. Interestingly, most of these regulatory proteins contain C2 domains, versatile protein modules with Ca2+-dependent and Ca2+-independent activities. However, the mechanism of regulation by these C2-domain proteins remains unclear. My research has focused on understanding the structural and functional properties of two types of C2-domain proteins, synaptotagmins and RIMs. With NMR spectroscopy and biochemical assays, we demonstrated that synaptotagmin 4 is a Ca2+ sensor in Drosophila but not in rat, in contrast to the prediction based on sequence alignments. X-ray crystallography revealed that changes in the orientations of critical Ca2+-ligands render the rat synaptotagmin 4 C2B domain unable to form full Ca2+-binding sites. We also analyzed the structural and biochemical properties of the RIM2 C2A domain. NMR spectroscopy and FRET experiments demonstrated no interaction between the RIM2 C2A domain and Ca2+, phospholipid, synaptotagmin 1, and SNAP-25. However, the crystal structure of RIM C2A domain exhibits a strikingly dipolar distribution of the surface electrostatic potential. Several lines of evidence from the crystal structure suggested a potential target binding site around the bottom 310-helix. With fluorescence microscopy and microfluidic channel technology, we demonstrated that synaptotagmin 1 binds simultaneously to phospholipids and the SNARE complex reconstituted in membranes in the presence of Ca2+, forming a quaternary SSCAP complex, and that the membrane penetration of synaptotagmin 1 into phospholipids is independent of the reconstituted SNARE complex. We also showed that synaptotagmin 1 displaces complexin from the reconstituted SNARE complex in the presence of Ca2+, and that the synaptotagmin 1 C2B domain is primarily responsible for SNARE binding. Moreover, NMR spectroscopy and site-directed mutagenesis studies yielded structural information of the potential binding interface, allowing us to use computational modeling and docking to generate a preliminary model of the SSCAP complex.Item Structural Studies of Integral Membrane Proteins Involved in GPCR Signaling and Sterol Homeostasis(2018-11-27) Clark, Lindsay D.; Rice, Luke M.; Rosenbaum, Daniel M.; Gardner, Kevin H.; Jiang, YouxingMembrane proteins are crucial molecules for cellular survival, and can take on multiple and diverse roles within the native membrane. In this dissertation, I will detail my efforts to understand and study two different types of membrane proteins. First, I will discuss my research developing and applying a strategy to use NMR spectroscopy to study specific receptors within the large family of G protein-coupled receptors. This strategy enabled the first methyl-TROSY experiments on a wild-type human GPCR, and have significant value for future drug discovery efforts on this important class of membrane proteins. Second, I will discuss my endeavors to understand the important role of the protein Scap, which can both sense and respond to differences in cholesterol levels within the ER membrane. Scap is a central player in the SREBP pathway, which is targeted by multiple classes of pharmaceuticals, including statins. Through efforts described in the second half of this dissertation, I have been able to demonstrate the first biochemical characterization of the full-length mammalian Scap/Insig complex, which has led to the first structural characterization of this important machinery. The long-term goal of both of these projects is aimed at having a more complete understanding of how these important membrane proteins respond to ligands and other environmental changes within their native cell membrane. This information will further our ability to diagnose and treat diseases ranging from insomnia and chronic pain to atherosclerosis and hypercholesterolemia.