Steven Owen Smith - US grants

Membrane proteins and protein complexes have in general eluded detailed structural characterization since many membrane systems and large macromolecules are inaccessible to traditional structural methods such as a solution NMR and X-ray diffraction. Solid-state NMR spectroscopy has developed into a versatile probe of molecular structure and dynamics, and is ideally suited for such systems. The proposed research from four principal investigators at Yale covers a broad range of active research in biochemistry and biophysics where the problems involve membrane systems or molecular complexes that require solid-state NMR methodology. The acquisition of a high-field solid-state NMR spectrometer is essential for addressing these problems, and will extend and complement the current methods for studying biomolecular structure and dynamics at Yale. (1) The first proposal on the visual pigment rhodopsin takes advantage of low-temperature solid-state NMR methods to study the photoreaction intermediates of rhodopsin. These experiments will provide critical new information on the location of protein charges in rhodopsin, and on the mechanism of light-transduction by the retinal chromophore. (2) Solid- state NMR studies are proposed for determining the mechanism of proton translocation by ubiquinone in mitochondrial and photosynthetic membranes. The research proposal describes a newly developed method for enhancing the rates of magnetization transfer between nuclear spin pairs and measuring intermolecular distances. These methods shall be used to establish the orientation of ubiquinone in energy-transducing membranes. (3) The third proposal uses deuterium NMR methods as a sensitive probe of the structure of phospholipids that are important in membrane fusion. These studies focus on anionic lipid-cation complexes that have been implicated in the initiation of fusion, a key process in cellular function. (4) Several recent advances in solid-state NMR methods are exploited in proposed research to study the dynamics of metal-ligand exchange in the active sites of metalloproteins. Exchange processes have prevented the observation of NMR resonances in solution. (5) The fifth proposal focuses on the structure of nucleotide-protein complexes. These studies are designed to bridge the two established structural methods, solution NMR and X-ray diffraction.

The proposed research applies recent advances in solid-state NMR methodology to study the mechanism of light-transduction by the visual pigment rhodopsin and proton translocation by ubiquinones. The rhodopsin studies are concerned with the origin of the red-shift in the pigment's visible absorption band and the mechanism of energy storage and conversion upon photoisomerization. Low temperature methods previously developed by the PI for obtaining solid-state 13C-NMR spectra of the photointermediates of bacteriorhodopsin will be used to study the structure and protein environment of the retinal chromophore in bathorhodopsin and metarhodopsin I and II. Specifically, these studies address the structure of the C6-C7, C10-C11, and C=N bonds of the retinal, and the location of protein charges that are thought to interact with the chromophore at positions C-12, C-13 and C-15. Solid-state NMR studies are also proposed on rhodopsin bearing nitroxide spin labels for obtaining information on the tertiary structure of the protein. These labels do not disrupt protein structure and provide a method for establishing the location of several of the transmembrane alpha-helices of the protein relative to the retinal. The solid-state NMR studies of ubiquinone address the location and orientation of this long polyisoprenoid coenzyme within mitochondrial and photosynthetic membranes. In these membranes, the quinone exists in both protein-bound and free membrane-diffusible states. Our strategy is to first characterize the population of the protein-bound quinones by deuterium and 13C solid-state NMR, and subsequently to investigate the diffusible membrane component responsible for proton translocation. Newly developed methods for enhancing spin exchange rates between 13C nuclei are proposed for localizing the quinone within the membrane. The ubiquinone studies are at a preliminary stage, but will provide a basis for solid-state NMR investigations of electron transport and photosynthetic proteins, similar in design to the studies on rhodopsin. The long-range objectives of this research are to develop methods for studying electrostatic an hydrogen- bonding interactions within membrane proteins, and to investigate mechanisms of ion transport.

This is a Shannon Award providing partial support for research projects that fall short of the assigned institute's funding range but are in the margin of excellence. The Shannon award is intended to provide support to test the feasibility of the approach; develop further tests and refine research techniques; perform secondary analysis of available data sets; or conduct discrete projects that can demonstrate the PI's research capabilities or lend additional weight to an already meritorious application. Further scientific data for the CRISP System are unavailable at this time.

Vitamin A (retinal) and coenzyme Q (ubiquinone) are small hydrophobic isoprenoids whose photochemical and redox properties have been exploited by membrane proteins. The proposed research focuses on the mechanism of light transduction by the retinal chromophore in the visual pigment rhodopsin and proton translocation by quinone molecules in mitochondrial and photosynthetic membranes. These studies take advantage of magic angle spinning NMR approaches for obtaining high-resolution structural data of membrane proteins in bilayer environments. Chemical shift measurements of the protein-bound retinal chromophore are proposed for determining the structure of the retinal binding site in rhodopsin and its photointermediates. Rotational resonance NMR measurements are proposed for determining C+C-C+C torsion angles along the retinal chain. Together, these studies address the mechanism for energy storage in the rhodopsin - >bathorhodopsin reaction, and the mechanism for proton transfer in the metarhodopsin I -> metarhodopsin II reaction. The proton transfer reaction is the key step in triggering the binding of the G-protein transducin. Similar measurements on the red, green and blue cone proteins will establish the mechanism of color regulation in visual pigments. NMR studies are proposed for determining the location and orientation of free ubiquinone and ubiquinol in membrane bilayers. NMR measurements of 13C-labeled quinone molecules in oriented membrane bilayers are planned that take advantage of the orientation dependence of the chemical shift and dipole-dipole interactions. NMR measurements of 2H-labeled quinones are proposed that take advantage of the sensitivity to motion of the 2H lineshape to characterize quinone dynamics. Comparative studies of quinones with different chain lengths (3-10 isoprene units) are aimed at addressing the role of chain length in determining quinone location and dynamics. Together, these studies address how quinones facilitate the transport of protons between protein components in energy transducing membranes. Finally, rotational-echo double resonance NMR measurements are planned for determining which residues form the quinone binding sites in the cytochrome bC1 complex. The rotational-echo experiment yields high- resolution distance constraints between 13C--labeled quinones and 15N- labeled protein groups with approximately 5 A. Such structural data is essential for establishing the key residues responsible for catalyzing the quinone redox chemistry.

DESCRIPTION (provided by applicant): The proposed research is to establish the mechanism for how the transmembrane and juxtamembrane regions of cytokine receptors and receptor tyrosine kinases couple ligand binding to receptor activation, and how viral membrane proteins activate these single transmembrane helix receptors in the absence of signaling ligands. The proposal has four specific aims: 1) to determine the structure of the E5 protein of bovine papillomavirus and the transmembrane region of the gp55p protein of Spleen Focus-Forming virus. These are viral membrane proteins that activate the PDGF-beta receptor and the erythropoietin (Epo) receptor, respectively, through transmembrane helix interactions. 2) to establish the structure of the transmembrane and juxtamembrane regions of the Epo and thrombopoietin receptors as isolated domains, and fused to the native extracellular domain. 3) to determine and compare the structures of the inhibitory juxtamembrane region of the native PDGF-beta receptor and the V536A constitutively active mutant. 4) to determine the structure of the complex between the viral proteins and the transmembrane juxtamembrane regions of these receptors by establishing key intermolecular contacts. The long term objectives of the research are to establish methods for determining the structure of membrane peptides and proteins in membrane bilayers and establishing how transmembrane helices associate in a sequence specific manner in hydrophobic membrane bilayers. These results will provide a rational route to the design of competitive non-peptide inhibitors to block constitutively active receptor dimers, and will provide a basis for engineering viral membrane proteins to specifically target oncogenic membrane receptors.

Joint NIH-NSF support is requested for the purchase of a 600 MHz wide bore NMR spectrometer. The proposed instrumentation will support on going research involving membranes and membrane proteins (Smith and McLaughlin), protein folding (Raleigh) and structural analysis of catalysts, biopolymers and other chemical systems (Grey). Several factors at Stony Brook assure the productive use of the requested instrumentation. . The research groups of Smith, Raleigh and Grey have extensive experience in solid-state NMR with expertise in biological NMR and methods development. . The requested instrumentation is a key component of an NMR facility that is being created through a structural biology initiative at Stony Brook. The NMR facility and the structural biology program have strong university support. . There is a strong research emphasis on membranes, membrane proteins and signal transduction at Stony Brook. Roughly 25 research groups participate in a weekly seminar and discussion series. . Facility management and technical support will be assured through the expertise of Dr. Martine Ziliox, currently the sold-state NMR applications manager at Bruker Instruments in the United States. She will relocate to Stony Brook and run the NMR facility.

DESCRIPTION (Adapted from abstract): Rhodopsin is the prototypical G protein-coupled receptor. The G-protein receptors are thought to have a common architecture built around seven transmembrane helices. Ligand binding in most GPCRs or retinal isomerization in the case of rhodopsin triggers a protein conformational change that allows binding of G proteins to the cytoplasmic surface of the receptor. The high resolution structure is not known for any member of this large receptor family. Moreover, there is no consistent mechanism for how ligand binding activates a GPCR. The pharmaceutical importance of this class of receptor is revealed in the estimate that 60% of the targets for all drugs sold today are GPCRs. In the last few years the investigators have made progress in establishing the structure of the retinal binding site in rhodopsin and how retinal isomerization triggers rhodopsin activation. Recent progress in the expression and purification of rhodopsin containing 13C and 15N labels has opened a new route for structural studies. Three specific aims form the core of this proposal. Structural studies are proposed for determining the retinal conformation and how it is packed in the protein interior. These studies involve establishing the orientation of the retinal in the protein binding pocket and the contact points between specific retinal carbons and residues in the protein binding site. NMR measurements are proposed for establishing the structure changes involved in rhodopsin activation. Glutamic acid, histidine and the retinal PSB linkage are thought to change protonation state upon formation of metarhodopsin 11. The investigators plan to measure changes in the protonation state of these residues during the rhodopsin photoreaction. Changes in retinal and protein structure will also be determined by measuring distances between specific retinal and protein isotopic labels. NMR studies are proposed for determining the structure and interactions of the C-terminal peptide of the a-subunit of transducin bound to metarhodopsin 11. Intrapeptide distances will be measured to determine the peptide conformation. Specific peptide-receptor distances will be measured by engineering unique sites into the loops and C-terminus of rhodopsin. Together these studies lay the foundation for establishing ligand conformation and binding contacts in the family of GPCRs, the long term objective of the proposed research.

[unreadable] DESCRIPTION (provided by applicant): The proposed research is to establish the structural basis for how basic-aromatic clusters in membrane-associated proteins are able to sequester specific lipids into lateral membrane domains, how they induce membrane curvature, and how they allow specific peptides to cross cell membranes. The proposal is focused primarily on the MARCKS protein, but also investigates the structure and binding of a wide range of additional basic-aromatic clusters in order to establish how membrane binding and penetration depends on protein sequence. The specific aims of the proposal are to determine 1) the location and conformation of the MARCKS (151-175) effector domain in membrane bilayers, 2) how the protein sequence determines the depth of penetration into bilayers, 3) whether and how basic-aromatic clusters sequester cholesterol, and 4) the role of basic-aromatic clusters in membrane curvature and peptide penetration. The research relies primarily on magic angle spinning NMR measurements of membrane multilayers and solution NMR of membrane bicelles. The NMR approaches are complemented by fluorescence techniques to investigate binding and membrane interactions. [unreadable] [unreadable] Basic-aromatic clusters may be a common mechanism for sequestering phosphoinositides and cholesterol in lateral domains. As a result, their importance in human health is significant. Understanding how the levels of phosphoinositides are regulated is central to signal transduction pathways which are involved in cell growth and differentiation, while understanding how the distribution of cholesterol is controlled in plasma membranes has relevance for cardiovascular and related diseases. The strategy is to progress from simple domains induced by the MARCKS (151-175) peptide to the more complex caveolar domains. The NMR measurements proposed will provide high resolution detail which is not available from crystallographic methods or more traditional solution NMR methods. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The Center for Structural Biology (CSB) was established in 1998 as an institutional and interdepartmental effort to introduce structural biology to the State University of New York at Stony Brook. The CSB is housed on the first and ground floors of the Centers for Molecular Medicine. The CSB is comprised of core faculty from three different departments in the University, which interact closely with each other and several faculties from various departments on campus. Besides supporting the research of the CSB members the center provides easy access to computational facilities for departmental faculty throughout the University. The aim of the current proposal is to upgrade the outdated and partially non-functional computational equipment that was purchased in 1998 and still forms the core facility in the CSB. The equipment requested consists of three major components: graphics workstations, a multi-processor computational server, and data storage and backup capabilities. The plan is to replace our current Silicon Graphics servers and workstations with a PC-based facility. The advantage is increased performance, lower cost for the equipment and lower cost for maintenance. The facility is supported strongly by the SUNY Stony Brook, which covers 50% of the salary for in the terms of salary for a Ph.D. level computational facility manager. The computational equipment requested will have a significant impact on the NIH supported research carried out by the core faculty of the Center for Structural Biology as well as multiple minor users from four departments on campus.

[unreadable] DESCRIPTION (provided by applicant): The requested instrumentation is for the first of the next-generation of Atomic Force Microscopes that can be used for high resolution structural studies of biological molecules in solution. [unreadable] [unreadable] The AFM instrument employs a non-oscillatory "tapping mode" that takes advantage of the advances in tip technology to surpass the resolution limits previously established for biomolecules. The new AFM operation is embedded in a computer program to provide sub-angstrom linear control of cantilever base and tip position, including programmed contact and programmed separation of the tip by a magnetic force ramp. Advantages include a compressive force as small as 20 piconewtons for detection of the sample surface, one touch per pixel and an integrated capability for chemical force mapping. The low compressive forces inherent in the new methodology facilitate the use of single-walled nanotubes of diameter 1 nm or less. [unreadable] [unreadable] The major users for the instrument are from Stony Brook University and Brookhaven National Laboratory. The research that will be supported by the instrument falls into three general areas: protein-DNA interactions, amyloid formation and inhibition, and membrane protein complexes and arrays. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): G protein-coupled receptors (GPCRs) are involved in most cellular processes and are the target of over 50% of the pharmaceuticals currently on the market. Despite their importance, the only known high-resolution structure of a GPCR is the crystal structure of the inactive state of the visual receptor rhodopsin. The goal of the proposed research is to establish the structure of metarhodopsin II, the activated state of rhodopsin. The experimental approach is to incorporate 13C labels into both the vitamin A (retinal) chromophore and the rhodopsin protein for measurements of internuclear 13C...13C distances using solid- state magic angle spinning NMR spectroscopy. The first three specific aims target different regions of the rhodopsin protein to address specific questions involving the activation mechanism. Distance measurements are planned of 1) retinal-protein contacts to establish the location of the retinal relative to transmembrane helices H5, H6, and H7, 2) helix-helix contacts to establish how transmembrane helices H5-H7 move relative to the H1-H4 core of rhodopsin, and 3) the extracellular and intracellular loops to establish their location relative to the retinal chromophore and the C- terminal peptide of the Galpha subunit of transducin. Together these studies are intended to establish how isomerization of the retinal chromophore is coupled to motion of the transmembrane helices and cytoplasmic loops. A final aim is to extend our structural studies to CCR5, the chemokine receptor in T-cells that serves as the co-receptor for HIV. The expression levels of CCR5 are now sufficient for solid-state NMR spectroscopy. We plan to establish the structure and location of inhibitors that bind to CCR5 and block HIV entry into T-cells. These studies will facilitate the rational design of inhibitors for the prevention of AIDS. Selective NMR measurements are also proposed that will allow comparisons to be made of the activated state structure of CCR5 with that of metarhodopsin II. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Amyloid deposits found in neurodegenerative diseases result from misfolding of cellular proteins. The challenge for developing specific inhibitors that block oligomer or fibril formation is that there are no high- resolution molecular structures that can guide the design. The proposal has three specific aims. The first aim is to use high-resolution solid-state NMR in combination with atomic force microscopy (AFM) to refine the structures that are emerging of Abeta oligomers and fibrils. The second aim is to design inhibitors based on the structural data from Aim 1 and to test their ability in vitro to block oligomer or fibril formation. The third aim is to assay the ability of these inhibitors to block the toxicity of Abeta oligomers and fibrils on neuronal cells. [unreadable] [unreadable] A new approach has been developed for obtaining high-resolution AFM images of Abeta soluble oligomers and fibrils in solution. The method takes advantage of a novel AFM controller that provides resolution in 'single touch' AFM experiments that surpasses the resolution currently available using commercial instruments. High resolution AFM of solution samples will allow us to follow the formation of Abeta oligomers, protofibrils and fibrils, and determine how designed inhibitors prevent fibrillization. [unreadable] [unreadable] We have developed structural models for the Abeta42 monomer, dimer, protofibril and fibril based on preliminary results from high resolution AFM and solid-state NMR. The structures show that when the b-strands have a parallel orientation and the amino acids are in-register with one another, the surface of the b-sheet has pronounced ridges and grooves. This architecture provides the key elements for the rational design of inhibitors to prevent fibril formation. Our template inhibitor peptide based on a rational design approach has the sequence GxFxGxF, where the bulky phenylalanine side chains of the inhibitor are predicted to pack against the glycines in the GxxxG motif of the amyloidogenic peptide. [unreadable] [unreadable] We will test the ability of the designed peptides to disrupt the formation of oligomers and fibrils by thioflavin T fluorescence, size exclusion chromatography, electron microscopy, AFM and solid-state NMR. We will also test the ability of our designed inhibitors to protect neurons from cell death induced by amyloid fibrils. We will focus on the Abeta42 peptide because of its higher ability to form aggregates than the shorter isoforms. Moreover, most gene mutations that are associated with the inherited forms of Alzheimer's disease result in an increase in the ratio of Abeta42 over Abeta40. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The proposed conference, Molecular Biophysics of Cellular Membranes, will be the ninth in a series of FASEB summer research conferences devoted to the study of model and biological membranes using biophysical approaches. The conference will be held July 1- 6, 2006 at the Vermont Academy in Saxtons River, Vermont. These conferences have provided a unique forum for addressing important questions of cell membrane biology that are best answered using biophysical techniques. The principal objective has always been to stimulate cross-pollination of ideas amongst a diverse group of membrane biophysicists, cell biologists, biochemists, and physiologists by bringing them together in an intimate setting to share the latest developments in their fields. The conference will have 9 oral sessions, 3 poster sessions, one workshop and 2 short oral presentations for the poster presenters. There are 32 confirmed speakers. Funding is also requested for the tenth conference in the series to held in the summer of 2008. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Cytokine receptors and receptor tyrosine kinases (RTKs) are large integral membrane proteins with single membrane-spanning helices. Representative high-resolution structures have been determined of the ligand binding domains for both classes of receptor, and of the intracellular kinase domain for the RTKs. However, structures of the transmembrane (TM) and juxtamembrane (JM) regions of these receptors are generally lacking. An emerging view of both receptor families is that pre-formed dimers with high-affinity binding sites are the physiologically important forms of the receptors. In these receptors, the intracellular domains are in close proximity, but remain inactive until ligand binding triggers a conformational change in the receptor dimer. A common element of both receptor families is that the intracellular JM region plays a key regulatory role. We hypothesize that a change in the orientation of the TM helices releases inhibitory constraints within the JM region allowing the receptor to switch from an inactive to an active conformation. We propose to show how the rotational orientation of the TM helices is coupled to changes in the relative orientation and/or structure of the JM regions of these biologically important single helix membrane proteins. A combination of biophysical methods will be used, including solid-state and solution NMR, fluorescence and infrared spectroscopy. The proposed studies on TM helix interactions are part of a long-range effort to understand how membrane proteins fold and function. The proposed studies on the JM regions of these receptors address questions involving the role of membrane lipids, such as the phosphoinositides, in regulating receptor activity. The significance of the proposed research is to provide a molecular basis for disease. Mutations and deletions in the TM and JM domains of the RTKs have been identified in a variety of human tumors that result in constitutive receptor activity. Mutations in the TM and JM domains of the Epo and Tpo receptors produce erythroleukemia and myeloproliferative disorders, respectively. The four specific aims of the grant are to determine the structure and function of the TM and JM regions of these single TM helix receptors in order to have a comprehensive view of the full receptor structure. PUBLIC HEALTH RELEVANCE: Membrane receptors are involved in most cellular processes and are the target of the majority of pharmaceuticals currently on the market. The aim of the research is to establish how receptors in the receptor tyrosine kinase and cytokine receptor families with a single transmembrane sequence transmit information across cell membranes. We propose to determine the structures of the membrane-spanning region of these receptors in their active and inactive conformations, and to establish the structural changes that result from disease-causing mutations.

DESCRIPTION (provided by applicant): A 500 MHz solid-state NMR spectrometer is requested in order to replace an aging 360 MHz NMR instrument that was purchased in 1989. This NMR instrument currently supports four areas of NIH-funded biomedical research at Stony Brook University that rely on high resolution solid-state NMR for structure-function studies. The first project focuses on the structure and mechanism of G protein-coupled receptors (Steven Smith). These receptors are important pharmaceutical targets. Solid-state NMR methods are being used to determine the structures of the active state of the visual pigment rhodopsin, the 22- adrenergic receptor with bound agonists and antagonists, and CCR5 with bound HIV entry inhibitors. The second project (Erwin London) concerns membrane rafts and membrane domain formation. Integral membrane proteins and extrinsic membrane-associated proteins both associate with and may serve to nucleate cholesterol-rich domains. NMR structural studies are used in combination with fluorescence spectroscopy to understand the mechanism of domain formation and protein association. One structural target is the domain forming protein, caveolin. Caveolin is the protein component of caveolae, cholesterol-rich domains responsible for membrane internalization. The second project (Stuart McLaughlin) deals with the structure and function transmembrane and membrane-associated proteins having clusters of basic and aromatic residues. One structural target is the epidermal growth factor (EGF) receptor, a member of the ErbB family of receptor tyrosine kinases. The receptor tyrosine kinases are cell-surface membrane receptors that mediate cell growth and differentiation, and are associated with a wide variety of human tumors when constitutively activated through mutation or overexpression. NMR structural studies are focused on how receptor activity is regulated. The fourth project (William Van Nostrand) targets the structure, formation and inhibition of amyloid fibrils involved in Alzheimer's disease and cerebral amyloid angiopathy (CAA). Solid-state NMR is used to follow the structural changes of amyloid A2 peptides from monomers to oligomers to fibrils. The focus is on the A242 peptide and the Dutch and Iowa mutants of the A240 peptide. PUBLIC HEALTH RELEVANCE: Over the past ten years, solid-state NMR spectroscopy has emerged as an effective method for determining the high-resolution structures of cellular components that are not amenable to the traditional approaches in structural biology, namely X-ray crystallography and solution NMR spectroscopy. These structures include amyloid fibrils associated with a wide range of neurodegenerative diseases (Alzheimer's disease, Parkinson's disease, prion diseases) and G protein-coupled receptors, the major target of drugs currently on the market. The requested instrumentation supports research that focuses on these biomedical targets.

DESCRIPTION (provided by applicant): Structure and Activation Mechanism of the Visual Pigment Rhodopsin Rhodopsin is a specialized G protein-coupled receptor (GPCR) found in vertebrate rod cells. Absorption of light by its 11-cis retinal chromophore leads to rapid photochemical isomerization and receptor activation. Structural changes on the extracellular side of rhodopsin induced by the retinal isomerization are coupled to motion of the membrane-spanning helices to create a G-protein binding pocket on the intracellular side of the receptor. The existing crystal structures of rhodopsin provide a high-resolution framework to study in detail the role of specific residues and motifs in receptor activation. Because of the high conservation of many of the key residues involved activation of rhodopsin, the emerging model indicates that rather than being unique, the visual receptors provide a basis for understanding the common structural and dynamic elements in the class A GPCRs. The general experimental strategy is to use solid-state NMR spectroscopy in combination with mutational, optical and biochemical methods to target specific regions in the inactive and active states of the receptor. The goal is to understand in atomic detail the interplay between specific signature, group-conserved and subfamily-conserved motifs in the activation mechanism of rhodopsin and derive the basis of a working model for the activation of other GPCRs. Three specific aims address structure-function questions involving regions on the extracellular side of the receptor (Aim 1), within the transmembrane (TM) core (Aim 2) and on the intracellular side of the receptor (Aim 3). In Aim 1, we describe two hydrogen-bonding networks that tether extracellular loop 2 (EL2) to the ends of the TM helices H5-H7. We propose NMR measurements to quantify the displacement of EL2 upon activation and to establish how this displacement is coupled to helix motion. In Aim 2, we target the conserved stable core of rhodopsin composed of interlocking signature and group-conserved residues. We hypothesize that H6 rotates in the conversion to Meta I and then tilts outward upon deprotonation of the retinal Schiff base and associated motion of EL2. In Aim 3, we focus on the G-protein and its interactions with residues on the intracellular surface of Meta I and Meta II. The experiments target the structural transitions between inactive and active complexes of rhodopsin with G1 peptide or G-protein. In addition, our studies address how specific mutations lead to retinal diseases through constitutive activation, receptor misfolding or stabilization of non-functional receptor conformations.

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is a neurodegenerative disease characterized by the accumulation of amyloid plaques in the brain. These plaques are composed of mostly A? peptides generated by proteolysis of the amyloid precursor protein (APP) by two proteases, ?- and ?-secretase. The primary cleavage product is an A? peptide with a length of 40 residues (A?40). However, proteolysis is not highly specific and ~10% of the cleavage products of APP are peptides with two additional amino acids (A?42). The A?42 peptide is more toxic than A?40, and is the principal component of amyloid plaques in the brain. The overarching goal of the proposed research is to establish the mechanism of inhibition for small molecule inhibitors that target neurotoxic A? oligomers in order to design more effective inhibitors. The approach is to combine structural methods with functional assays to determine A?42 structure-function relationships in three specific aims. The first aim is to determine the structure and toxicity of the soluble oligomers and fibrils of A?42 using a suite of methods including solution and solid-state nuclear magnetic resonance (NMR) spectroscopy, single touch atomic force microscopy and Fourier transform infrared (FTIR) spectroscopy. The second aim is to determine the structure of membrane-bound oligomers and the dynamics of oligomer- membrane interactions. Single molecule total internal reflection fluorescence microscopy will be used to establish the association-dissociation rates and distribution of A?42 bound to membrane bilayers. FTIR spectroscopy will be used to characterize the changes in secondary structure as a function of membrane composition. Solution-state NMR and solid-state NMR spectroscopy will be used to follow specific structural markers identified in Aim 1 that are unique to the oligomers, protofibrils and fibrils. The third aim is to determine the mechanism of interaction of small molecule, peptide and protein inhibitors with A? oligomers and fibrils. The small molecule inhibitors include the natural products, curcumin and resveratrol. The peptide inhibitors are designed on the basis of the structure of the A? fibrils. The protein inhibitors are derived from fragments of the myelin basic protein, which we have shown is a natural A? inhibitor in brain white matter. An improved understanding of A?-inhibitor interactions will impact the design of inhibitors to the soluble oligomers. The goal is to establish 1) how the neurotoxic soluble oligomers differ in structure from membrane-bound oligomers and A?42 fibrils, 2) how the addition of two amino acids changes the structure of the A?42 oligomers and fibrils compared to the less toxic A?40 form, and 3) how inhibitors bind to A?42 and prevent toxicity. PUBLIC HEALTH RELEVANCE: Alzheimer's disease is associated with the deposition of fibrillar plaques in the human brain that are composed primarily of a 42-amino acid amyloid-? peptide (A?42). The proposed research is to investigate the molecular structure of A?42 and to develop inhibitors that reduce its toxicity.

? DESCRIPTION (provided by applicant): There are two distinct processes involved in the deposition of amyloid in the brain during aging. Accumulation of the amyloid ?-protein (A?) in the brain parenchyma is the hallmark of Alzheimer's disease (AD), while accumulation of the A? protein in the cerebrovascular network is a condition known as cerebral amyloid angiopathy (CAA). There are familial mutations associated with both conditions. For AD, many of the mutations reside in the A? amyloid precursor protein and are responsible for the increased production of A?42 over the more prevalent A? 40 form of the peptide. For CAA, familial CAA disorders result from specific mutations in the A? peptides themselves, including the Dutch-type (E22Q) and Iowa-type (D23N) mutations. Despite the highly fibrillogenic nature of Dutch and Iowa mutant A? peptides, fibrillar A? is restricted to the cerebral vasculature in these familil disorders. Recent evidence suggests the parenchymal plaque amyloid is distinct from cerebral vascular amyloid. However, there is a poor understanding as to why either amyloid forms, and it is not known whether there are unique structural motifs that promote the distinct pathological consequences leading to dementia. The focus of this proposal is to fill this critical void in knowledge. Accordingly, the overall hypothesis of this proposal is that the A? peptides forming parenchymal and vascular amyloid have distinct structures that determine their location and pathology. To address this hypothesis we propose four specific aims. First, we plan to isolate parenchymal plaque amyloid and cerebral vascular amyloid from post mortem brain tissue of AD and familial CAA cases. Parallel studies will be undertaken on unique transgenic mouse models of AD and CAA. The studies on transgenic mice provide a direct method to assess the level of parenchymal and vascular amyloid, and the ability to engineer mutations. The isolated amyloid will serve as seeds to nucleate fibril growth for in vitro studies in Aim 2, which will reval the distinct structural signatures of cerebral vascular and parenchymal plaque amyloid deposits. Next, we will generate a collection (library) of homogeneous fibrils isolated from brain tissue. Fluorescence, infrared and NMR spectroscopy will be used to determine the structural features of these fibrils. Preliminary results show the A? peptides in parenchymal amyloid adopt ? - sheet structure with parallel, in-register ?-strands, whereas the ?-sheets in vascular amyloid have anti-parallel structure. Third, differences in cell toxicity and activation of different fibri (and oligomer) forms will be assessed using neuronal, vascular and microglial cell cultures. Preliminary results reveal profound differences of different A? fibrils on microglial activation. Fourth, we will assess the influence of A? inhibitors on different A? fibril structures. We wil first test the ability of known inhibitors to dissociate the different fibril structures isolated from brin tissue. Next, we will design dual-site inhibitors that combine elements of known inhibitors starting with designed, synthetic inhibitors that target the GxxxG sequences in the hydrophobic C-terminus of A? and inhibitors based on fragments of the myelin basic protein that interact with the N-terminus of A?.

? DESCRIPTION (provided by applicant): Cerebrovascular accumulation of the amyloid ß-protein (Aß), a condition known as cerebral amyloid angiopathy (CAA), is an important driver of vascular cognitive impairment and dementia (VCID) and is a common comorbidity of patients with Alzheimer's disease (AD). CAA can promote VCID through a number of mechanisms including chronic inflammation, hypoperfusion and ischemia, loss of vessel wall integrity and hemorrhage. In addition to its prevalence in AD, several related familial CAA disorders result from specific mutations that reside within the Aß peptide sequence of the Aß precursor protein including the Dutch-type (E22Q) and Iowa-type (D23N) mutations. Despite the highly fibrillogenic nature of Dutch mutant and Iowa mutant Aß peptides, fibrillar Aß is restricted to te cerebral vasculature in these familial disorders. Recent evidence suggests the cerebral vascular amyloid is distinct from parenchymal plaque amyloid. However, there is a poor understanding as to why cerebral vascular amyloid forms and its unique structural features that promotes distinct pathological consequences leading to VCID. Thus, the focus of this proposal is to fill this critica void in knowledge. Accordingly, the overall hypothesis of this proposal is that fibrillar amyloid i cerebral blood vessels possesses distinct structural features compared to parenchymal fibrillar amyloid and unique anti-parallel structures, enhanced by CAA mutations, drives the cerebral vascular specific deposition of amyloid in brain. To address this hypothesis we propose three specific aims. First, we will determine the structure, assembly and membrane interactions of wild-type and the Dutch and Iowa CAA mutants of Aß in solution and model membrane systems that drive their compartmental deposition. Second, we will determine how familial CAA variants of Aß chronologically influence the structural features and assembly of wild-type Aß in the brains of transgenic mice. Third, we will isolate parenchymal plaque amyloid and cerebral vascular amyloid from post mortem brain tissue of AD cases, sporadic CAA cases and familial CAA cases and investigate their ability to promote assembly of wild-type and CAA mutant Aß peptides. These important studies will reveal the distinct structural signatures of cerebral vascular and parenchymal plaque amyloid deposits in human disease. Presently, there are no reliable biomarkers or effective therapies specifically for CAA and VCID. These deficiencies are complicated by our lack of understanding of the unique structural attributes of cerebral vascular amyloid and its early-stage oligomeric precursors, and their distinctive features and processes compared to parenchymal plaque amyloid. The present proposal will seek to fill this critical void in our knowledge and will advance our understanding of the pathogenesis of CAA and provide the basis for the future development of novel therapeutic interventions and diagnostic markers for CAA and VCID.

Vascular cognitive impairment & dementia (VCID) is defined as a form of dementia that is triggered by damage to cerebral blood vessels or cerebrovascular disease. Cerebral amyloid angiopathy (CAA), which is accumulation of amyloid ß-protein (Aß) within and along primarily small and medium-sized arteries and arterioles of the brain and in the cerebral microvasculature, is a common cerebral vascular condition that can cause VCID in the elderly. Not surprisingly, with the involvement of Aß, CAA is the most common vascular comorbidity found in the brains of Alzheimer's disease (AD) patients. Although there is evidence that both parenchymal plaque amyloid and cerebral microvascular amyloid can contribute to dementia in patients with AD and related disorders, there is growing recognition that the latter is a potent driver of cognitive impairment. Yet, the reasons as to why cerebral vascular amyloid forms and its contribution to downstream pathologies and early cognitive impairment remain unclear. Altered copper homeostasis has been considered an important factor in the neurodegenerative diseases. Earlier findings suggest that copper may play an important role in the formation of amyloid deposits and in subsequent neuronal dysfunction and cognitive impairment. However, relatively little is known about the accumulation of copper in cerebral vascular amyloid deposits, which are associated with early-onset VCID. Thus, the overall hypothesis of our proposal is that copper plays a role in driving fibrillar amyloid assembly in CAA and that the subsequent accumulation of copper in the cerebrovascular amyloid deposits promotes downstream pathologies and early- onset cognitive impairment. In order to test this hypothesis we propose to three specific aims. First, we will determine if vascular amyloid deposits exhibit high levels of copper compared to parenchymal amyloid plaques in post mortem human brain tissue samples of AD, sporadic CAA and familial CAA patients and in transgenic mouse models. Second, we will investigate the effects of copper on Aß fibril assembly. Third, we will determine the effects of increasing or reducing copper levels on the development of CAA, downstream pathologies and cognitive impairment in Tg-SwDI mice. Currently, there are no effective therapies or reliable biomarkers specifically for CAA. These deficiencies are complicated by our lack of understanding of the assembly and unique structural attributes of cerebral vascular amyloid and their distinctive features that lead to CAA formation and subsequent pathologies. The present proposal, focused on the role of copper in these events, will seek to fill this critical void in our knowledge and will advance our understanding of the pathogenesis of CAA and provide insight into the development of novel diagnostic markers and potential therapeutic interventions for CAA and VCID.

The light-activated visual receptor rhodopsin has provided the foundation for understanding the structure and mechanism of G protein-coupled receptors (GPCRs). Nevertheless, there remain fundamental unanswered questions about how these receptors work. Here, we target several basic questions that are relevant for understanding their mechanism(s) of activation. The approach is primarily through structural measurements using solid-state NMR spectroscopy. The existing crystal structures of these receptors provide a high- resolution framework to study in detail the role of specific residues and motifs in receptor activation. Because of the high conservation of residues between the visual and ligand-activated GPCRs, the emerging consensus is that rather than being unique, the visual receptors provide a basis for understanding the common structural and dynamic elements in these receptors. The general experimental strategy is to use solid-state NMR spectroscopy in combination with mutational, optical and biochemical methods to target specific regions in the inactive and active states of the dim-light receptor, rhodopsin. The goal is to understand in atomic detail the interplay between specific signature, group-conserved and subfamily-conserved motifs in the activation mechanism of rhodopsin and establish a common basis for the activation of other GPCRs. Four specific aims address structure-function questions involving regions on the extracellular side of rhodopsin (Aim 1) and within the transmembrane (TM) core and on the intracellular side of the receptor (Aim 2). In Aim 1, we will establish the role of Trp6.48 ? a key residue that mediates retinal isomerization and Schiff base deprotonation with the conserved TM core of the receptor. In Aim 2, we address how retinal Schiff base deprotonation leads to activation. The working model is that there are two triggers, one electrostatic and one steric in nature. We target the conserved TM core of rhodopsin composed of interlocking signature and group- conserved residues. The working model is that the TM core is composed of two packing clusters and two activation switches. These provide stable and flexible elements to the receptor, respectively. In Aim 3, we focus on the G protein and its interactions with residues on the intracellular surface of the active Meta II intermediate. In this aim, we address the role of the membrane environment in receptor stability and activation. Finally, in Aim 4 we use the information garnered above and from past studies to determine the basis for two retinal diseases, congenital stationary night blindness and autosomal dominant retinitis pigmentosa.

Alzheimer's disease is associated with the deposition of amyloid in the brain during aging. Since the correlation between amyloid formation and AD was originally made, it has been recognized that there are many subtypes and forms that the disease can take. For example, accumulation of the amyloid ?-protein (A?) in the brain parenchyma is the hallmark of Alzheimer's disease (AD). Nevertheless, there is a poor understanding as to why amyloid forms, and it is not known whether there are unique structural motifs that promote the distinct pathological consequences leading to dementia. The focus of this proposal is to fill this critical void in knowledge. Accordingly, the overall hypothesis of this proposal is that the A? peptides forming amyloid with distinct subtypes have distinct structures that determine their location and pathology. To address this hypothesis we propose two specific aims. First, we plan to isolate amyloid from different subtypes of post mortem brain tissue of late-onset AD and early- onset familial AD. We plan to compare five different amyloid plaque subtypes: typical AD, atypical AD, cotton wool, early-onset AD (EOAD) and very early-onset AD (VEOAD). The last two subtypes are associated with familial AD mutations. Clinical information is available concerning age and gender, age of onset and duration of disease, course and symptoms of the disease, medication and ApoE genotype. In the EOAD and VEOAD cases genetic testing was performed for APP, PSEN 1, PSEN2 and tau. For most cases, biomarkers in CSF and neuroimaging results are available. The isolated amyloid will serve as seeds to nucleate fibril growth for in vitro studies. The structure and polymorphism of the fibrils will be assessed by complementary structural approaches including solid-state NMR spectroscopy, Fourier transform infrared spectroscopy, transmission electron microscopy, and atomic force microscopy. Using the amyloid isolated from the five different subtypes of AD, we will assess the biofunctional consequences of the different strains using three approaches. First, we will assess the differences in the inflammatory response and cell toxicity due to different fibril forms using microglial cell cultures. Second, we will determine the influence of amyloid strains on promoting neuroinflammation. Third, we will determine the influence of amyloid strains on assembly and propagation in rat brain. The overall objective is to correlate pathologies (biofunctional consequences) of different amyloid subtypes between cell culture, rat brain and human brain, and to relate these pathologies with specific structural characteristics of the A? fibrils that are associated with the isolated amyloid from each subtype.