Steven Smith - US grants

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.

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.

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.

structural biology; membrane proteins; protein sequence; dimer; intermolecular interaction; protein structure; MHC class II antigen; CD3 molecule; lipid bilayer membrane; hydrogen bond; ionic bond; conformation; growth factor receptors; T cell receptor; protein reconstitution; infrared spectrometry; peptide chemical synthesis; nuclear magnetic resonance spectroscopy; protein purification; interferometry;

Quantitative and mathematical approaches to biology are becoming more prevalent in both industrial and academic research. However, there remain significant barriers between the mathematical and life sciences, reinforced by the reality that college students in these programs are not typically exposed to the other discipline beyond the freshman year. This project will establish a program to train undergraduates at the interface of mathematics and biology, through a combination of course work and research experience. The program has a focus on team-based learning, with biology and math majors working together on projects jointly led by faculty from the departments of Applied Mathematics & Statistics and various Life Sciences departments.

Each year, a cohort of undergraduate students will be recruited to the program, half drawn from majors in the mathematical sciences, and half from majors in the life sciences. These students will be grouped into research teams (including both math and biology majors) that will engage in a summer of full-time research; the research projects will continue through the following year, with the students receiving academic credit. The research projects will all involve components both of mathematics and of experimental biology, and will be jointly supervised by faculty from both disciplines. As an introduction to mathematical approaches in biology, a pair of new courses will be offered: one with a focus on the analysis of experimental data, and the other with a focus on the simulation of biological systems. These courses will provide a foundation in both the theory and application of mathematical biology, with an emphasis on how mathematical models can be integrated with experimental techniques.

This combination of team-based learning, multidisciplinary research and mentoring, and novel course work, will provide a solid foundation for students to apply mathematical approaches to biological questions. This initiative will broadly impact undergraduate education at this university, serve as a model for change at other colleges and universities, and influence the broader scientific community by graduating students with a multi-disciplinary background who will take this perspective with them in their future careers.

? DESCRIPTION (provided by applicant): The major technological and analytical advances in human brain imaging achieved as part of the Human Connectome Projects (HCP) enable examination of structural and functional brain connectivity at unprecedented levels of spatial and temporal resolution. This information is proving crucial to our understanding of normative variation in adult brain connectivity. It is now timely to use the tools and analytical approaches developed by the HCP to understand how structural and functional wiring of the brain develops. Using state-of-the art HCP imaging approaches will allow investigators to push our currently limited understanding of normative brain development to new levels. This knowledge will critically inform prevention and intervention efforts targeting well known public health concerns (e.g., neurological and psychiatric disorders, poverty). The majority of developmental connectivity studies to date have used fairly coarse resolution, have not been multi-modal in nature, and few studies have used comparable methods to assess individuals across a sufficiently wide age range to truly capture developmental processes (e.g., early childhood through adolescence). Here we propose a consortium of five sites (Harvard, Oxford, UCLA, University of Minnesota, Washington University), with extensive complimentary expertise in brain imaging and neural development, including many of the investigators from the adult and pilot lifespan HCP efforts. Our synergistic integration of advances from the HARVARD-MGH and WU-MINN-OXFORD HCPs with cutting edge expertise in child and adolescent brain development will enable major advances in our understanding of the normative development of human brain connectivity. The resultant unique resource will provide rich, multimodal data on several biological and cognitive constructs that are of critical importance to health and well-being across this age range and allow a wide range of investigators in the community to gain new insights about brain development and connectivity. Aim 1 will be to optimize existing HCP Lifespan Pilot project protocols on the widely available Prisma platform to respect practical constraints in studying healthy children and adolescents over a wide age range and will also collect a matched set of data on the original Skyra and proposed Prisma HCP protocols to serve as a linchpin between the past and present efforts. Aim 2 will be to collect 1500 high quality neuroimaging and associated behavioral datasets on healthy children and adolescents in the age range of 5-21, using matched protocols across sites, enabling robust characterization of age-related changes in network properties including connectivity, network integrity, response properties during tasks, and behavior. Aim 3 will be to collect and analyze longitudinal subsamples, task, and phenotypic measures that constitute intensive sub-studies of inflection points of health-relevant behavioral changes within specific developmental phases. Aim 4 will capitalize on our success in sharing data in the HCP, and use established tools, platforms and procedures to make all data publically available through the Connectome Coordinating Facility (CCF).

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.

? 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.

This award is an experimental effort to characterize rare but strong disturbances in the Earth's atmosphere that are often visible to the naked eye as large bands of faint optical emission called airglow that extend from horizon to horizon. These disturbances are atmospheric waves that are known as mesospheric bores. These waves are characterized by an extensive propagating "step-like" frontal region of enhanced or depleted airglow followed a train of waves. Mesospheric bores exhibit large brightness amplitudes (typically >10%) and can transport large amounts of energy and momentum through the mesosphere-lower-thermosphere (MLT) region. The wave dynamics significantly affects the large-scale temperature and circulation structure of the MLT and higher atmospheric regions. Understanding the full scope of wave effects on the MLT region is important for obtaining a complete physical picture of atmospheric processes.

This work will utilize the data from eight existing ground-based optical imaging systems, together with a suite of co-located multi-diagnostic instrumentation and satellite data, to investigate the global and seasonal occurrence characteristics of mesospheric bores and other frontal gravity waves in the MLT region. The effort will:
1) conduct a global survey and characterization of these waves and an estimate of the momentum flux contributing to the MLT using the Boston University all-sky imager network, and
2) utilize co-located ground-based multi-diagnostic measurements and satellite measurements to supplement the imager data in order to fully characterize both the propagation environment and the gravity wave propagation modes.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.