2000 — 2004 |
Weiss, Shimon |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Development of Q-Dots as Biological Probes @ University of Calif-Lawrenc Berkeley Lab
The long-term goal of this Bioengineering Research Partnerships is to develop semiconductor nanocrystals fluorescent probes (q- dots) technology that will provide biomedical research with better tools for diagnosis of diseases and biomedical techniques and instrumentation necessary for basic research of cellular and molecular structure and fundamental life processes. This includes q-dot probe synthesis, bio-conjugation techniques, dedicated optical instrumentation and unique imaging methodologies. We will develop optimized protocols for q-dot synthesis with desired optical, physical and chemical properties. Various spectroscopic and structural measurements will be used to fully characterize q-dots. This information will be fed back into the synthesis for optimization of the desired properties. Bio-conjugation schemes and labeling protocols will be developed for biomolecules and fixed and living cells. The utility and the new possibilities opened-up by q-dot technology will be demonstrated by studying protein trafficking and assembly in living cells and by physically mapping genes. The movements of secretory granule membranes during recycling will be tracked in living cells. Actin-based locomotion and mitotic spindle assembly will be imaged in real-time in cell- extracts. Molecular mechanism of synaptic transmitter release will be studied by following vesicle dynamics and protein trafficking in the synaptic apparatus. We will also physically map large number of distinct markers on chromosomes and combed DNA molecules and monitor the kinetics of chromosome pairing during meiotic prophase. All these demonstrations rely on the unique photophysical properties of q-dots, enabling new experiments and measurements to be performed and significant new biology to be revealed.
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1 |
2001 — 2003 |
Weiss, Shimon |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Single Molecule Protein and Biopolymer Folding Dynamics @ University of California Los Angeles
The objective of this proposal is to study protein folding using single-molecule fluorescence methodology. Understanding protein folding is fundamental to deciphering how the genetic code is translated into functional protein units. Improper protein folding is related to complex aggregation phenomena that cause diseases such to the prion family of diseases and Alzheimer's disease. We will develop single-molecule fluorescence methodology to follow conformational dynamics of individual fluorescently-labeled proteins and other biopolymers. In contrast to ensemble methods, single-molecule methods provide information on fluctuations, distributions and time-trajectories of observables that are obscured in ensemble measurements. In particular, single-molecule methods are free from synchronization requirements that are impossible to achieve with ensemble methods. We will use single-pair fluorescence resonance energy transfer (spFRET) as reporter of the distance between two amino acid residues in the polypeptide chain, or the site-to-site distance of a fluctuating biopolymer. The recovered distance information will be used as a reaction coordinate of protein folding, and as a reporter of conformational dynamics. We will emphasize early events in the folding pathway, especially focusing on the "fast-collapse" of the polypeptide chain when exposed to specific solvent conditions. We will address both equilibrium and non-equilibrium reaction conditions, and study molecules that are (i) diffusing in solution, or (ii) immobilized on surfaces and in gels. We will develop single-molecule, continuous flow, fast-mixing methods (for diffusing molecules) and rapid liquid-exchange methods (for immobilized molecules). Our measurements will combine fluorescence-intensity ratiometric methods with fluorescence-lifetime methods to provide unprecedented sensitivity and temporal resolution that cover many orders of magnitude. Advanced data acquisition, data analysis and signal processing algorithms will be developed for a variety of samples, experimental formats and reaction conditions. A theoretical framework will be constructed to accurately describe the results of our experiments. We will: (i) develop single-molecule methods with spatial and temporal resolution relevant to protein folding; and (ii) use homopolymeric single-stranded DNA (ssDNA) as a model system for method development. We will combine the proposed methodology with existing single-molecule fluorescence methodology to study the folding of protein chymotrypsin inhibitor 2 (CI2). In particular, we will address: (1) early events of the folding pathway, such as fast collapse; (2) late events of the folding pathway, such as formation of native protein contacts; and (3) effects of amino acid substitutions on early and late events of the folding pathway.
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1 |
2002 — 2003 |
Weiss, Shimon |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
High Performance Photon-Counting Imager @ University of California Los Angeles
DESCRIPTION (provided by applicant): The objectives of this proposal are to develop a high performance photon counting imager optimized for ultrahigh sensitivity fluorescence spectroscopy and imaging and to develop new methodologies and protocols that take advantage of its unique capabilities. The detector will be capable of registering single photons with high-resolution in space and time at high-count rates and high quantum efficiency. It will be suitable for imaging and spectroscopy of single molecules, molecular complexes and macromolecules in living cells and tissues with increased sensitivity, signal to noise and signal to background, while providing multi-parametric, high information-content from each detected photon. They dub this detector H33D (pronounced `heed', for: high-spatial resolution, high-temporal resolution and high count-rate 3-dimensional detector). The targeted detector performance: (i) a spatial resolution of 100 microm x 100 microm per pixel (spatially resolvable units) with a detector dimension (25 mm diameter) allowing at least 256 x 256 resolution elements (pixels), (ii) 100 picoseconds (ps) temporal resolution, (iii) a maximum count rate of 10(5) Hertz (100 kHz) per single pixel and 5.10(7) Hertz (50 MHz) over the whole detector and (iv) a quantum efficiency (QE) of > 40 percent @ 600nm. The proposed work is "high risk, high impact" and fits well the structure of the R21/R33 phased innovation mechanism. They will develop 3 generations of H33D detectors. The specifications/milestones for the pilot phase are already superior to those of current commercial detectors. They anticipate that these milestones will be met within 12 months. In the expanded development phase (R33), they will build two more generations. To deliver on the milestones, they will need to develop high QE photocathode (feasibility unknown, high risk); low-gain, moderate-resolution spatial detection (proof of principle exists); time-stamping of simultaneous spatially-separable events (feasibility unknown, high risk); high-count rate at high timing resolution (feasibility good). The development of the H33D would open new windows in multi-parameters studies of single molecules (simultaneous acquisition of lifetime, fluorescence spectrum and polarization), in time-resolved wide-field imaging of in vivo cellular processes or in vitro enzymatic reactions and high-throughput fluorescence correlation spectroscopy (FCS). They will demonstrate proof-of-principle biological applications building on other funded projects in their laboratory: (i) Time-correlated imaging and spectroscopy of semiconductor nanocrystals (NCs); (ii) Digital time-gated imaging of NCs in live fibroblast cells; (iii) Fluorescence lifetime imaging (FLIM) of live cells; (iv) Line-confocal time-resolved spectral imaging of cells; (v) Single-pair FRET conformational dynamics of surface-immobilized RNA polymerase molecules; (vi) Microfluidic applications; (vii) Multiconfocal fluorescence correlation spectroscopy (FCS); (viii) High frame-rate cell imaging.
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1 |
2002 — 2003 |
Zhang, Xiang Weiss, Shimon Ho, Chih-Ming (co-PI) [⬀] Yablonovitch, Eli (co-PI) [⬀] |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Mri: 3d Nano Manufacturing of Novel Photonic Structures @ University of California-Los Angeles
This Major Research Instrumentation (MRI) award provides funds for the purchase of a scanning electron microscope and a Fourier transformation infrared spectrometer. This equipment will be used to support research on development of three-dimensional nanomanufacturing technologies and on those technologies' applications in nanophotonic structures and devices. Most nanophotonic structures with designed functionalities are complicated and three-dimensional, therefore three-dimensional nanomanufacturing technologies are essential. The scanning electron microscope can provide images with high resolution, and the Fourier transformation infrared spectrometer can measure the essential optical properties of photonic structures and devices. These structural and optical properties will provide important information and guidance for the development and optimization of nanomanufacturing processes. The research conducted using this equipment is interdisciplinary and involves the collaborative efforts of researchers from various departments. This will likely lead to advances in the sciences and technologies of nanomanufacturing, photonic material, and devices. By creating a tight-nit environment of mechanical engineers, electrical engineers, and chemists, a new generation of engineers with interdisciplinary knowledge and research experience will be trained through participating in these research activities. The advances in nanomanufacturing and education will benefit society by helping the United States maintain a competitive edge in this high technology industry and prepare a high quality workforce for the nation's economy and security.
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1 |
2004 |
Weiss, Shimon |
S10Activity Code Description: To make available to institutions with a high concentration of NIH extramural research awards, research instruments which will be used on a shared basis. |
One-Photon/Two-Photon/Lifetime Confocal Microscope: Genetics, Cancer @ University of California Los Angeles |
1 |
2004 |
Weiss, Shimon |
S10Activity Code Description: To make available to institutions with a high concentration of NIH extramural research awards, research instruments which will be used on a shared basis. |
One-Photon/Two-Photon/Lifetime Confocal Microscope: Protein Studies @ University of California Los Angeles |
1 |
2004 |
Weiss, Shimon |
S10Activity Code Description: To make available to institutions with a high concentration of NIH extramural research awards, research instruments which will be used on a shared basis. |
One-Photon/Two-Photon/Lifetime Confocal Microscope: Nanotechnology @ University of California Los Angeles |
1 |
2004 |
Weiss, Shimon |
S10Activity Code Description: To make available to institutions with a high concentration of NIH extramural research awards, research instruments which will be used on a shared basis. |
One-Photon/Two-Photon/Lifetime Confocal Microscope @ University of California Los Angeles
DESCRIPTION (provided by applicant): Understanding the properties of biological and artificial systems on the nanometer scale requires not only the ability to manipulate, fabricate, synthesize and assemble them with precise atomic/molecular level control, but also the ability to characterize and probe them with state-of-the-art nanoscale spectroscopic and imaging tools. Fluorescence microscopy offers many advantages for probing nanoscale systems. It is non-invasive and provides imaging in three dimensions; it has high sensitivity (down to the single molecule level), and allows the observation of molecular- and organelle-specific signals. We propose the acquisition of a combined One-Photon / Two-Photon / Lifetime Confo(;al Laser Scanning Microscope (1P/2P/Lifetime CLSM) to support research in artificial and biological nanoscale system assemblies conducted at the California Nano-Systems Institute (CNSI) at UCLA. The combined 1P/2P/Lifetime CLSM will allow the characterization of biological and artificial systems with the highest spatial and temporal resolutions achievable. This advanced optical tool would be particularly useful for: (1) The study dynamic interactions of biological macromolecules in living cells; (2) The visualization of multiple fluorescent markers at high resolution in a single optical plane (optical plane sectioning) and reconstruction of 3D images from consecutive sections; (3) The visualization and 3D sectioning of multiple fluorescent markers embedded in scattering media and tissues with high signal to noise and minimal photo-bleaching. The 1P/2P/Lifetime CLSM will be use to address outstanding questions covering a broad spectrum of topics in Chemistry, Biology, Physiology and Material Sciences. We propose to use the advanced performances of the 1P/2P/Lifetime CLSM in the following research projects: (1) The targeting of semiconductor nanocrystal probes in live cells, (2) The detection of conformational changes in voltage dependent ion channels and (3) The detection of structural and functional abnormalities in the transverse tubular system of normal and dystrophic muscle fibers. The acquisition of the 1P/2P/Lifetime CLSM would considerably enhance the nanoscale science performed at the CNSI and our understanding of the central functions of the cell.
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1 |
2004 |
Weiss, Shimon |
S10Activity Code Description: To make available to institutions with a high concentration of NIH extramural research awards, research instruments which will be used on a shared basis. |
One-Photon/Two-Photon/Lifetime Confocal Microscope: Muscle Research @ University of California Los Angeles |
1 |
2004 — 2005 |
Weiss, Shimon |
P41Activity Code Description: Undocumented code - click on the grant title for more information. |
Single Molecule Protein &Biopolymer Folding Dynamics @ University of California Los Angeles
model design /development
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1 |
2005 — 2010 |
Weiss, Shimon |
P41Activity Code Description: Undocumented code - click on the grant title for more information. R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Single-Molecule Fluorescence Analysis of Transcription @ University of California Los Angeles
Anti-Oncogenes; Antioncogenes; Behavior; Biochemical Genetics; CRISP; Cancers; Cells; Complex; Computer Retrieval of Information on Scientific Projects Database; Condition; DNA; DNA Damage Repair; DNA Recombination; DNA Repair; DNA Replication; DNA Synthesis; DNA biosynthesis; DNA recombination (naturally occurring); DNA-Dependent RNA Polymerases; DNA-Directed RNA Polymerase; Defect; Deoxyribonucleic Acid; Detection; Development; E coli; EC 2.7.7.6; Electromagnetic, Laser; Emerogenes; Ensure; Equilibrium; Escherichia coli; Family; Fluorescence; Functional RNA; Funding; Gene Expression; Gene Products, RNA; Gene Transcription; General Transcription Factor Gene; Generations; Genes, Cancer Suppressor; Genes, Onco-Suppressor; Genetic Recombination; Genetic Transcription; Genetic, Biochemical; Grant; Heterogeneity; Individual; Institution; Investigators; Label; Lasers; Life; Malignant Neoplasms; Malignant Tumor; Methods; Modeling; Molecular; Monitor; NIH; National Institutes of Health; National Institutes of Health (U.S.); Non-Coding; Non-Coding RNA; Nucleoproteins; Nucleoside-triphosphate[{..}]RNA nucleotidyltransferase (DNA-directed); Oncogenes, Recessive; Oncogenes-Tumor Suppressors; Pathway interactions; Polymerase; Process; Promoter; Promoters (Genetics); Promotor; Promotor (Genetics); Proto-Oncogene, Transcription Factor; RNA; RNA Expression; RNA Metabolism[{..}] Processing and Transport; RNA Polymerases; RNA Processing; RNA, Non-Polyadenylated; Radiation, Laser; Recombination; Recombination, Genetic; Research; Research Personnel; Research Resources; Researchers; Resources; Ribonucleic Acid; Site; Source; Structure; Time; Transcription; Transcription Regulation; Transcription factor genes; Transcription, Genetic; Transcriptional Control; Transcriptional Regulation; Translations; Tumor Suppressing Genes; Tumor Suppressor Genes; United States National Institutes of Health; Unscheduled DNA Synthesis; Work; balance; balance function; human disease; insight; malignancy; member; movie; neoplasm/cancer; oncosuppressor gene; pathway; single molecule; single-molecule FRET; single-molecule fluorescence resonance energy transfer; smFRET; tool; transcription factor
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1 |
2006 — 2010 |
Weiss, Shimon Michalet, Xavier |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Idbr: Collaborative Research: Development of a Time-Resolved Photon-Counting Imager For Biology @ University of California-Los Angeles
This is one of two awards supporting a collaborative project aimed at development of a new time-resolved single-photon imager optimized for ultrahigh sensitivity fluorescence spectroscopy and microscopy. The detector will be capable of registering individual (single) photons with high quantum efficiency and time resolution, and with high spatial resolution in two-dimensions (2D) at high-count rates. The instrument will be suitable for imaging and spectroscopy of single molecules, molecular complexes and macromolecules in living cells and tissues with increased sensitivity, signal-to-noise ratio and signal-to-background ratio, while providing multi-parametric, high information content from each detected photon (2D position, microscopic and macroscopic time of arrival with respect to an excitation by a train of short laser pulses, or wavelength and polarization anisotropy). The device has been dubbed the "H33D detector" for High-spatial resolution, High-temporal resolution and High count-rate 3-Dimensional detector. Targeted detector performance is: (i) quantum efficiency (QE) of > 35 % @ 600nm. (ii) spatial resolution of 50 um x 50 um, allowing at least 256 x 256 resolution elements (pixels), (iii) temporal resolution of 150 picoseconds, and (iv) maximum count rate of 100 kHz per single pixel and 5 MHz over the whole detector. The development of the H33D detector comprises two complementary aspects: 1) a new detector design based on an initial prototype, and 2) new biological imaging and spectroscopy applications. The new detector design consists of a fast GaAs photocathode mounted in front of a stack of microchannel plates and a cross-strip anode. The readout electronics will be designed, built and tested by one of the collaborating groups. The other collaborating group will build the optical instrumentation and software needed for it. Two instruments will be built; the first, a non-optimized prototype, will be installed initially in a campus shared-use facility at UCLA, and eventually transferred to the California NanoSystems Institute Advanced Light Microscopy/Spectroscopy Shared Facility. Through these placements, the instrument will be available for use by a number of independent investigators and students. The experience gained from such use will be helpful in development of a second, more optimized instrument. Broader impacts of the project derive from the involvement of students in instrument construction and testing, and the availability of the instrument by a large number of investigators in a shared facility.
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1 |
2006 — 2010 |
Weiss, Shimon |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Multimodal Qdot Probes For Bioimaging of Cells and Tumors in Small Animals @ University of California Los Angeles
DESCRIPTION (provided by applicant): Quantum dots (qdots) have emerged as new fluorescent, non-isotopic labels that are thought to have unmatched potentials as novel intravascular probes for both diagnostic (e.g., biological imaging) and therapeutic purposes (e.g., drug delivery). This application capitalizes on the progress we have made in the first 5 years of BRP funding, aiming to bring qdots one step closer to routine use in pre-clinical and clinical models. Towards this goal, we will perform detailed validation studies of targeted qdots in living mice models that are already impacting clinical management of cancer patients by using small animal imaging technologies including multiphoton microscopy and fluorescence tomography. During the next 5 years of this project, we will (1) develop and bioconjugate new multimodal contrasting qdot agents;(2) develop new optical instruments for near infrared (NIR) qdot detection and (3) perform validation studies of targeted qdots in cells and animals. We will focus on cancer where no single imaging agent can provide adequate information for diagnosis, prognosis and treatment decisions. Rather, a better understanding of the biological behavior and potential of malignant cells is inherent to new instrumentations and probes that will allow multiplex imaging of panels of targets/markers. The availability of NIR qdots with several output wavelengths, coupled with tumor marker-specific engineered antibodies, peptides or nucleic acids, will streamline multicolor/ marker imaging of cells and tumors. We will develop panels of qdot probes useful for karyotyping cancerous cells and for gene expression profiling towards a combination of genes that would be diagnostic or prognostic markers for cancer progression. Using cancer models, we will visualize the bio-distribution of qdots at all scales, from the level of the whole animal body down to nanometer resolution using a single probe by combining the micro-PET and optical modalities of antibody-functionalized NIR qdots. We will also asses toxicity and size effects on biodistribution of these probes. Our multidisciplinary team of 5 investigators with expertise in physics, chemistry, materials sciences, bioengineering, pharmacology, imaging and cancer biology should help to more rapidly validate this new exciting class of imaging probes for eventual clinical applications. Lay abstract: Physicians can now monitor tumors and malignant cells deep within our body. Our research aims at validating new sensitive probes (known as Quantum Dots, or qdots) for non-invasive tumor imaging in animal models. The unique properties of qdots might afford in the future earlier diagnosis and better management of disease in humans.
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1 |
2006 — 2012 |
Kelly, Jeffery Pande, Vijay Bakajin, Olgica (co-PI) [⬀] Weiss, Shimon Johnson, Arthur |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Fibr: How Do Proteins Fold Into Their Native and Functional Structures in-Vitro and in the Physiological Milue of the Living Cell? @ University of California-Los Angeles
In all living cells, proteins self-assemble or fold into precise, three dimensional structures having unique functions from a linear polypeptide chain assembled by the ribosome (the enzyme responsible for protein synthesis). The precise folding of a protein is dictated by its DNA sequence but researchers have not yet deciphered the rules for encoding structure by sequence ("the protein folding problem"). Addressing this problem is crucial to understanding how gene sequence variation translates into variation in protein and cell function. The delicate balance of forces which controls and guides the structural dynamics of the folding process is highly sensitive to environmental conditions inside the cell. The goal of this project is to synergistically apply cutting-edge methodologies, including single molecule spectroscopy, ultra-fast microfluidics mixing, photo-induced electron transfer, non-natural amino-acid labeling, mitochondrial protein transport, chemical peptide synthesis and simulation modeling using distributed and super-computing systems, to the study of protein folding under conditions that mimic the natural folding environment inside the living cell. The consortium of researchers will study the unfolded state of three different proteins in simple solutions (in-vitro) under a variety of conditions, and while the proteins are being made directly on the ribosome itself. By comparing such studies to protein folding experiments conducted within the crowded environment of the mitochondrial matrix (a mimic for the intracellular folding environment), this project seeks to understand the major differences between in-vitro and in-vivo folding environments and the effects of such differences on protein folding mechanisms.
This project will have broad impacts on the field of cellular biology through the development of novel tools and methods as well as a general approach for studying complex biological processes on the molecular level. An outreach program will target the dissemination of these research tools to faculty and students from underrepresented institutions, and the enhancement of scientific and technological knowledge at the secondary education level. This project represents an interdisciplinary collaboration of researchers led by Shimon Weiss, at the University of California-Los Angeles with subawards to Stanford University (Vijay Pande), Texas A&M University (Arthur Johnson), University of California-Davis (Olgica Bakajin), Michigan State University (Lisa Lapidus) and Scripps Research Institute (Jeff Kelly). A large number of students and postdoctoral fellows will receive advanced training in conceptual and technical aspects of research at the interface of chemistry, biophysics, and simulation.
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1 |
2006 |
Weiss, Shimon |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Single-Molecule Fluoresence Analysis of Transcription @ University of California Los Angeles
DESCRIPTION (provided by applicant): Transcription by Escherichia coli RNA polymerase (RNAP), a well-characterized member of the multisubunit RNAP family, involves several mechanistic steps inaccessible to methods that study static structures or molecular ensembles. To understand transcription mechanisms, it is necessary to uncover and analyze dynamic, transient, and non-equilibrium steps along the transcription pathway. Single-molecule detection (SMD) is a new set of tools that can stand up to this challenge by monitoring the real-time behavior of individual transcription complexes. We have developed single-molecule Fluorescence Resonance Energy Transfer (smFRET) combined with alternating-laser excitation in order to study the structure and dynamics of transcription complexes. We propose to use this method to understand transcription by analyzing poorly-characterized transitions in transcription complexes; several of these transitions are extremely important for transcriptional regulation, since they form the steps where transcription factors control gene expression. We propose to focus on multistep transitions: the transitions occurring on the path from RNA polymerase to the formation of RNA polymerase-promoter open complex, the transitions occurring on the path from RNA polymerase-promoter open complex to initial transcribing complexes, and transitions occurring on the path from initial transcribing complexes to a mature elongation complex. The results of the proposed work will allow direct observation of structural and mechanistic heterogeneity of transcription complexes; validate or disprove models proposed after decades of genetic, biochemical, and structural analysis of transcription that were not validated experimentally; and will allow generation of real-time, molecular "movies" of individual, functional RNAP molecules operating on DNA. The high homology of E. coli RNAP polymerase with its eukaryotic counterparts ensures that mechanistic insights obtained from the proposed work will be directly extrapolated to eukaryotic transcription and will greatly enhance understanding of transcription-associated human diseases, such as various forms of cancer, (since numerous oncogenes and tumor-suppressor genes are transcription factors), developmental defects, and other pathological conditions. The proposed methods are applicable to the analysis of nucleoprotein complexes present in DNA replication, DNA recombination, DNA repair, RNA processing and RNA translation, and when combined with advances in site-specific labeling, will allow the study of such processes in living cells.
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1 |
2007 — 2010 |
Weiss, Shimon |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
High-Resolution, High Speed, High-Throughput 3-Dimensional Detector For Biology @ University of California Los Angeles
DESCRIPTION (provided by applicant): The objectives of this proposal are to develop a high-performance photon-counting 3-dimensional imager (2 spatial, 1 temporal) for biology and new methodologies and protocols taking advantage of its new capabilities. This detector will be optimized for ultrahigh sensitivity fluorescence spectroscopy in the visible and near-infrared spectral range (500-850 nm). It will have: (i) a high detection efficiency based on a fast GaAs photocathode: ~35 %, (ii) will register single-photons with high spatial resolution using a cross-strip anode design: 35 um, and (iii) high temporal resolution using microchannel plates: 250 ps, and (iv) be capable of both high local and global couting rates (100 kHz, 20 MHz). These characteristics are condensed in the acronym "H33D" (pronounced heed) for High-spatial, High-temporal resolution, and High throughput (the 3 "H'"s, hence H3) 3-Dimensional detector. The H33D detector will be suitable for a variety of biomedical applications, such as imaging and spectroscopy of single molecules, molecular complexes and macromolecules in living cells and tissues, as well as in vivo animal imaging, some of which will be studied during the proposed 4 year span of this research proposal. The last application will be a first step towards using the H33D detector for biomedical imaging of fluorescent molecular probes in human patients with unprecedented sensitivity. These characteristics of our interdisciplinary research proposal regrouping scientists with a biophysical/biochemical background and scientists with a physics/instrumentation background are in perfect match with the vision expressed during the Biomedical Imaging Symposium 'Visualizing the Future of Biology and Medicine" organized by the BECON in 1999, and fits particularly well within the mission of the NIGMS and the NIBIB, who are part of BRG program announcement PA-02-011. Relevance of this research to public health: The richer information provided by the H33D detector will lead to a better understanding of cellular processes at the molecular level, a prerequisite step to design intelligent counter-measures aimed at curing abnormal cellular behavior with medical compounds. The long-term application of the H33D detector for live animal and human imaging will provide a much needed high- sensitivity instrument for diagnosing and monitoring the evolution of diseases at the molecular level.
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1 |
2007 — 2010 |
Grunstein, Michael (co-PI) [⬀] Weiss, Shimon Bentolila, Laurent |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Mri: Acquisition of a Stimulated Emission Depletion (Sted) Microscope For Nanoscopic Resolution of Biological Samples @ University of California-Los Angeles
The Chemistry Department at the University of California-Los Angeles will acquire a stimulated emission depletion confocal laser scanning microscope (STED-CLSM) with this award from the Major Research Instrumentation (MRI) program. The requested microscope displays resolution down to 28 nm in the focal plane, a 10x improvement over conventional light microscopes. The instrument will be used to develop multi-color inorganic, stable, quantum rods as novel STED probes, decipher the structure of chromatin and its packaging into chromosomes in the cell, study cell signaling, viral and bacterial infection pathways, neural plasticity and many other important biological questions.
STED microscopy provides an alternative to electron microscopy because it capitalizes on the well-established advantages of fluorescence microscopy (sensitivity, molecular specificity, genetically encoded probes, live cells, ease of operation). The STED concept relies on a purely physical phenomenon, stimulated emission, coupled with smart optics, to sharpen the confocal excitation spot, resulting in much more detailed, nanometer resolved images. Bridging the gap between electron and diffraction-limited light microscopy, a STED nanoscope should be a powerful tool for unraveling the relationship between structure and function in cell biology. Indeed, many outstanding problems lay in the nanometer scale, such as the organization of chromosomes, the assembly of large protein complexes and viral structures, organelle structures, as well as applications to non-biological nano-scale devices.
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1 |
2008 — 2010 |
Weiss, Shimon |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Multipixel Hybrid Photon-Counting Detector For High-Throughput Single-Molecule As @ University of California Los Angeles
DESCRIPTION (provided by applicant): The long-term objective of this project is to provide the biomedical research community with a new device for high-throughput single-molecule fluorescence assays. The domains of applications of these methods range from basic research to high-throughput diagnostics and drug screening. The goal of this research proposal is to develop an integrated system for multiplexed, high-throughput single-molecule assays. This system will be based on a new type of single-photon counting capable mutichannel hybrid photodetector (HPD) recently developed by Hamamatsu Photonics Corporation. We will incorporate this detector into a multiple excitation/multiple detection spot confocal microscope and demonstrate its capabilities in actual single-molecule assays. We expect this detector to find applications not only in fundamental research applications using single-molecule methods, but also in the emerging fields of high-throughput single-molecule diagnostics and drug screening. The specific aims of this research proposal are: (1) to develop an electronic interface and data acquisition system (including software) for the multichannel hybrid detector developed by Hamamatsu. (2) to integrate this system into a multiplexed single-molecule fluorescence excitation/detection confocal microscope. (3) to demonstrate the capabilities of this system for multiplexed, high-throughput single- molecule assays, by performing multiplexed fluorescence correlation spectroscopy (FCS) measurements on model systems of biological relevance as well as multiplexed single-molecule FRET measurements using an alternate laser excitation (ALEX) scheme.
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1 |
2009 — 2012 |
Weiss, Shimon |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Single Molecule Studies of Prokaryotic and Eukaryotic Transcription Machineries @ University of California Los Angeles
DESCRIPTION (provided by applicant): This proposal seeks to unravel unresolved questions in the mechanisms and regulation of prokaryotic transcription initiation, elongation and termination, as well as in early stages of eukaryotic transcription initiation. Single molecule fluorescence spectroscopic and microscopic methods will be used to detail the sequence, magnitude, kinetics and order of conformational transitions and intermediates along the basal transcription cycle pathway. Transient protein-DNA and protein-protein interactions (and their induced conformations) of the prokaryotic transcription machinery's subunits and of some of the eukaryotic transcription machinery's subunits will be investigated. The mechanisms of prokaryotic and eukaryotic transcription regulation will also be explored using several transcription factors as model systems. Several single molecule assay formats, implemented on freely diffusing transcription complexes and on immobilized complexes will be utilized to address mechanistic and kinetic questions respectively. The proposed studies are expected to elucidate the role of conformational transitions and biomolecular interactions in transcription and its regulation, and the tools developed for these studies could be generalized for the study of regulatory circuits and other biomolecular machineries. The proposed studies of prokaryotic transcription will provide a basis for understanding antibacterial drugs (through inhibition of conformations) and therefore will provide relevant insight and assays for drugs design and screening. The proposed studies of eukaryotic transcription will lay a foundation for understanding mechanisms of transcription related human diseases and will aid in designing and screening therapeutics agents for these diseases. PUBLIC HEALTH RELEVANCE: The proposed work will unravel the detailed mechanism of the first and most important step in gene expression, i.e. transcription and transcription regulation. It will pave the way for detailed molecular and structural understanding of the action of antibacterial agents. It will also provide answers for several long standing and unresolved questions regarding the early stages of eukaryotic transcription initiation and regulation. The latter will provide detailed molecular understanding of the causes for transcription related diseases and will provide tools for drug design and screening that will prevent, manage and cure these diseases.
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1 |
2013 — 2018 |
Weiss, Shimon |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Collaborative Research: Elucidating Pre-Initiation Complex Assembly and Transcription Initiation by Pol-Ii Using Advanced Single Molecule and Microfluidic Methods @ University of California-Los Angeles
Intellectual Merit: RNA polymerase II (also called pol II) is an essential enzyme that catalyzes the transcription of DNA into messenger RNA (and most other forms of RNA), an important first step in gene expression. This project will elucidate key structural and functional aspects of the assembly of the Pre-Initiation Complex (PIC) of pol II and the mechanism of transcription initiation, i.e. the mechanistic steps involved in starting the transcription of a new gene. Sophisticated solution-based single-molecule fluorescence techniques, combined with microfluidics, will be applied to individual complexes in order to study how pol II assembles at a promoter (gene start site) and how it performs its catalytic function. Because single-molecule approaches remove ensemble and temporal averaging, information about the order and the kinetics of assembly will be unraveled.
Broader Impacts: The "nano-biotechnology revolution" and the emergence of single molecule biophysics and super-resolution imaging have opened up tremendous opportunities in the investigation of processes that define life. One such process, fundamental to the "Central Dogma of Biology," is transcription. The approach for studying this basic process to be taken in this research could break conceptual and practical barriers that have hindered progress, and offers potential to catalyze discovery in many new ways. The tools and methods to be developed, as well as the general approaches pursued, will have greatest impact if distributed to a broad audience of receptive and innovative young scientists. Through educational and outreach programs, the project will expose a young audience (high school and college students), as well as more mature scientists, to novel research tools and methodologies that could be used to study many other biological processes and molecular machines.
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1 |
2016 — 2018 |
Neuhauser, Daniel (co-PI) [⬀] Weiss, Shimon |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Eager: Measuring Near-Field Nanoplasmonics Fields Using Super-Resolved Far-Field Optics @ University of California-Los Angeles
With support from the Chemical Measurement and Imaging Program, Professors Weiss and Neuhauser at the University of California-Los Angeles are developing a new imaging tool to measure local surface plasmon field intensity near nanometer-sized structures. It is known that sometimes when the incoming light illuminates a surface immobilized with small metal features, the electrons in the metal can oscillate back and forth together and form a "wave" ? the so called "surface plasmon". Surface plasmon is an important optical phenomenon and has been widely used in many real world applications, including the dark red color in medieval stained-glass windows that are seen in an old buildings ?the color comes from the visible light interacting with gold nanoparticles embedded in the glass. In order to better utilize the surface plasmon phenomenon, it is important to understand how it is distributed around imperfect nano-structures. Modern science advancement allows scientists to estimate the distribution of surface plasmon field intensity with computer simulation programs, but direct measurements of such field intensity, especially around imperfectly prepared nano-structures, are challenging and have not been fully realized. Professors Weiss and Neuhauser are developing a way to directly measure surface plasmon intensity near a small surface structure by monitoring the blinking rate of certain types of inorganic particles. This method would allow them to map the field intensity at a very high spatial resolution. It is also very fast and inexpensive as compared with other methods currently in development. During this 18-month grant period, both groups are focusing on (1) placing the inorganic particles around nanometer-sized features on a surface and (2) studying how the placement of these particles may be used to map the local EM field intensity. They are applying this imaging technique to study how molecules move near a surface or how a reaction happens on a metal nanoparticle. The graduate students in two groups are involved in both experimental and theoretical components of research. Both professors are also actively engaged in encouraging talented high school student to be enrolled in graduate programs, in particular from underrepresented minority groups.
The ability to simultaneously superresolve plasmonic field strengths over a large region is unique and desirable. Such approach will deepen the understanding of and control over plasmonic systems, and will broaden the impact of plasmonics. The novel probing technology Professors Weiss and Neuhauser are working uses the dependence of the blinking statistics in quantum dots on the electric field strength to resolve plasmonic field strengths well below the diffraction limit. The methods negate complications typical of localizing dipole emitters near a metallic nanostructure. A theoretical framework based on modeling of the quantum dots response with time-dependent density functional theory in deterministic or stochastic variants is also used to construct simplified building blocks. A computationally simplified building-blocks based modeling then allow simulations of a very large number of quantum dots and plasmonic structures simultaneously, mimicking the on-going experimental systems. By optimizing the theoretical and experimental tools developed here, the detailed electric field map of ~100x100 micrometer-squared size regions may be measured in quick succession. The imaging method, if successful, could benefit many applications that rely on the ability to measure field strengths below the diffraction limit, ranging from biology, to high speed integrated circuits, to optical computing. Additionally, the software developed for the experiments and for the theory studies provides an approachable tool for analyzing and predicting field strengths in heterogeneous regions. Professors Weiss and Neuhauser intend to disseminate the research tools developed to a broad community through freely available software packages.
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1 |
2018 — 2021 |
Neuhauser, Daniel (co-PI) [⬀] Weiss, Shimon |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Simultaneous Characterization of Near-Field Nanoplasmonic Structure and Function Using Super-Resolved Far-Field Optics: Solving the Inverse Problem @ University of California-Los Angeles
With support from the Chemical Measurement and Imaging Program in the Division of Chemistry, Professors Shimon Weiss and Daniel Neuhauser at University of California-Los Angeles are studying light-matter interaction at the nanoscale. Specifically, the study explores how light-emitting nanoparticles or molecules interact with metal nanostructures underneath. The gained knowledge could find applications in high resolution imaging in cells, high-speed integrated circuits, and quantum information science. Professors Weiss and Neuhauser work closely with graduate, undergraduate and high school students by providing them multidisciplinary training opportunities. They also plan to make their software broadly available to other scientists who are working on super resolution imaging.
Professors Weiss and Neuhauser merge super-resolution techniques, wide-field single photon detector, and multiscale simulations to understand the coupling strength of point emitters to plasmonic nanostructures. A novel probing technology is used to simultaneously resolve plasmonic structure and field strengths well below the diffraction limit, exploiting the dependence of the blinking statistics of quantum dots on the electric field strength for Stochastic Optical Fluctuation Imaging (SOFI). An additional layer of polarized excitation and emission is used to help understand emitter-metal coupling/scattering strengths in close proximity. They then plan to implement the method to solve the inverse problem, where structure and function can be simultaneously measured without any a priori knowledge of the underlying system.
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.
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1 |
2018 — 2020 |
Weiss, Shimon |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Eager: Methodology Development For 3d Atomic-Scale Structural Dynamics Movies of Enzymes @ University of California-Los Angeles
Capturing the dynamic structure of a biological machine performing its function remains the holy grail of biology. Conventional tools provide only structural "snapshots" of stable states along the reaction pathway. This project aims to develop methods that will capture a full movie of a macromolecule's structure as it works. The research tools developed will be applicable to a wide variety of biological systems, and will be disseminated by means of publications, tutorials, conference presentations and the like, to accelerate their adoption. The education component of the project will emphasize the use of single molecule biophysics and computational tools for dynamic structure determination. It will provide high school, undergraduate, graduate students and postdocs training in state-of-the-art single molecule biophysics research, and offer leadership experiences for graduate students and postdocs as well.
Specifically, this project will study the structure of the transcription bubble in a simple bacterial transcription system during initiation as a test case for developing new technological concepts. The existence of certain intermediates in bacterial transcription suggests common features of the process exist across the different domains of life and reflects a conserved regulatory mechanism; thus, the findings will have broad utility. For the purpose of dynamic structure determination, the project will combine experimental and theoretical tools, including multi-spot single-molecule FRET measurements, fast single-molecule microfluidic mixing, molecular dynamics simulations and time-resolved single-photon detection to acquire data on- and perform molecular modeling of- the transcription bubble during initiation.
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.
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1 |
2018 — 2022 |
Weiss, Shimon Taatjes, Dylan [⬀] |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Dynamic Regulation of Initiation and Pausing of Transcription by Human Rna Polymerase Ii @ University of Colorado At Boulder
The human genome contains about 3 billion base pairs of DNA that provide essential instructions for cells to respond to developmental or environmental cues. A key step in the response is transcribing the information in DNA to intermediary RNA molecules that supply another layer of information. Transcribing DNA to RNA is a highly regulated process, but exactly how it is controlled remains an enigma. The goal of this project is to decipher this problem by bringing together an experienced team of investigators to take a multi-faceted approach to catalyze discovery in new ways. The research plan will integrate educational and outreach programs at University of Colorado-Boulder and University of California-Los Angeles to expose high school, college students, and postdoctoral scholars to high-impact collaborative research. The tools, strategies, and methodologies to be implemented will be applicable to the study of other biological processes and molecular machines.
The questions addressed in this project are fundamentally important for understanding the molecular control of eukaryotic transcription. The human RNA polymerase II transcription machinery will be studied in detail using cutting-edge methods developed initially through analysis of bacterial RNA polymerase. Highly regulated stages in gene expression, including transcription initiation and pausing, will be the focus. This collaborative project will merge solution-based single-molecule fluorescence techniques with detailed functional assays enabled by biochemical reconstitution of the entire 4.5 MDa human transcription apparatus. Two key questions will be addressed: How is RNA polymerase II transcription dynamically regulated by specific protein factors? What role does RNA polymerase II backtracking play in regulating initiation and pausing? An important aspect of the work will be to test and validate mechanistic findings with in vitro and cell-based approaches.
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.
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0.955 |
2019 — 2022 |
Weiss, Shimon Shan, Shu-Ou [⬀] |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Probe the Conformational Dynamics of a Protein Targeting Machine At Single Molecule Resolution @ California Institute of Technology
Observing the dynamic movements of molecular machines in action and deciphering their roles in biological function are the frontiers of life science research. This project aims to elucidate the molecular movements in a protein targeting machine, the signal recognition particle (SRP), as it delivers ~30% of newly synthesized proteins to the correct biological membrane in the cell. The project will generate valuable reagents, tools, and assays that are useful to many other researchers. The results of the research will be disseminated through publications in academic journals as well as animated movie illustrations that will be available to the general public. The education component of the proposal will emphasize the training of graduate students and postdoctoral scholars in multidisciplinary biochemical and biophysical research. It will also expose high school and undergraduate students to state-of-the-art research tools and provide leadership experience for the graduate students and postdocs.
More specifically, this project will combine the expertise of the Shan lab in mechanistic biochemistry and the Weiss lab in biophysics to decipher the dynamic motions that drive co-translational protein targeting by SRP at single molecule resolution. The recent works by the Shan lab indicated the presence of multiple largescale conformational rearrangements in SRP during the protein targeting cycle. This project will develop solution-based single molecule fluorescence assays to directly observe the conformational rearrangements in SRP during targeting, decipher the molecular forces that drive them, and understand their regulation by the GTPase cycle of SRP and by spatial and temporal signals in the pathway. The results will elucidate the molecular basis for the spatiotemporal control of this targeting machine, and reveal generalizable principles that underlie the action of nucleotide-driven macromolecular machines in biology.
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.
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0.936 |
2019 — 2021 |
Weiss, Shimon |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Structural Dynamics of Rnap-Promoter Complex in Late Transcription Initiation @ University of California Los Angeles
PROJECT SUMMARY: Bacterial transcription initiation and promoter escape are highly-regulated early steps of gene expression. A critical initiation step occurs when the 5'-end of the nascent RNA clashes with region 3.2 of the promoter specificity factor ?70 (?R3.2) occluding the RNA exit channel. Then, either the occluded channel is cleared to facilitate RNA forward translocation through the RNA exit channel and RNAP to escape the promoter, or the nascent RNA back-translocates into the NTP entry channel, leading to its abortive release. We have recently shown that a fraction of RNAPs get stabilized in a long-lived paused backtracked intermediate during initiation. We have also shown that even after removal of ?R3.2 from the RNA exit channel by the nascent transcript, transcription kinetics is still slower than expected for elongation. Therefore, we hypothesize an additional promoter escape-intermediate further slows down the transition from initiation to elongation, and that both intermediates have regulatory roles. In Aim 1.A, we will elucidate the structures of the transcription initiation complex in these states by using multiple experimentally-derived intramolecular distances as spatial constraints on coarse-grained simulations. In Aim 1.B, we will define the molecular determinants controlling the abundance of these late initiation intermediates. Specifically, we will examine the sequence and order in which ?70 regions are removed from the RNA exit channel during promoter escape for different promoters. In Aim 1.B we hypothesize that: (1) displacement of ?R3 & ?R4 during promoter escape follows a two-step process; (2) the bulge formed in the scrunched DNA template strand of the transcription bubble assists in removal of these ? regions from the RNA exit channel by projecting into the channel. We recently discovered that an excessive number of RNAPs stall at promoters of many genes in vivo that are essential for stress-response and that stalling is enhanced under hyperosmotic conditions in a ?greA/?greB E. coli strain (unpublished). In Aim 2 we will test whether pausing in initiation occurs in live bacteria and serves as a regulatory intermediate for stress response. We will test this hypothesis by high-resolution (1-2 nt) chromosomal DNA mapping & footprinting in vivo techniques. We will also develop in vivo smFRET transcription bubble size assay to test whether pausing in initiation occurs in the bacterial cell through a mechanism similar to that studied in Aim 1. This project will significantly advance the field of transcription for the following reasons: (1) antibiotic resistance is a serious public health concern. Elucidating the mechanisms of bacterial gene regulation is crucial for the development of effective antimicrobial therapy; (2) the conservation of many features of RNAP structure & function from bacteria to humans facilitates modeling of transcription mechanisms for eukaryotic enzymes; (3) the structure of paused-backtracked RNAP in initiation has not yet been determined. Therefore, delineating the spatial rearrangements of ?70 regions blocking the RNA exit channel for different promoters will provide valuable insight into the mechanism of promoter escape.
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1 |
2022 — 2026 |
Weiss, Shimon Shan, Shu-Ou [⬀] |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Fidelity and Regulation of Signal Recognition Particle @ California Institute of Technology
Understanding the cellular ‘mail distribution’ system of newly synthesized proteins is of fundamental importance for understanding life itself and for harnessing and engineering organisms to solve societal needs such as food and clean energy. In addition to the basic understanding and support for societal needs, this project will provide excellent opportunities for science education and the associated challenges that emerged from the COVID-19 pandemic. The results of the research will be disseminated through publications in academic journals and presentations at scientific conferences, as well as lecture forums that directly interface with the general public. The education component of the project will emphasize the training of graduate students and postdoctoral scholars in multidisciplinary biochemical and biophysical research. It will also expose high school and undergraduate students to state-of-the-art research tools and provide leadership experience for the graduate students and postdocs. Finally, the project will generate valuable reagents, tools, and assays that are useful to many other researchers.<br/><br/>Eukaryotic cells are highly compartmentalized, with each organelle harboring a unique set of proteins that endow the organelle its structure and functions. To maintain the higher order structure and proper functioning of the cell, all newly synthesized proteins need to localize to their correct cellular destinations. To accomplish this, cells have evolved a universally conserved protein targeting machine, signal recognition particle (SRP), to recognize and deliver newly synthesized proteins to the appropriate cellular membrane. This project aims to understand how SRP is regulated in the cell to ensure the organelle specificity of protein localization, and how its complex with the SRP receptor and the nascent protein is disassembled at the target membrane to allow efficient insertion of nascent proteins. The proposed project will combine the expertise of the Shan lab in mechanistic biochemistry and the Weiss lab in biophysics to decipher the sophisticated spatiotemporal regulation of SRP. The results will reveal generalization principles that ensure the proper functioning of the “mail delivery” system in the cell.<br/><br/>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.
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0.936 |