| NSF Org: |
MCB Division of Molecular and Cellular Biosciences |
| Recipient: |
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| Initial Amendment Date: | March 19, 2021 |
| Latest Amendment Date: | September 22, 2021 |
| Award Number: | 2117998 |
| Award Instrument: | Standard Grant |
| Program Manager: |
Marcia Newcomer
mnewcome@nsf.gov (703)292-2357 MCB Division of Molecular and Cellular Biosciences BIO Directorate for Biological Sciences |
| Start Date: | April 1, 2021 |
| End Date: | September 30, 2021 (Estimated) |
| Total Intended Award Amount: | $300,000.00 |
| Total Awarded Amount to Date: | $86,444.00 |
| Funds Obligated to Date: |
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| History of Investigator: |
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| Recipient Sponsored Research Office: |
1500 HORNING RD KENT OH US 44242-0001 (330)672-2070 |
| Sponsor Congressional District: |
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| Primary Place of Performance: |
Kent OH US 44240-0001 |
| Primary Place of
Performance Congressional District: |
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| Unique Entity Identifier (UEI): |
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| Parent UEI: |
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| NSF Program(s): | Molecular Biophysics |
| Primary Program Source: |
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| Program Reference Code(s): |
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| Program Element Code(s): |
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| Award Agency Code: | 4900 |
| Fund Agency Code: | 4900 |
| Assistance Listing Number(s): | 47.074 |
ABSTRACT
This research will develop molecular tools that allow the study of membrane proteins (MPs), which are among the most important, but least understood components of cells. All cells are surrounded by lipid membranes that are almost impermeable to water, salts or nutrients that cells need. For this reason, many membrane proteins (MPs) are inserted into the membranes that control cellular functions such as material transport, sensing, intercellular communication, cell adhesion, and energy conversion. MPs are also the targets for many therapeutic drug molecules. Knowledge of the molecular structure of MPs is necessary to understand the underlying molecular mechanisms of their function and can guide the development of therapeutic drugs for many common diseases. However, MPs are difficult to study and therefore the molecular structure of most MPs is still unknown. The goal of this project is to develop broadly applicable new tools using DNA nanotechnology that will facilitate solving MP structures with cryo-electron microscopes. This project will provide research opportunities for graduate, undergraduate and high school students from underrepresented minority groups through several established programs, which will inform them about experimental research and potential career opportunities. Finally, introductory physics courses will be strengthened by developing modules that underline interdisciplinary aspects and applications of physical principles, and an interdisciplinary molecular biophysics course will be developed. These efforts will train the next generation of diverse, interdisciplinary leaders in STEM research and technology.
Single-particle cryo-electron microscopy (cryo-EM) is becoming the standard method for MP structure determination, but several experimental challenges have prevented solving more than 1% of human MP structures so far. The overall goal of this project is to establish DNA-lipid nanodiscs (DLNs) as a radically new customizable nanoscale lipid bilayer mimetic for single-particle cryo-EM of MPs. This DNA nanotechnology-based approach will overcome existing limitations of established bilayer mimetics and offer unprecedented control over structural, chemical and physical design parameters that could transform MP research. Such a paradigm shift involves significant risks and requires substantial exploratory development efforts. First, strategies will be developed to increase synthesis yields and to prevent lipid bilayer aggregation. Next, MP expression, purification and co-reconstitution of MPs in DLNs need to be established. Then, freezing conditions, additives and other sample preparation parameters need to be iteratively optimized to produce high quality grids for imaging. It is expected that the same or better resolutions can be achieved than with established bilayer mimetics, while providing new functionalities and unprecedented programmability. It is expected that DNA-lipid nanodiscs will initiate new research in structural biology, pharmacology, virology and bio-catalysis and therefore enhance the understanding of common diseases. The methods and models developed in this research will be made publicly available to benefit the larger scientific community researching the mechanisms of actions of drugs and vaccines.
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.
PROJECT OUTCOMES REPORT
Disclaimer
This Project Outcomes Report for the General Public is displayed verbatim as submitted by the Principal Investigator (PI) for this award. Any opinions, findings, and conclusions or recommendations expressed in this Report are those of the PI and do not necessarily reflect the views of the National Science Foundation; NSF has not approved or endorsed its content.
Background and Motivation
Membrane proteins (MPs) are found in the membranes of all cells. They are key molecules for many cellular functions and play important roles in most diseases. Knowledge of the molecular structure of these proteins, preferentially in their native lipid bilayer environment, is necessary to understand the underlying biophysical and molecular mechanisms of their function. Cryo-electron microscopy has become one of the most powerful methods for structure determination of membrane proteins but remains a highly challenging procedure. The overall goal of this EAGER project was to establish DNA-lipid nanodiscs (DLNs) as a radically new customizable nanoscale lipid bilayer mimetic that provides a native environment for studying MPs. Although other nanometer-sized bilayer systems exist, this DNA nanotechnology-based approach will overcome some of their existing limitations and offers unprecedented control over structural, chemical and physical design parameters.
Intellectual Merit
Towards this goal, this project has improved the laboratory’s established strategies to synthesize the DLNs and developed new chemistries and nanostructure designs. These new approaches allow to produce more complex nanostructures with a higher yield and purity which will be needed for high-resolution structure determination of MPs embedded in the DLNs. The mechanism through which lipids assemble inside of DLNs or other bilayer systems are poorly understood. The programmability of DNA nanostructures allows to experimentally test different design hypotheses and further our understanding of the assembly mechanisms which will benefit the larger field of membrane biophysics and structural biology.
As its ultimate outcome, this project will enable to perform radically new experiments, allow to study MPs that are currently difficult to approach and transform MP research.
Broader Impacts
The PI developed and teaches an introductory course on molecular biophysics for undergraduate and graduate students. The interdisciplinary course focusses on single-molecule biophysics and physical, chemical and biological foundations. In the course, key methods and techniques are being introduced. The background for this research was integrated into the educational material. The course will help building a diverse community of students, teachers and researchers for this highly interdisciplinary research field.
The program offered partial or full support for the training of three graduate and two undergraduate students. These efforts will train the next generation of diverse, interdisciplinary leaders in STEM research and technology.
Last Modified: 12/10/2021
Modified by: Thorsten L Schmidt
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