
NSF Org: |
MCB Division of Molecular and Cellular Biosciences |
Recipient: |
|
Initial Amendment Date: | January 27, 2020 |
Latest Amendment Date: | January 27, 2020 |
Award Number: | 1951425 |
Award Instrument: | Standard Grant |
Program Manager: |
Elizabeth Sztul
esztul@nsf.gov (703)292-0000 MCB Division of Molecular and Cellular Biosciences BIO Directorate for Biological Sciences |
Start Date: | February 1, 2020 |
End Date: | January 31, 2024 (Estimated) |
Total Intended Award Amount: | $983,837.00 |
Total Awarded Amount to Date: | $983,837.00 |
Funds Obligated to Date: |
|
History of Investigator: |
|
Recipient Sponsored Research Office: |
3112 LEE BUILDING COLLEGE PARK MD US 20742-5100 (301)405-6269 |
Sponsor Congressional District: |
|
Primary Place of Performance: |
MD US 20742-5141 |
Primary Place of
Performance Congressional District: |
|
Unique Entity Identifier (UEI): |
|
Parent UEI: |
|
NSF Program(s): |
Cellular Dynamics and Function, Molecular Biophysics |
Primary Program Source: |
|
Program Reference Code(s): |
|
Program Element Code(s): |
|
Award Agency Code: | 4900 |
Fund Agency Code: | 4900 |
Assistance Listing Number(s): | 47.074 |
ABSTRACT
This project will probe the mechanism of lipid movement between cellular compartments known as organelles. Recently, strong evidence indicates that most lipid transport occurs with the help of lipid transport proteins that can facilitate the formation of organelle contacts known as membrane contact sites. The oxysterol binding homologue (Osh) proteins of yeast are an example of lipid transport proteins that form membrane contact sites and serve as an excellent model for lipid exchange between organelles. Since cells can be used to produce chemicals for commercial use, understanding lipid exchange and ways to modify it could improve production of chemicals. This project will provide scientific training for researchers with focused recruitment from underrepresented groups. Project concepts and results will be used in educational outreach for minority-serving high school summer programs, courses, and developing hands-on protein engineering activities for the general public.
Osh proteins were originally thought to transport sterols but have recently been shown to be important in the transport of signaling lipids, such as phosphatidylinositols. This project integrates experimental and computational techniques to elucidate membrane contact site formation and probe lipid exchange by first studying membrane-binding regions of Osh proteins and then the mechanism the full-length protein uses to form membrane contact sites. The binding of the lipid-packing sensor of Osh proteins to model membranes (planar and curved) will be probed using enhanced sampling simulations, neutron diffraction, and high-resolution field-cycling NMR. Other domains of Osh4 interacting with membranes will be studied with a focus on how this protein might conformationally change to facilitate membrane contact sites. Ultimately, the mechanism of the full-length Osh4 protein to form membrane contact sites and lipid transfer will be probed by developing new computational tools for dual-membrane binding and the use of field-cycling NMR and fluorescence techniques. This proposal is jointly funded by the Cellular Dynamics and Function cluster and Molecular Biophysics cluster in the Division of Molecular and Cellular Biosciences.
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.
PUBLICATIONS PRODUCED AS A RESULT OF THIS RESEARCH
Note:
When clicking on a Digital Object Identifier (DOI) number, you will be taken to an external
site maintained by the publisher. Some full text articles may not yet be available without a
charge during the embargo (administrative interval).
Some links on this page may take you to non-federal websites. Their policies may differ from
this site.
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.
This NSF grant focused on researching the mechanism of a protein (Osh4) involved in lipid exchange between two organelles in yeast cells. This was a combined computational and wet lab effort to probe binding motifs on Osh4 and provide insight on the mechanism this protein uses to binding simultaneously to two membrane organelles. The thermodynamics and structure of the ALPS motif on Osh4 binding to model membranes was probed. The ALPS motif was found to fold to an a-helix with the presence of a negatively charged lipid with relatively strong affinity. Another distal membrane-active domain on Osh4 was probed and used to develop a model for how Osh4 binds to two membranes. Our microsecond simulations suggest a dual-membrane binding motif where the core of the Osh4 interacts with one membrane and an arm that unwinds from the protein to reach out and bind to an opposing membrane. Overall, this project produced 9 peer-reviewed published research papers, one currently under review, and two to be submitted in 2024. This grant trained one high school student, 9 undergraduates, 4 graduate students and a postdoctoral fellow. Specific areas of research and findings are as follows:
- Molecular dynamics (MD) simulations probed the ALPS binding structure and binding affinity to model lipid bilayers and complemented x-ray diffraction, Trp-fluorescence, and isothermal calorimetry measurements
- The ALPS motif was found to be more helical in hydrophobic solvent compared to water and mostly helical with the increased concentration of a negatively charged lipid
- MD simulations with recent force field updates on the full-length Osh4 resulted in a balanced driving force for single membrane binding (electrostatic and dispersion)
- Recombinant expression and purification procedures were significantly improved
- Protocols were developed to perform fast field-cycling nuclear magnetic resonance to measure the protein-lipid interactions
- Developed a dual-membrane model for Osh4 based on targeted structures from the arm-like motif and is stable on the ms timescale
Last Modified: 05/22/2024
Modified by: Jeffery B Klauda
Please report errors in award information by writing to: awardsearch@nsf.gov.