Award Abstract # 1951425
Studies on the Protein-assisted Mechanism for Intracellular Membrane Contact Sites

NSF Org: MCB
Division of Molecular and Cellular Biosciences
Recipient: UNIVERSITY OF MARYLAND, COLLEGE PARK
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: FY 2020 = $983,837.00
History of Investigator:
  • Jeffery Klauda (Principal Investigator)
    jbklauda@umd.edu
  • Amy Karlsson (Co-Principal Investigator)
Recipient Sponsored Research Office: University of Maryland, College Park
3112 LEE BUILDING
COLLEGE PARK
MD  US  20742-5100
(301)405-6269
Sponsor Congressional District: 04
Primary Place of Performance: University of Maryland College Park
MD  US  20742-5141
Primary Place of Performance
Congressional District:
04
Unique Entity Identifier (UEI): NPU8ULVAAS23
Parent UEI: NPU8ULVAAS23
NSF Program(s): Cellular Dynamics and Function,
Molecular Biophysics
Primary Program Source: 01002021DB NSF RESEARCH & RELATED ACTIVIT
Program Reference Code(s): 7465
Program Element Code(s): 111400, 114400
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

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Allsopp, Robert and Pavlova, Anna and Cline, Tyler and Salyapongse, Aria M. and Gillilan, Richard E. and Di, Y. Peter and Deslouches, Berthony and Klauda, Jeffery B. and Gumbart, James C. and Tristram-Nagle, Stephanie "Antimicrobial Peptide Mechanism Studied by Scattering-Guided Molecular Dynamics Simulation" The Journal of Physical Chemistry B , v.126 , 2022 https://doi.org/10.1021/acs.jpcb.2c03193 Citation Details
Allsopp, Robert J. and Klauda, Jeffery B. "Impact of PIP2 Lipids, Force Field Parameters, and Mutational Analysis on the Binding of the Osh4s 6 7 Domain" The Journal of Physical Chemistry B , v.125 , 2021 https://doi.org/10.1021/acs.jpcb.0c10393 Citation Details
Feng, Shasha and Wang, Ruixing and Pastor, Richard W. and Klauda, Jeffery B. and Im, Wonpil "Location and Conformational Ensemble of Menaquinone and Menaquinol, and ProteinLipid Modulations in Archaeal Membranes" The Journal of Physical Chemistry B , v.125 , 2021 https://doi.org/10.1021/acs.jpcb.1c01930 Citation Details
Fernandes, Joshua B. and Yu, Yalun and Klauda, Jeffery B. "Molecular dynamics simulations of the human ocular lens with age and cataract" Biochimica et Biophysica Acta (BBA) - Biomembranes , v.1864 , 2022 https://doi.org/10.1016/j.bbamem.2022.184025 Citation Details
Hsieh, Min-Kang and Yu, Yalun and Klauda, Jeffery B. "All-Atom Modeling of Complex Cellular Membranes" Langmuir , v.38 , 2022 https://doi.org/10.1021/acs.langmuir.1c02084 Citation Details
Karmakar, Sharmistha and Klauda, Jeffery B. "Modeling the membrane binding mechanism of a lipid transport protein Osh4 to single membranes" Biophysical Journal , v.121 , 2022 https://doi.org/10.1016/j.bpj.2022.03.001 Citation Details
Klauda, Jeffery B. "Considerations of Recent All-Atom Lipid Force Field Development" The Journal of Physical Chemistry B , v.125 , 2021 https://doi.org/10.1021/acs.jpcb.1c02417 Citation Details
Konakbayeva, Dinara and Karlsson, Amy J "Strategies and opportunities for engineering antifungal peptides for therapeutic applications" Current Opinion in Biotechnology , v.81 , 2023 https://doi.org/10.1016/j.copbio.2023.102926 Citation Details
Kuba, Jacob Olondo and Yu, Yalun and Klauda, Jeffery B. "Estimating localization of various statins within a POPC bilayer" Chemistry and Physics of Lipids , v.236 , 2021 https://doi.org/10.1016/j.chemphyslip.2021.105074 Citation Details
Yu, Yalun and Klauda, Jeffery B. "Symmetric and Asymmetric Models for the Arabidopsis thaliana Plasma Membrane: A Simulation Study" The Journal of Physical Chemistry B , v.125 , 2021 https://doi.org/10.1021/acs.jpcb.1c04704 Citation Details

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

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