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Title: Method for measuring the unbinding energy of strongly-bound membrane-associated proteins

Abstract

Here, we describe a new method to measure the activation energy for unbinding (enthalpy ΔH*u and free energy ΔG*u) of a strongly-bound membrane-associated protein from a lipid membrane. It is based on measuring the rate of release of a liposome-bound protein during centrifugation on a sucrose gradient as a function of time and temperature. The method is used to determine ΔH*u and ΔG*u for the soluble dengue virus envelope protein (sE) strongly bound to 80:20 POPC:POPG liposomes at pH 5.5. ΔH*u is determined from the Arrhenius equation whereas ΔG*u is determined by fitting the data to a model based on mean first passage time for escape from a potential well. The binding free energy ΔGb of sE was also measured at the same pH for the initial, predominantly reversible, phase of binding to a 70:30 PC:PG lipid bilayer. The unbinding free energy (20 ± 3 kcal/mol, 20% PG) was found to be roughly three times the binding energy per monomer, (7.8 ± 0.3 kcal/mol for 30% PG, or est. 7.0 kcal/mol for 20% PG). This is consistent with data showing that free sE is a monomer at pH 5.5, but assembles into trimers after associating with membranes. Furthermore, this newmore » method to determine unbinding energies should be useful to understand better the complex interactions of integral monotopic proteins and strongly-bound peripheral membrane proteins with lipid membranes.« less

Authors:
; ; ; ; ; ; ; ; ; ;
Publication Date:
Research Org.:
Sandia National Lab. (SNL-NM), Albuquerque, NM (United States)
Sponsoring Org.:
USDOE National Nuclear Security Administration (NNSA)
OSTI Identifier:
1406383
Alternate Identifier(s):
OSTI ID: 1397547; OSTI ID: 1411226
Report Number(s):
SAND2017-11814J
Journal ID: ISSN 0005-2736; S0005273616302474; PII: S0005273616302474
Grant/Contract Number:  
AC04-94AL85000
Resource Type:
Published Article
Journal Name:
Biochimica et Biophysica Acta. Biomembranes
Additional Journal Information:
Journal Name: Biochimica et Biophysica Acta. Biomembranes Journal Volume: 1858 Journal Issue: 11; Journal ID: ISSN 0005-2736
Publisher:
Elsevier
Country of Publication:
Netherlands
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY

Citation Formats

La Bauve, Elisa, Vernon, Briana C., Ye, Dongmei, Rogers, David M., Siegrist, Cathryn M., Carson, Bryan D., Rempe, Susan B., Zheng, Aihua, Kielian, Margaret, Shreve, Andrew P., and Kent, Michael S. Method for measuring the unbinding energy of strongly-bound membrane-associated proteins. Netherlands: N. p., 2016. Web. doi:10.1016/j.bbamem.2016.07.004.
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La Bauve, Elisa, Vernon, Briana C., Ye, Dongmei, Rogers, David M., Siegrist, Cathryn M., Carson, Bryan D., Rempe, Susan B., Zheng, Aihua, Kielian, Margaret, Shreve, Andrew P., & Kent, Michael S. Method for measuring the unbinding energy of strongly-bound membrane-associated proteins. Netherlands. https://doi.org/10.1016/j.bbamem.2016.07.004
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La Bauve, Elisa, Vernon, Briana C., Ye, Dongmei, Rogers, David M., Siegrist, Cathryn M., Carson, Bryan D., Rempe, Susan B., Zheng, Aihua, Kielian, Margaret, Shreve, Andrew P., and Kent, Michael S. Tue . "Method for measuring the unbinding energy of strongly-bound membrane-associated proteins". Netherlands. https://doi.org/10.1016/j.bbamem.2016.07.004.
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@article{osti_1406383,
title = {Method for measuring the unbinding energy of strongly-bound membrane-associated proteins},
author = {La Bauve, Elisa and Vernon, Briana C. and Ye, Dongmei and Rogers, David M. and Siegrist, Cathryn M. and Carson, Bryan D. and Rempe, Susan B. and Zheng, Aihua and Kielian, Margaret and Shreve, Andrew P. and Kent, Michael S.},
abstractNote = {Here, we describe a new method to measure the activation energy for unbinding (enthalpy ΔH*u and free energy ΔG*u) of a strongly-bound membrane-associated protein from a lipid membrane. It is based on measuring the rate of release of a liposome-bound protein during centrifugation on a sucrose gradient as a function of time and temperature. The method is used to determine ΔH*u and ΔG*u for the soluble dengue virus envelope protein (sE) strongly bound to 80:20 POPC:POPG liposomes at pH 5.5. ΔH*u is determined from the Arrhenius equation whereas ΔG*u is determined by fitting the data to a model based on mean first passage time for escape from a potential well. The binding free energy ΔGb of sE was also measured at the same pH for the initial, predominantly reversible, phase of binding to a 70:30 PC:PG lipid bilayer. The unbinding free energy (20 ± 3 kcal/mol, 20% PG) was found to be roughly three times the binding energy per monomer, (7.8 ± 0.3 kcal/mol for 30% PG, or est. 7.0 kcal/mol for 20% PG). This is consistent with data showing that free sE is a monomer at pH 5.5, but assembles into trimers after associating with membranes. Furthermore, this new method to determine unbinding energies should be useful to understand better the complex interactions of integral monotopic proteins and strongly-bound peripheral membrane proteins with lipid membranes.},
doi = {10.1016/j.bbamem.2016.07.004},
journal = {Biochimica et Biophysica Acta. Biomembranes},
number = 11,
volume = 1858,
place = {Netherlands},
year = {Tue Nov 01 00:00:00 EDT 2016},
month = {Tue Nov 01 00:00:00 EDT 2016}
}
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https://doi.org/10.1016/j.bbamem.2016.07.004
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