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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 ΔG b 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.more » 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.« less

Authors:
 [1];  [1];  [1];  [2];  [1];  [1];  [1];  [3];  [3];  [4];  [1]
  1. Sandia National Lab. (SNL-NM), Albuquerque, NM (United States)
  2. Univ. of South Florida, Tampa, FL (United States)
  3. Albert Einstein College of Medicine, Bronx, NY (United States)
  4. Univ. of New Mexico, Albuquerque, NM (United States)
Publication Date:
Research Org.:
Sandia National Lab. (SNL-NM), Albuquerque, NM (United States)
Sponsoring Org.:
USDOE National Nuclear Security Administration (NNSA)
OSTI Identifier:
1406383
Report Number(s):
SAND2017-11814J
Journal ID: ISSN 0005-2736; 658298
Grant/Contract Number:
AC04-94AL85000
Resource Type:
Journal Article: Published Article
Journal Name:
Biochimica et Biophysica Acta. Biomembranes
Additional Journal Information:
Journal Volume: 1858; Journal Issue: 11; Journal ID: ISSN 0005-2736
Publisher:
Elsevier
Country of Publication:
United States
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. United States: N. p., 2016. Web. doi:10.1016/j.bbamem.2016.07.004.
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. United States. doi:10.1016/j.bbamem.2016.07.004.
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. 2016. "Method for measuring the unbinding energy of strongly-bound membrane-associated proteins". United States. doi:10.1016/j.bbamem.2016.07.004.
@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 = {United States},
year = 2016,
month = 7
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record at 10.1016/j.bbamem.2016.07.004

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  • 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 modelmore » based on mean first passage time for escape from a potential well. The binding free energy ΔG b 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.« less
  • Here, we describe a new method to measure the activation energy required to remove a strongly-bound membrane-associated protein from a lipid membrane (anchoring energy). 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 was used to determine anchoring energy for the soluble dengue virus envelope protein (sE) strongly bound to 80:20 POPC:POPG liposomes at pH 5.5. We also measured the binding energy of sE at the same pH for the initial, predominantly reversible, phase of binding to a 70:30 PC:PG lipidmore » bilayer. The anchoring energy (37 +/- 1.7 kcal/mol, 20% PG) was found to be much larger than the binding energy (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. But, trimerization alone is insufficient to account for the observed difference in energies, and we conclude that some energy dissipation occurs during the release process. This new method to determine anchoring energy should be useful to understand the complex interactions of integral monotopic proteins and strongly-bound peripheral membrane proteins with lipid membranes.« less
  • We have assessed the binding of (alpha-/sup 32/P)GTP to platelet proteins from cytosolic and membrane fractions. Proteins were separated by NaDodSO/sub 4//PAGE and electrophoretically transferred to nitrocellulose. Incubation of the nitrocellulose blots with (alpha-/sup 32/P)GTP indicated the presence of specific and distinct GTP-binding proteins in cytosol and membranes. Binding was prevented by 10-100 nM GTP and by 100 nM guanosine 5'-(gamma-thio)triphosphate (GTP(gamma S)) or GDP; binding was unaffected by 1 nM-1 microM ATP. One main GTP-binding protein (29.5 kDa) was detected in the membrane fraction, while three others (29, 27, and 21 kDa) were detected in the soluble fraction. Twomore » cytosolic GTP-binding proteins (29 and 27 kDa) were degraded by trypsin; another cytosolic protein (21 kDa) and the membrane-bound protein (29.5 kDa) were resistant to the action of trypsin. Treatment of intact platelets with trypsin or thrombin, followed by lysis and fractionation, did not affect the binding of (alpha-/sup 32/P)GTP to the membrane-bound protein. GTP(gamma S) still stimulated phospholipase C in permeabilized platelets already preincubated with trypsin. This suggests that trypsin-resistant GTP-binding proteins might regulate phospholipase C stimulated by GTP(gamma S).« less
  • A new method is described for the purification of a membrane bound glycoprotein, the kappa opioid receptor from human placental tissue. The method uses preparative slab-gel electrophoresis in the presence of the non-denaturing detergent CHAPS. A linear relationship between log molecular weight and SDS PAGE electrophoretic mobility of known molecular weight markers, in the presence of CHAPS, is observed. Using this method, we were able partially to purify an /sup 3/H-etorphine binding glycoprotein, from placental villus tissue, with an apparent molecular weight range of 60-70,000. The iodinated glycoprotein migrates in SDS PAGE with an apparent molecular weight of 63,000. Thismore » method may be useful for the isolation of membrane bound proteins, especially when an affinity ligand is not available.« less