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

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:
Report Number(s):
SAND2017-11814J
Journal ID: ISSN 0005-2736; 658298
Grant/Contract Number:
AC04-94AL85000
Type:
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
Research Org:
Sandia National Lab. (SNL-NM), Albuquerque, NM (United States)
Sponsoring Org:
USDOE National Nuclear Security Administration (NNSA)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
OSTI Identifier:
1406383
Alternate Identifier(s):
OSTI ID: 1397547; OSTI ID: 1411226