Abstract
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. 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.
Original language | English |
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Pages (from-to) | 2753-2762 |
Number of pages | 10 |
Journal | Biochimica et Biophysica Acta - Biomembranes |
Volume | 1858 |
Issue number | 11 |
DOIs | |
State | Published - Nov 1 2016 |
Externally published | Yes |
Funding
Sandia National Laboratories is a multi-program laboratory managed and operated by Sandia Corporation, a wholly owned subsidiary of Lockheed Martin Corporation, for the United States Department of Energy's National Nuclear Security Administration under contract DE-AC04-94AL85000. This work was supported by the Laboratory Directed Research and Development program at Sandia National Laboratories , the Defense Threat Reduction Agency - Joint Science and Technology Office for Chemical and Biological Defense (SBR), and by NIH grant R01 AI075647 (to M.K.). This work was performed, in part, at the Center for Integrated Nanotechnologies, a U.S. Department of Energy, Office of Basic Energy Sciences user facility at Los Alamos National Laboratory (Contract DE-AC52-06NA25396 ) and Sandia National Laboratories (Contract DE-AC04-94AL85000 ).
Keywords
- Binding energy
- Dengue virus envelope protein
- Integral monotopic proteins
- Lipid membrane-associated proteins
- Liposome–protein coflotation/sedimentation
- Unbinding energy