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

• Published in: Biochimica et biophysica acta 2016-11, Vol.1858 (11), p.2753-2762
• Main Authors: 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.
• Format: Article
• Language: English
• Subjects: Animals; BASIC BIOLOGICAL SCIENCES; Binding energy; Cells, Cultured; Dengue Virus - chemistry; Dengue virus envelope protein; Drosophila melanogaster; Hydrogen-Ion Concentration; INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; Integral monotopic proteins; Kinetics; Lipid Bilayers - chemistry; Lipid membrane-associated proteins; Liposome–protein coflotation/sedimentation; Phosphatidylcholines - chemistry; Phosphatidylglycerols - chemistry; Protein Binding; Thermodynamics; Unbinding energy; Unilamellar Liposomes - chemistry; Viral Envelope Proteins - chemistry
• ISSN: 0005-2736; 0006-3002; 1879-2642; 1878-2434
• DOI: 10.1016/j.bbamem.2016.07.004

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 pH5.5. 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±3kcal/mol, 20% PG) was found to be roughly three times the binding energy per monomer, (7.8±0.3kcal/mol for 30% PG, or est. 7.0kcal/mol for 20% PG). This is consistent with data showing that free sE is a monomer at pH5.5, 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. [Display omitted] •A method to measure the unbinding energy of strongly bound membrane-associated proteins is proposed•The method is based on measuring the rate of release of liposome-bound protein during centrifugation on a sucrose gradient.•The method was used to determine the free energy for unbinding of the dengue virus envelope protein from POPC-POPG membranes.•The unbinding free energy was found to be roughly three times the binding free energy•The results are consistent with the hypothesis that sE initially inserts as a monomer but then associates to form trimers.