title: Method for measuring the unbinding energy of strongly-bound membrane-associated proteins.
abstract: We describe a new method to measure the activation energy for unbinding (enthalpy ΔH*uand 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*uand ΔG*ufor the soluble dengue virus envelope protein (sE) strongly bound to 80:20 POPC:POPG liposomes at pH5.5. ΔH*uis determined from the Arrhenius equation whereas ΔG*uis determined by fitting the data to a model based on mean first passage time for escape from a potential well. The binding free energy ΔGbof 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.
authors.affiliation: Sandia National Laboratories, Albuquerque, NM 87185.
authors.forename: Elisa La
authors.initials: E
authors.lastname: Bauve
authors.affiliation: Sandia National Laboratories, Albuquerque, NM 87185.
authors.forename: Briana C
authors.initials: BC
authors.lastname: Vernon
authors.affiliation: Sandia National Laboratories, Albuquerque, NM 87185.
authors.forename: Dongmei
authors.initials: D
authors.lastname: Ye
authors.affiliation: Department of Chemistry, University of South Florida, 4202 E Fowler Av, Tampa, FL 33620.
authors.forename: David M
authors.initials: DM
authors.lastname: Rogers
authors.affiliation: Sandia National Laboratories, Albuquerque, NM 87185.
authors.forename: Cathryn M
authors.initials: CM
authors.lastname: Siegrist
authors.affiliation: Sandia National Laboratories, Albuquerque, NM 87185.
authors.forename: Bryan D
authors.initials: BD
authors.lastname: Carson
authors.affiliation: Sandia National Laboratories, Albuquerque, NM 87185.
authors.forename: Susan B
authors.initials: SB
authors.lastname: Rempe
authors.affiliation: Department of Cell Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461.
authors.forename: Aihua
authors.initials: A
authors.lastname: Zheng
authors.affiliation: Department of Cell Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461.
authors.forename: Margaret
authors.initials: M
authors.lastname: Kielian
authors.affiliation: Center for Biomedical Engineering and Department of Chemical and Biological Engineering, University of New Mexico, Albuquerque, NM 87131.
authors.forename: Andrew P
authors.initials: AP
authors.lastname: Shreve
authors.affiliation: Sandia National Laboratories, Albuquerque, NM 87185.
authors.forename: Michael S
authors.initials: MS
authors.lastname: Kent
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D003716:Dengue Virus / Q000737:chemistry; D004331:Drosophila melanogaster; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry*; D010713:Phosphatidylcholines / Q000737:chemistry*; D010715:Phosphatidylglycerols / Q000737:chemistry*; D011485:Protein Binding; D013816:Thermodynamics; D053835:Unilamellar Liposomes / Q000737:chemistry*; D014759:Viral Envelope Proteins / Q000737:chemistry*
chemical_list: D008051:Lipid Bilayers; D010713:Phosphatidylcholines; D010715:Phosphatidylglycerols; D053835:Unilamellar Liposomes; D014759:Viral Envelope Proteins; C060037:1-palmitoyl-2-oleoylglycero-3-phosphoglycerol; C028694:1-palmitoyl-2-oleoylphosphatidylcholine
keywords: Binding energy; Dengue virus envelope protein; Integral monotopic proteins; Lipid membrane-associated proteins; Liposome–protein coflotation/sedimentation; Unbinding energy