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

Elisa La Bauve, Briana C. Vernon, Dongmei Ye, David M. Rogers, Cathryn M. Siegrist, Bryan D. Carson, Susan B. Rempe, Aihua Zheng, Margaret Kielian, Andrew P. Shreve, Michael S. Kent

Research output: Contribution to journalArticle

1 Scopus citations

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 languageEnglish (US)
Pages (from-to)2753-2762
Number of pages10
JournalBiochimica et Biophysica Acta - Biomembranes
Volume1858
Issue number11
DOIs
StatePublished - Nov 1 2016

Keywords

  • Binding energy
  • Dengue virus envelope protein
  • Integral monotopic proteins
  • Lipid membrane-associated proteins
  • Liposome–protein coflotation/sedimentation
  • Unbinding energy

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Cell Biology

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