Method for Measuring the Unbinding Energy of Strongly-bound Membrane-associated Proteins

Authors

Elisa La Bauve, Sandia National Labs
Briana C. Vernon, Sandia National Labs
Dongmei Ye, Sandia National Labs
David M. Rogers, University of South FloridaFollow
Cathryn M. Siegrist, Sandia National Labs
Bryan D. Carson, Sandia National Labs
Susan B. Rempe, Sandia National Labs
Aihua Zheng, Albert Einstein College of Medicine
Margaret Kielian, Albert Einstein College of Medicine
Andrew P. Shreve, University of New Mexico
Michael S. Kent, Sandia National Labs

Document Type

Article

Publication Date

2-16-2016

Keywords

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

Digital Object Identifier (DOI)

https://doi.org/10.1016/j.bbamem.2016.07.004

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 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. 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.

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Was this content written or created while at USF?

Yes

Citation / Publisher Attribution

Biochimica et Biophysica Acta (BBA) - Biomembranes, v. 1858, issue 11, p. 2753-2762

This article is the post-print author version.

Scholar Commons Citation

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.; and Kent, Michael S., "Method for Measuring the Unbinding Energy of Strongly-bound Membrane-associated Proteins" (2016). Chemistry Faculty Publications. 28.
https://digitalcommons.usf.edu/chm_facpub/28