Measuring membrane protein stability under native conditions

Yu Chu Chang, James U. Bowie

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31 Citations (Scopus)

Abstract

The thermodynamic stability of proteins is typically measured at high denaturant concentrations and then extrapolated back to zero denaturant conditions to obtain unfolding free energies under native conditions. For membrane proteins, the extrapolations are fraught with considerable uncertainty as the denaturants may have complex effects on the membrane or micellar structure. We therefore sought to measure stability under native conditions, using a method that does not perturb the properties of the membrane or membrane mimetics. We use a technique called steric trapping to measure the thermodynamic stability of bacteriorhodopsin in bicelles and micelles. We find that bacteriorhodopsin has a high thermodynamic stability, with an unfolding free energy of ~11 kcal/mol in dimyristoyl phosphatidylcholine bicelles. Nevertheless, the stability is much lower than predicted by extrapolation of measurements made at high denaturant concentrations. We investigated the discrepancy and found that unfolding free energy is not linear with denaturant concentration. Apparently, long extrapolations of helical membrane protein unfolding free energies must be treated with caution. Steric trapping, however, provides a method for making these measurements.

Original languageEnglish
Pages (from-to)219-224
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume111
Issue number1
DOIs
Publication statusPublished - 2014
Externally publishedYes

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Keywords

  • Membrane protein folding
  • Steric trap

ASJC Scopus subject areas

  • General

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