Evidence that bilayer bending rigidity affects membrane protein folding

Paula J. Booth, M. Louise Riley, Sabine L. Flitsch, Richard H. Templer, Amjad Farooq, A. Rachael Curran, Neil Chadborn, Penny Wright

Research output: Contribution to journalArticle

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Abstract

The regeneration kinetics of the integral membrane protein bacteriorhodopsin have been investigated in a lipid-based refolding system. Previous studies on bacteriorhodopsin regeneration have involved detergent- based systems, and in particular mixed dimyristoylphosphatidylcholine (DMPC)/CHAPS micelles. Here, we show that the short chain lipid dihexanoylphosphatidylcholine (DHPC) can be substituted for the detergent CHAPS and that bacteriorhodopsin can be regenerated to high yield in mixed DMPC/DHPC micelles. Bacteriorhodopsin infolding kinetics are measured in the mixed DMPC/DHPC micelles. Rapid, stopped flow mixing is employed to initiate refolding of denatured bacterioopsin in SDS micelles with mixed DMPC/DHPC micelles and time-resolved fluorescence spectroscopy to follow changes in protein fluorescence during folding. Essentially identical refolding kinetics are observed for mixed DMPC/CHAPS and mixed DMPC/DHPC micelles. Only one second-order retinal/apoprotein reaction is identified, in which retinal binds to a partially folded apoprotein intermediate, and the free energy of this retinal binding reaction is found to be the same in both types of mixed micelles. Formation of the partially folded apoproptein intermediate is a rate-limiting step in protein folding and appears to be biexponential. Both apparent rate constants are found to be dependent on the relative proportion of DMPC present in the mixed DMPC/DHPC micelles as well as on the pH of the aqueous phase. Increasing the DMPC concentration should increase the bending rigidity of the amphiphilic bilayer, and this is found to slow the rate of formation of the partially folded apoprotein intermediate. Increasing the mole fraction of DMPC from 0.3 to 0.6 slows the two apparent rate constants associated with formation of this intermediate from 0.29 and 0.031 to 0.11 and 0.013 s-1, respectively. Formation of the intermediate also slows with increasing pH, from 0.11 and 0.013 s-1 at pH 6 to 0.033 and 0.0053 s-1 at pH 8. Since this pH change has no known effect on the phase behavior of lecithins, this is more likely to represent a direct effect on the protein itself. Thus, it appears to be possible to control the rate-limiting process in bacterioopsin folding through both bilayer bending rigidity and pH.

Original languageEnglish
Pages (from-to)197-203
Number of pages7
JournalBiochemistry
Volume36
Issue number1
DOIs
StatePublished - Jan 7 1997
Externally publishedYes

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Dimyristoylphosphatidylcholine
Protein folding
Protein Folding
Rigidity
Micelles
Membrane Proteins
Bacteriorhodopsins
Apoproteins
Detergents
Kinetics
Regeneration
Rate constants
Lipids
Lecithins
Fluorescence Spectrometry
Fluorescence spectroscopy
Phase behavior
Free energy
1,2-hexanoylphosphatidylcholine
Proteins

ASJC Scopus subject areas

  • Biochemistry

Cite this

Booth, P. J., Riley, M. L., Flitsch, S. L., Templer, R. H., Farooq, A., Curran, A. R., ... Wright, P. (1997). Evidence that bilayer bending rigidity affects membrane protein folding. Biochemistry, 36(1), 197-203. https://doi.org/10.1021/bi962200m

Evidence that bilayer bending rigidity affects membrane protein folding. / Booth, Paula J.; Riley, M. Louise; Flitsch, Sabine L.; Templer, Richard H.; Farooq, Amjad; Curran, A. Rachael; Chadborn, Neil; Wright, Penny.

In: Biochemistry, Vol. 36, No. 1, 07.01.1997, p. 197-203.

Research output: Contribution to journalArticle

Booth, PJ, Riley, ML, Flitsch, SL, Templer, RH, Farooq, A, Curran, AR, Chadborn, N & Wright, P 1997, 'Evidence that bilayer bending rigidity affects membrane protein folding', Biochemistry, vol. 36, no. 1, pp. 197-203. https://doi.org/10.1021/bi962200m
Booth PJ, Riley ML, Flitsch SL, Templer RH, Farooq A, Curran AR et al. Evidence that bilayer bending rigidity affects membrane protein folding. Biochemistry. 1997 Jan 7;36(1):197-203. https://doi.org/10.1021/bi962200m
Booth, Paula J. ; Riley, M. Louise ; Flitsch, Sabine L. ; Templer, Richard H. ; Farooq, Amjad ; Curran, A. Rachael ; Chadborn, Neil ; Wright, Penny. / Evidence that bilayer bending rigidity affects membrane protein folding. In: Biochemistry. 1997 ; Vol. 36, No. 1. pp. 197-203.
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