Evidence That Bilayer Bending Rigidity Affects Membrane Protein Folding

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 refolding 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.

Lipid Spontaneous Curvatures Estimated from Temperature-Dependent Changes in Inverse Hexagonal Phase Lattice Parameters: Effects of Metal Cations

“…This has been shown to play a critical role in the energetics of many of the lipid-protein interactions that occur in biological membranes 1,2 . For example, curvature elastic stress, which arises from a system being forced to adopt a curvature that is different from c 0 , has been shown to modulate the activity of many membrane interacting proteins 3,4 and drives the folding of other membrane bound proteins 5,6 . These observations underpin the intrinsic curvature hypothesis, which postulates that cells regulate the composition of their biological membranes so that the net curvature elastic stress is under homeostatic control [7][8][9][10] .…”

Building a Thermostable Membrane Protein

“…Some conformations may be more susceptible to inactivation than others. It has been suggested that membrane protein structure is significantly influenced by ordering imposed by the bilayer (17)(18)(19)(20). Thus, diminishing conformational restraints by extraction into a less ordered detergent micelle could increase conformational flexibility.…”

Secondary Structure of Bacteriorhodopsin Fragments

“…This lipid/detergent system is, however, inappropriate for CD measurements, since the high absorbance of CHAPS obscures key CD bands in the far UV range associated with protein secondary structure. Thus, in the present study a lipid-based refolding system was employed, in which the lipid DHPC is substituted for CHAPS (31). In the case of native BR from purple membrane (32) or eBR a near quantitative regeneration of the chromophore has been observed in DMPC/DHPC micelles.…”

“…Reconstitution of the native structure has also been accomplished with complementary combinations of recombinant or proteolytic fragments and/or synthetic peptides, comprising one or more of the TM regions (24 -30). Furthermore, the folding process of native BR has been analyzed by time-resolved spectroscopic techniques (31,32). Third, recombinant variants of BR can be produced in sufficient quantities for the necessary biophysical measurements, using heterologous or homologous expression systems (reviewed in Ref.…”