Lipid bilayer composition modulates the unfolding free energy of a knotted α-helical membrane protein

Abstract

α-Helical membrane proteins have eluded investigation of their thermodynamic stability in lipid bilayers. Reversible denaturation curves have enabled some headway in determining unfolding free energies. However, these parameters have been limited to detergent micelles or lipid bicelles, which do not possess the same mechanical properties as lipid bilayers that comprise the basis of natural membranes. We establish reversible unfolding of the membrane transporter LeuT in lipid bilayers, enabling the comparison of apparent unfolding free energies in different lipid compositions. LeuT is a bacterial ortholog of neurotransmitter transporters and contains a knot within its 12-transmembrane helical structure. Urea is used as a denaturant for LeuT in proteoliposomes, resulting in the loss of up to 30% helical structure depending upon the lipid bilayer composition. Urea unfolding of LeuT in liposomes is reversible, with refolding in the bilayer recovering the original helical structure and transport activity. A linear dependence of the unfolding free energy on urea concentration enables the free energy to be extrapolated to zero denaturant. Increasing lipid headgroup charge or chain lateral pressure increases the thermodynamic stability of LeuT. The mechanical and charge properties of the bilayer also affect the ability of urea to denature the protein. Thus, we not only gain insight to the long-sought-after thermodynamic stability of an α-helical protein in a lipid bilayer but also provide a basis for studies of the folding of knotted proteins in a membrane environment.

How bilayer properties influence membrane protein folding

“…Although most studies focused on the effects of electrostatic or mechanical interactions in isolation, a growing number demonstrate that both are critical. 20,64,74,75,100,[107][108][109] Lipid mixtures may be necessary to balance the disparate effects of electromechanical properties on membrane proteins. The bilayer must contain enough bilayer lipids to maintain structural integrity, but enough nonbilayer lipids to allow for essential destabilizing events like budding, mitosis, and fusion.…”

“…73 Increasing the DOPG fraction in DOPC/DOPG vesicles increases the unfolding free energy of LeuT. 74 KcsA tetramer stability also increases with negative charge, with DOPE/DOPG liposomes optimally stabilizing the KcsA tetramer. 75 Replacing DOPG with DOPC decreases KcsA's melting temperature, while replacement with the negatively charged DOPA has no effect.…”

“…The unfolding free energy of LeuT was found to increase as the PE content in PC liposomes increased. 74 Increasing the DOPE fraction in DOPE/DOPC vesicles increased the lifetime of alamethicin channels and the denaturation temperature for LacY. 64,103 Adding PE to PC vesicles stabilized the GpATM dimer, while adding lysoPC (and thus lowering chain lateral pressure) destabilized it.…”

Proteolysis mediated by the membrane‐integrated ATP‐dependent protease FtsH has a unique nonlinear dependence on ATP hydrolysis rates

“…In comparison to water‐soluble substrates, ATP‐dependent degradation of membrane proteins poses a general thermodynamic challenge, that is, dislocation of hydrophobic TM segments from the membrane to the proteolytic active sites located in the cytosol (Δ G dislocation = 50–100 kcal/mol per TM), as seen in the degradation by FtsH, the endoplasmic reticulum‐associated degradation by the Hrd1 ubiquitin ligase complex/Cdc48 (an AAA+ enzyme)/26S proteasome system and the dislocation of mislocalized tail‐anchored membrane proteins from the mitochondrial outer membranes by the membrane‐anchored AAA+ enzyme Msp1 . The conformational stabilities of membrane proteins can also be substantial (Δ G o U = 3–12 kcal/mol) . Nonetheless, we always observed a sigmoidal dependence with thresholds, regardless of substrate stability and membrane localization, indicating that the high degree of cooperativity among ATP hydrolysis events is an intrinsic feature of FtsH‐mediated protein degradation.…”

CDtoolX, a downloadable software package for processing and analyses of circular dichroism spectroscopic data

“…CDtool has been widely used as a processing and analysis tool in CD and SRCD studies on diverse samples, including proteins, nucleic acids and even chiral small molecules, as well as for examining samples in different physical forms such as monolayers, fibers, emulsions, and oriented samples . Some recent examples of its use in different applications include: protein folding/unfolding, thermal stability profiles, comparisons of wild type and mutant proteins, monitoring continuous flow dynamics measurements, identifying similarities in protein evolutionary relationships, comparisons of new spectra with downloaded Protein Circular Dichroism Data Bank (PCDDB) components, demonstrations of fidelity of folding of expressed proteins, instrumentation comparisons and calibrations, as well as “on‐the‐fly” displays, data processing and analyses at SRCD beamlines . All of these types of studies are still compatible with CDtoolX.…”