Effects of temperature and pressure on the lateral organization of model membranes with functionally reconstituted multidrug transporter LmrA.
To contribute to the understanding of membrane protein function upon application of pressure, we investigated the influence of hydrostatic pressure on the conformational order and phase behavior of the multidrug transporter LmrA in biomembrane systems. To this end, the membrane protein was reconstituted into various lipid bilayer systems of different chain length, conformation, phase state and heterogeneity, including raft model mixtures as well as some natural lipid extracts. In the first step, we determined the temperature stability of the protein itself and verified its reconstitution into the lipid bilayer systems using CD spectroscopic and AFM measurements, respectively. Then, to yield information on the temperature and pressure dependent conformation and phase state of the lipid bilayer systems, generalized polarization values by the Laurdan fluorescence technique were determined, which report on the conformation and phase state of the lipid bilayer system. The temperature-dependent measurements were carried out in the temperature range 5-70 degrees C, and the pressure dependent measurements were performed in the range 1-200 MPa. The data show that the effect of the LmrA reconstitution on the conformation and phase state of the lipid matrix depends on the fluidity and hydrophobic matching conditions of the lipid system. The effect is most pronounced for fluid DMPC and DMPC with low cholesterol levels, but minor for longer-chain fluid phospholipids such as DOPC and model raft mixtures such as DOPC/DPPC/cholesterol. The latter have the additional advantage of using lipid sorting to avoid substantial hydrophobic mismatch. Notably, the most drastic effect was observed for the neutral/glycolipid natural lipid mixture. In this case, the impact of LmrA incorporation on the increase of the conformational order of the lipid membrane was most pronounced. As a consequence, the membrane reaches a mechanical stability which makes it very insensitive to application of pressures as high as 200 MPa. The results are correlated with the functional properties of LmrA in these various lipid environments and upon application of high hydrostatic pressure and are discussed in the context of other work on pressure effects on membrane protein systems.
Periasamy
Nagarajan
N
Dortmund University of Technology, Physical Chemistry I - Biophysical Chemistry, D-44227 Dortmund, Germany.
Teichert
Holger
H
Weise
Katrin
K
Vogel
Rudi F
RF
Winter
Roland
R
D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D002942:Circular Dichroism; D048191:Lactiplantibacillus plantarum / Q000737:chemistry / Q000378:metabolism; D008051:Lipid Bilayers / Q000737:chemistry* / Q000378:metabolism; D008823:Micelles; D018625:Microscopy, Atomic Force; D008954:Models, Biological; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000737:chemistry* / Q000378:metabolism*; D044367:Phase Transition; D011312:Pressure; D017433:Protein Structure, Secondary; D013696:Temperature
D001426:Bacterial Proteins; D008051:Lipid Bilayers; D008823:Micelles; D027425:ATP-Binding Cassette, Sub-Family C Proteins