Probing the spontaneous membrane insertion of a tail-anchored membrane protein by sum frequency generation spectroscopy

Abstract

In addition to providing a semipermeable barrier that protects a cell from harmful stimuli, lipid membranes occupy a central role in hosting a variety of biological processes, including cellular communications and membrane protein functions. Most importantly, protein-membrane interactions are implicated in a variety of diseases and therefore many analytical techniques were developed to study the basis of these interactions and their influence on the molecular architecture of the cell membrane. In this study, sum frequency generation (SFG) vibrational spectroscopy is used to investigate the spontaneous membrane insertion process of cytochrome b(5) and its mutants. Experimental results show a significant difference in the membrane insertion and orientation properties of these proteins, which can be correlated with their functional differences. In particular, our results correlate the nonfunctional property of a mutant cytochrome b(5) with its inability to insert into the lipid bilayer. The approach reported in this study could be used as a potential rapid screening tool in measuring the topology of membrane proteins as well as interactions of biomolecules with lipid bilayers in situ.

Figure 1

(A) ssp and ppp polarized SFG amide I signals of Cyt-b5 in a dDMPC/dDMPC lipid bilayer at 25°C. The dependence of the ppp/ssp ratio with respect to the helical tilt angle is shown in Supporting information. Thus, from the experimentally measured ppp/ssp ratio, it is possible to calculate the tilt angle of an alpha helix from an SFG experiment. (B) A proposed model of Cyt-b5 describing its orientation and topology in lipid bilayers.

Figure 2

(A) ssp and ppp polarized SFG amide I signals of a mutant-Cyt-b₅ in a dDMPC/dDMPC lipid bilayer at 25 °C. (B) The dependence of the experimentally measured tilt angle of the transmembrane helix on the number of residues in the linker region of the protein.

Figure 3

(A) ppp polarized SFG amide I band of a 8-deletion mutant-Cyt-b5 in a dDMPC/dDMPC lipid bilayer as a function of temperature. The increase in the intensity of ppp polarized SFG amide I band indicates a reorientation of the protein. The intensity of ppp polarized SFG amide I band at 45° is lower compared to that of 40°, which can be attributed to the desorption of protein from the lipid bilayer surface. (B) Tilt angle of a 8-deletion mutant-Cyt-b₅ as a function of temperature determined using SFG ppp/ssp signal strength ratio.