Isotope-edited IR spectroscopy for the study of membrane proteins

Abstract

Fourier transform infrared (FTIR) spectroscopy has long been a powerful tool for structural analysis of membrane proteins. However, because of difficulties in resolving contributions from individual residues, most of the derived measurements tend to yield average properties for the system under study. Isotope editing, through its ability to resolve individual vibrations, establishes FTIR as a method that is capable of yielding accurate structural data on individual sites in a protein.

Figure 1

FTIR spectra of the influenza A M2 transmembrane domain (25 amino acids) reconstituted in lipid bilayers [23]. The spectra shown are of samples with no label (gray line), a single ¹³C¹⁶O label (dashed line) or a single ¹³C¹⁸O label (black line). The location of the unlabeled and labeled peaks are shown.

Figure 2

Schematic representation of the effect of helix geometry upon the dichroisms of three arbitrary vibrations shown in blue, yellow and green. In the left model, the three different vibration transition dipole moments are all equally aligned with respect to the z axis and hence exhibit the same dichroism. However, tilting the helix by the angle β in the middle structure now results in the green mode having the highest dichroism because it is the least tilted with respect to the z axis. Finally, rotation about the helix director by the angle ω reverses the dichroism ratio, causing the blue mode to exhibit the highest dichroism.

Figure 3

Structure of the $\text{C}\mathrm{\alpha }\text{-H}\cdots \text{O}\text{C}$ bond predicted to take place in the transmembrane domain of human glycophorin A [31] and measured experimentally using isotope-edited FTIR [30^••].

Figure 4

2D IR spectrum of the transmembrane helical bundle formed by the T-cell receptor CD3-ζ component reconstituted in lipid bilayers. The spectrum exhibits cross peaks between the amide I band of the protein and the lipid head-group ester carbonyl, providing proof that peptide backbone and membrane head-groups are strongly coupled.

Figure 5

FTIR spectra of the transmembrane domain of SARS coronavirus E protein reconstituted in phospholipid bilayers [47] in the amide I and amide II regions. The spectra were obtained after flushing air saturated with H₂O (solid line) or D₂O (dotted line) over the sample. Both spectra were normalized so as to adjust the absorption of the amide I mode (1658 cm⁻¹) to 1.0 OD. Note the reduction that takes place in the amide II mode (1545 cm⁻¹) upon D₂O exchange.