Membrane Peptides

2D IR spectroscopy is an exciting new tool for quantitatively studying protein structure and dynamics. Much of our work has been devoted to applying 2D IR spectroscopy to membrane bound polypeptides, because these systems are very difficult to study with standard structural tool such as x-ray crystallography and high-resolution NMR spectroscopy. Even solid-state NMR spectroscopy is difficult to apply to dynamical systems and systems for which little sample is available. Moreover, isotope labeling is non-perturbative, unlike spin-labels used in EPR.

We have learned that the amide I mode of 1-13C18O isotope labeled residues is very sensitive to local electrostatic disorder of its surrounding environment. In membrane bound systems, electrostatic disorder is strongest near the lipid headgroups where water permeates into the membrane, and weakest in the middle of the membrane where water is prohibited. As a result, the 2D lineshapes of the amide mode, indicated in Figure 1, provides information on the position of the residue in the bilayer. By measuring multiple labels, one can obtain the secondary structure, orientation and depth of a membrane peptides.

2D IR of an isotope-labeled membrane peptide

Figure 1. 2D IR spectrum of 1-13C18O isotope-labeled peptide in lipid vesicle.

For example, the peptide ovispirin binds parallel to the bilayer and weaves through varying densities of lipid components, ions, and solvent. The 2D IR diagonal linewidth, as the isotope label is placed further along the peptide backbone, oscillates with a period reflecting the peptide’s alpha-helical secondary structure (Figure 2). The diagonal linewidth has contributions from both the homogeneous and inhomogeneous linewidth. Using 2D lineshape analysis, we extract out the inhomogeneous width, which has the strongest relation to hydration. For ovispirin, it turns out that the hydrophilic residues that lie in the membrane interface have a larger inhomogenous width than hydrophobic residues that face the membrane interior. Most notably, the diagonal linewidth per residue closely follows the depth predicted from a helical wheel representation.

Ovispirin helix diagram

Figure 2. Alpha-helical wheel diagram for the antimicrobial peptide ovispirin, and 2D IR diagonal linewidth of isotope labels placed along the backbone of ovispirin.

The correlation between diagonal linewidth and residue depth agrees with our earlier studies on the CD3ζ and M2 peptides. Thus, 2D IR spectroscopy provides a unique means of probing secondary structures in membrane bound polypeptides and proteins in a method similar to EPR but without perturbing residues and with the ability to study kinetics.