2D IR Line Shapes Probe Ovispirin Peptide Conformation and Depth in Lipid Bilayers

Abstract

We report a structural study on the membrane binding of ovispirin using 2D IR line shape analysis, isotope labeling, and molecular dynamics simulations. Ovispirin is an antibiotic polypeptide that binds to the surfaces of membranes as an alpha-helix. By resolving individual backbone vibrational modes (amide I) using 1-(13)C=(18)O labeling, we measured the 2D IR line shapes for 15 of the 18 residues in this peptide. A comparison of the line shapes reveals an oscillation in the inhomogeneous line width that has a period equal to that of an alpha-helix (3.6 amino acids). The periodic trend is caused by the asymmetric environment of the membrane bilayer that exposes one face of the alpha-helix to much stronger environmental electrostatic forces than the other. We compare our experimental results to 2D IR line shapes calculated using the lowest free energy structure identified from molecular dynamics simulations. These simulations predict a periodic trend similar to the experiment and lead us to conclude that ovispirin lies in the membrane just below the headgroups, is tilted, and may be kinked. Besides providing insight into the antibiotic mechanism of ovispirin, our procedure provides an infrared method for studying peptide and protein structures that relies on the natural vibrational modes of the backbone. It is a complementary method to other techniques that utilize line shapes, such as fluorescence, NMR, and ESR spectroscopies, because it does not require mutations, the spectra can be quantitatively simulated using molecular dynamics, and the technique can be applied to difficult-to-study systems like ion channels, aggregated proteins, and kinetically evolving systems.

Do guanidinium and tetrapropylammonium ions specifically interact with aromatic amino acid side chains?

“…For an inhomogeneously broadened IR band, its diagonal 2D IR peak at a relatively short waiting time (i.e., T) would assume an elliptical contour (37). As shown ( Fig.…”

Electrostatic frequency shifts in amide I vibrational spectra: Direct parameterization against experiment

“…Nevertheless, to move the field forward, the comparison of these models with experimental data must, in our view, become significantly more rigorous, particularly as applications to experimental data interpretation become increasingly demanding. In the past, direct comparison between simulated and experimental spectra has been generally limited to analysis of peak widths 41,66 (rather than frequencies) or is accompanied by arbitrary peak shifts of up to 30 cm −1 in order to improve agreement between simulation and experiment. 11,26,39,40 A particular difficulty appears to be the assignment of 13 44,45,67 or assumed 12,41,42 in various applications in the literature.…”

2D IR spectroscopy reveals the role of water in the binding of channel-blocking drugs to the influenza M2 channel

“…One noticeable difference between the spectra of Ala30 and Gly34 is that the Ala30 peak is blue-shifted by ∼5 cm −1 compared to the Gly34 band. Noting that backbone amide vibrational frequencies are susceptible to the electrostatic environment inside a protein, 30,47,48 the frequency difference between the Ala30 and the Gly34 bands is not unexpected, considering that the Ala30 residues are one turn further away from the charged His37 sidechains.…”

“…When used in conjunction with isotope labeling, one can obtain residue-by-residue information on hydration dynamics, as has been demonstrated with experiment and theory over the past decade on several membrane protein domains, including CD3zeta, M2, and ovispirin. [29][30][31][32] In this study, we use a double mutant (D44N, R45A) 31,33,34 of the transmembrane domain of the M2 channel, with the pore lining carbonyls of its Ala30 and Gly34 residues isotopically labeled (i.e., 12 C= 16 O to 13 C= 18 O) to serve as site-specific IR probes. [35][36][37][38] The use of this double mutant avoids any potential complexity arising from the 2D IR signals of arginine and aspartic acid, which overlap with those of the 13 C= 18 O groups.…”