Ion Channels and Membrane Proteins

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 13C=18O 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, 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.

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. 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.

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.

More recently, we have used our isotope labeling stragey and 2D IR spectroscopy to investigate the equilibrium ion occupancy of KcsA, a prokaryotic potassium channel. By isotope labeling specific residues in the selectivity filter of the channel, two elongated features were resolved, indicative of two structural conformations with a statistical weighting. These spectra were reproduced with molecular dynamics to determine the ion occupancy revealed by 2D IR spectroscopy. The spectra and simulations indicated that the ion occupancy corresponds to the knock-on model with water.

Selected Publications
Ryan MJ, Gao L, Valiyaveetil FI, Zanni MT, and Kananenka A. Probing Ion Configurations in the KcsA Selectivity Filter with Single-Isotope Labels and 2D IR Spectroscopy J. Am. Chem. Soc.

Birdsall ER, Petti MK, Saraswat V, Ostrander JS,  Arnold MS, Zanni MT. Structure Changes of a Membrane Polypeptide under an Applied Voltage Observed with Surface-Enhanced 2D IR Spectroscopy. J.Phys. Chem. Lett.
Kratochvil, H. T.; Carr, J. K.; Matulef, K.; Annen, A. W.; Li, H.; Maj, M.; Ostmeyer, J.; Serrano, A. L.; Raghuraman, H.; Moran, S. D.; et al. Instantaneous Ion Configurations in the K + Ion Channel Selectivity Filter Revealed by 2D IR Spectroscopy. Science. 2016, 353, 1040–1044.
Kratochvil, H. T.; Maj, M.; Matulef, K.; Annen, A. W.; Ostmeyer, J.; Perozo, E.; Roux, B.; Valiyaveetil, F. I.; Zanni, M. T. Probing the Effects of Gating on the Ion Occupancy of the K+ Channel Selectivity Filter Using Two-Dimensional Infrared Spectroscopy. J. Am. Chem. Soc. 2017, 139, 8837–8845.
Ghosh, A.; Wang, J.; Moroz, Y. S.; Korendovych, I. V.; Zanni, M.; DeGrado, W. F.; Gai, F.; Hochstrasser, R. M. 2D IR Spectroscopy Reveals the Role of Water in the Binding of Channel-Blocking Drugs to the Influenza M2 Channel. J. Chem. Phys. 2014, 140, 235105.