IR-spectroscopy of KcsA binding states

Most of our today’s understanding on the function of channels is based on static crystallography data and electrophysiology. However, it is increasingly believed that in order to fully understand the mechanism that lead to the extreme ion selectivity and transport properties of ion channels, one has to account the fast dynamics and coupling of the system on the atomic scales.

Figure 1: Location of the potassium channel KcsA in the cell membrane of bacteria. The schematic illustration on the right shows the changes in strength and direction of vibrational coupling inside the filter depending on the ion species, as found by the study. @David S. Goodsell & RCSB Protein Data Bank.

To investigate these ideas, we have developed a methodology with our collaborators which enables us to visualize changes in conformation and coupling of the selectivity filter on an atomistic scale. This paves the way to study the dynamics of the selectivity filter using time resolved spectroscopy in the IR regime with atomistic precision.

Atomistic resolution in FTIR experiments is usually achieved by placing an isotope label at a specific position inside the protein which assignes a part of the deconvoluted spectrum to one single peptide unit of all aminoacids inside the protein. We show, that it is possible to assign the molecular origins of the vibrational modes of a big part of the protein by applying molecular and spectroscopic modeling.

Individual peptide groups and their associated amide I transition dipoles are identified in structures sampled from MD simulations. Individual peptide groups and their associated amide I transition dipoles are identified in structures sampled from MD simulations. A local mode Hamiltonian is parametrized from the structure using the molecular electric fi eld to set the diagonal site frequencies and o ff -diagonal couplings between sites. The delocalized eigenstates and their corresponding transition dipole moments are used to calculate the IR spectrum and use doorway modes to visualize the vibrations. — Figure 2: Individual peptide groups and their associated amide I transition dipoles are identified in structures sampled from MD simulations. Individual peptide groups and their associated amide I transition dipoles are identified in structures sampled from MD simulations. A local mode Hamiltonian is parametrized from the structure using the molecular electric field to set the diagonal site frequencies and off -diagonal couplings between sites. The delocalized eigenstates and their corresponding transition dipole moments are used to calculate the IR spectrum and use doorway modes to visualize the vibrations.

As outlined in the schematic above (Fig. 2), we obtain the location of all Amide I site vibrations inside the protein from structural ensembles generated by molecular dynamics simulations. Each oscillators’ site frequency (ω) is sensitive to its local environment and can be coupled with adjacent oscillators. This leads to delocalized vibrations which strongly dependent on the underlying structure of the protein and ligands (Na or K). With this information the IR-spectrum of the protein is calculated and matched to the experimentally obtained measurements (Fig. 3), linking the structural to the spectral information. Furthermore, Doorway mode analysis reveals the peptide units which contribute most to a narrow spectral window (spectral windows are colored in Fig 3, results in Fig. 4).

Figure 3: Comparison of the experimental and calculated spectra for KcsA in KCl buffer, a), and the difference spectra, b). Peaks 1, 2 and 3 are highlighted in red, green, blue respectively. The sidechain region is highlighted in orange; Sidechains were not included in the spectral modeling.

With this approach we can qualitatively reproduce key features of the experimentally determined spectra of KcsA in KCl or NaCl buffer and in the difference spectra (see Fig. 3: Regions 1, 2, 3). Doorway mode analysis of the most pronounced spectral features revealed which amino acids contribute most to specific parts (i.e. Region 1) of the spectra and how strongly they are vibrationally coupled to each other.

Figure 4: Visualizations of calculated doorway modes identified in the text presented as side and top (looking down pore) view of the extended filter region. Color intensity indicates the amplitude of the CO vibrational motion, while the color indicates its relative sign. Green arrows indicate the direction of the transition dipole moment for the mode. Circles on the right show indicate the overall displacement of the selectivity filter carbonyls in that strand.

We found, that differences in the spectra between K⁺ and Na⁺ are determined not only from structural differences in the selectivity filter but also from the pore helices surrounding this region (Fig. 4: Helix Mode 1660 cm^-1). The results suggest that the role of the pore helices may not only act as a mediator between activation gate and selectivity filter but that it is necessary to include the influence of the surrounding helices in discussing selectivity and transport in KcsA. Subtle changes in the vibrational couplings of these vibrations might have a role in determining the energetics and dynamics of ion binding.

Relevant publication:
Paul Stevenson, Christoph Götz, Carlos R. Baiz, Jasper Akerboom, Andrei Tokmakoff, and Alipasha Vaziri
Visualizing KcsA Conformational Changes upon Ion Binding by Infrared Spectroscopy and Atomistic Modeling
J. Phys. Chem. B 119(18), 5824–5831 (2015).
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