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1. Site-specific 2D IR spectroscopy: a general approach for the characterization of protein dynamics with high spatial and temporal resolution

   https://doi.org/10.1039/C8CP06146G  

   Ramos, Sashary; Horness, Rachel E.; Collins, Jessica A.; Haak, David; Thielges, Megan C. (January 2019, Physical Chemistry Chemical Physics)

   The conformational heterogeneity and dynamics of protein side chains contribute to function, but investigating exactly how is hindered by experimental challenges arising from the fast timescales involved and the spatial heterogeneity of protein structures. The potential of two-dimensional infrared (2D IR) spectroscopy for measuring conformational heterogeneity and dynamics with unprecedented spatial and temporal resolution has motivated extensive effort to develop amino acids with functional groups that have frequency-resolved absorptions to serve as probes of their protein microenvironments. We demonstrate the full advantage of the approach by selective incorporation of the probe p -cyanophenylalanine at six distinct sites in a Src homology 3 domain and the application of 2D IR spectroscopy to site-specifically characterize heterogeneity and dynamics and their contribution to cognate ligand binding. The approach revealed a wide range of microenvironments and distinct responses to ligand binding, including at the three adjacent, conserved aromatic residues that form the recognition surface of the protein. Molecular dynamics simulations performed for all the labeled proteins provide insight into the underlying heterogeneity and dynamics. Similar application of 2D IR spectroscopy and site-selective probe incorporation will allow for the characterization of heterogeneity and dynamics of other proteins, how heterogeneity and dynamics are affected by solvation and local structure, and how they might contribute to biological function. 

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2. Water inside the Selectivity Filter of a K ⁺ Ion Channel: Structural Heterogeneity, Picosecond Dynamics, and Hydrogen Bonding

   https://doi.org/10.1021/jacs.3c11513  

   Ryan, Matthew J; Gao, Lujia; Valiyaveetil, Francis I; Kananenka, Alexei A; Zanni, Martin T (January 2024, Journal of the American Chemical Society)

   Water inside biological ion channels regulates the key properties of these proteins such as selectivity, ion conductance, and gating. In this Article we measure the picosecond spectral diffusion of amide I vibrations of an isotope labeled KcsA potassium channel using two-dimensional infrared (2D IR) spectroscopy. By combining waiting time (100 - 2000 fs) 2D IR measurements of the KcsA channel including 13C18O isotope labeled Val76 and Gly77 residues with molecular dynamics simulations, we elucidated the site-specific dynamics of water and K+ ions inside the selectivity filter of KcsA. We observe inhomogeneous 2D lineshapes with extremely slow spectral diffusion. Our simulations quantitatively reproduce the experiments and show that water is the only component with any appreciable dynamics, whereas K+ ions and the protein are essentially static on a picosecond timescale. By analyzing simulated and experimental vibrational frequencies, we find that water in the selectivity filter can be oriented to form hydrogen bonds with adjacent, or non-adjacent carbonyl groups with the reorientation timescales being three times slower and comparable to that of water molecules in liquid, respectively. Water molecules can reside in the cavity sufficiently far from carbonyls and behave essentially like “free” gas-phase-like water with fast reorientation times. Remarkably, no interconversion between these configurations were observed on a picosecond timescale. These dynamics are in stark contrast with liquid water that remains highly dynamic even in the presence of ions at high concentrations. 

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3. Probing Ion Configurations in the KcsA Selectivity Filter with Single-Isotope Labels and 2D IR Spectroscopy

   https://doi.org/10.1021/jacs.3c05339  

   Ryan, Matthew J.; Gao, Lujia; Valiyaveetil, Francis I.; Zanni, Martin T.; Kananenka, Alexei A. (August 2023, Journal of the American Chemical Society)

   The potassium ion (K+) configurations of the selectivity filter of the KcsA ion channel protein are investigated with two-dimensional infrared (2D IR) spectroscopy of amide I vibrations. Single 13C–18O isotope labels are used, for the first time, to selectively probe the S1/S2 or S2/S3 binding sites in the selectivity filter. These binding sites have the largest differences in ion occupancy in two competing K+ transport mechanisms: soft-knock and hard-knock. According to the former, water molecules alternate between K+ ions in the selectivity filter while the latter assumes that K+ ions occupy the adjacent sites. Molecular dynamics simulations and computational spectroscopy are employed to interpret experimental 2D IR spectra. We find that in the closed conductive state of the KcsA channel, K+ ions do not occupy adjacent binding sites. The experimental data is consistent with simulated 2D IR spectra of soft-knock ion configurations. In contrast, the simulated spectra for the hard-knock ion configurations do not reproduce the experimental results. 2D IR spectra of the hard-knock mechanism have lower frequencies, homogeneous 2D lineshapes, and multiple peaks. In contrast, ion configurations of the soft-knock model produce 2D IR spectra with a single peak at a higher frequency and inhomogeneous lineshape. We conclude that under equilibrium conditions, in the absence of transmembrane voltage, both water and K+ ions occupy the selectivity filter of the KcsA channel in the closed conductive state. The ion configuration is central to the mechanism of ion transport through potassium channels. 

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4. Path to a 100 kHz 2D IR spectrometer based on a tunable Er fiber front end

   https://doi.org/10.1364/OE.566440  

   Luther, Bradley M; Guerrieri, Luke; Hall, Sarah; Williams, Devin R; Krummel, Amber T (January 2025, Optics Express)

   2D mid-IR spectroscopy is a powerful tool for revealing structure and dynamics in chemical systems. Recently systems operating at 100 kHz have been shown to have improved signal to noise as compared to 1 kHz Ti:Sa systems, as well as faster collection rates allowing more challenging experiments such as 2D IR microscopy. We present a tunable, 100 kHz mid-IR source for 2D IR spectroscopy based on an Er fiber front end which allows for the generation of both pump and signal beams for a three-stage magnesium doped periodically poled lithium niobate (PPLN) optical parametric chirped pulse amplifier (OPCPA). The use of a Raman shift in a photonic crystal fiber (PCF) allows for the PPLN seed to be easily tuned from 1660 nm to 1760nm. Difference frequency generation (DFG) of the PPLN outputs in a zinc germanium phosphate (ZGP) crystal generates mid-IR outputs tunable from 4 µm to 6.5 µm and supporting pulse durations of 42-65 fs, respectively. 

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5. Local Crowd, Local Probe: Strengths and Drawbacks of Azidohomoalanine as a Site-Specific Crowding Probe

   https://doi.org/10.1021/acs.jpcb.4c00712  

   Shirley, Joseph C; Baiz, Carlos R (June 2024, The Journal of Physical Chemistry B)

   Every residue on a protein can be characterized by its interaction with water, in lack or in excess, as water is the matrix of biological systems. Infrared spectroscopy and the implementation of local azidohomoalanine (AHA) probes allow us to move beyond an ensemble or surface-driven conceptualization of water behavior and toward a granular, site-specific picture. In this paper, we examined the role of crowding in modulating both global and local behavior on the β-hairpin, TrpZip2 using a combination of Fourier-transform infrared spectroscopy (FTIR) spectroscopy, two-dimensional infrared (2D IR) spectroscopy, and molecular dynamics simulations. We found that, at the amino acid level, crowding drove dehydration of both sheet and turn peptide sites as well as free AHA. However, the subpicosecond dynamics showed highly individualized responses based on the local environment. Interestingly, while steady-state FTIR measurements revealed similar responses at the amino-acid level to hard versus soft crowding (dehydration), we found that PEG and glucose had opposite stabilizing and destabilizing effects on the protein secondary structure, emphasizing an important distinction in understanding the impact of crowding on protein structure as well as the role of crowding across length scales. 

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