Search

[Display omitted] • Solution IR spectroscopy of biomolecules requires very thin cells. • Knowledge of their actual thickness is challenging. • Solution IR spectra contain interferences non-trivially related to the sample thickness. • We modelled the interferences with Fresnel equations. • We obtain the sample thickness by comparing experimental and modelled interferences in the Fourier domain. Absorption spectra of aqueous samples measured by transmission need to be acquired using very thin cells (5–50 μm) when targeting the mid-infrared (mid-IR) region due to the strong background absorbance of liquid water. The thickness of the cell used controls the pathlength of the light through the sample, a value needed to transform absorption spectra into molar absorption coefficient spectra, or to determine solute concentrations from absorption spectra. The most accurate way to determine the thickness of an empty cell (i.e., filled with air) is from the period of an interference pattern, known as interference fringes, that arises when the cell is placed perpendicular to the path of light in the spectrometer. However, this same approach is not directly applicable to determine the thickness of a cell filled with an aqueous solution, due partially to the smaller amplitude of the interference fringes but fundamentally caused by its complex waveform, with a wavenumber-dependent oscillation period. Here, using Fresnel equations, we derived analytical expressions to model interference fringes in absorption spectra obtained by transmission, which are also valid for aqueous samples. We also present a novel Fourier-based analysis of the interference fringes that, in combination with the derived analytical expressions, allowed us to determine the pathlength of aqueous samples with an error below ∼ 50 nm. We implemented this novel approach to analyze interference fringes as a Live Script running in the software Matlab. As an application, we measured the absorption spectra of a 97 mM solution of MES buffer at pH 3.4 and pH 8.4 using cells of various nominal thicknesses (6, 25 and 50 μm), whose actual thicknesses were determined using the present approach. The derived molar absorption coefficient spectrum for both the acidic and basic forms of MES were virtually identical regardless of the cell, indicating that the determined thicknesses were likely very accurate. These results illustrate the utility of the present methodology in obtaining accurate molar absorption coefficient spectra of water-soluble molecules in the mid-IR region. [ABSTRACT FROM AUTHOR]

Infrared difference spectroscopy probes vibrational changes of proteins upon their perturbation. Compared with other spectroscopic methods, it stands out by its sensitivity to the protonation state, H-bonding, and the conformation of different groups in proteins, including the peptide backbone, amino acid side chains, internal water molecules, or cofactors. In particular, the detection of protonation and H-bonding changes in a time-resolved manner, not easily obtained by other techniques, is one of the most successful applications of IR difference spectroscopy. The present review deals with the use of perturbations designed to specifically change the protein between two (or more) functionally relevant states, a strategy often referred to as reaction-induced IR difference spectroscopy. In the first half of this contribution, I review the technique of reaction-induced IR difference spectroscopy of proteins, with special emphasis given to the preparation of suitable samples and their characterization, strategies for the perturbation of proteins, and methodologies for time-resolved measurements (from nanoseconds to minutes). The second half of this contribution focuses on the spectral interpretation. It starts by reviewing how changes in H-bonding, medium polarity, and vibrational coupling affect vibrational frequencies, intensities, and bandwidths. It is followed by band assignments, a crucial aspect mostly performed with the help of isotopic labeling and site-directed mutagenesis, and complemented by integration and interpretation of the results in the context of the studied protein, an aspect increasingly supported by spectral calculations. Selected examples from the literature, predominately but not exclusively from retinal proteins, are used to illustrate the topics covered in this review. [ABSTRACT FROM AUTHOR]

Mid-IR spectroscopy is a powerful and label-free technique to investigate protein reactions. In this study, we use quantum-cascade-laser-based dual-comb spectroscopy to probe protein conformational changes and protonation events by a single-shot experiment. By using a well-characterized membrane protein, bacteriorhodopsin, we provide a comparison between dual-comb spectroscopy and our homebuilt tunable quantum cascade laser (QCL)-based scanning spectrometer as tools to monitor irreversible reactions with high time resolution. In conclusion, QCL-based infrared spectroscopy is demonstrated to be feasible for tracing functionally relevant protein structural changes and proton translocations by single-shot experiments. Thus, we envisage a bright future for applications of this technology for monitoring the kinetics of irreversible reactions as in (bio-)chemical transformations. [ABSTRACT FROM AUTHOR]

Fourier transform infrared (FT-IR) difference absorption spectroscopy is a common method for studying the structural and dynamical aspects behind protein function. In particular, the 2800–1800 cm−1 spectral range has been used to obtain information about internal (deuterated) water molecules, as well as site-specific details about cysteine residues and chemically modified and artificial amino acids. Here, we report on the presence of ghost bands in cryogenic light-induced FT-IR difference spectra of the protein bacteriorhodopsin. The presence of these ghost bands can be particularly problematic in the 2800–1900 cm−1 region, showing intensities similar to O–D vibrations from water molecules. We demonstrate that they arise from second harmonics from genuine chromophore bands located in the 1400–850 cm−1 region, generated by double-modulation artifacts caused from reflections of the IR beam at the sample and at the cryostat windows back to the interferometer (inter-reflections). The second-harmonic ghost bands can be physically removed by placing an optical filter of suitable cutoff in the beam path, but at the cost of losing part of the multiplexing advantage of FT-IR spectroscopy. We explored alternatives to the use of optical filters. Tilting the cryostat windows was effective in reducing the intensity of the second harmonic artifacts but tilting the sample windows was not, presumably by their close proximity to the focal point of the IR beam. We also introduce a simple numerical post-processing approach that can partially, but not fully, correct for second-harmonic ghost bands in FT-IR difference spectra. [ABSTRACT FROM AUTHOR]

We have developed a spectrometer based on tunable quantum cascade lasers (QCLs) for recording time-resolved absorption spectra of proteins in the mid-infrared range. We illustrate its performance by recording time-resolved difference spectra of bacteriorhodopsin in the carboxylic range (1800–1700 cm − 1 ) and on the CO rebinding reaction of myoglobin (1960–1840 cm − 1 ), at a spectral resolution of 1 cm − 1 . The spectrometric setup covers the time range from 4 ns to nearly a second with a response time of 10–15 ns. Absorption changes as low as 1 × 10 − 4 are detected in single-shot experiments at t > 1 μs, and of 5 × 10 − 6 in kinetics obtained after averaging 100 shots. While previous time-resolved IR experiments have mostly been conducted on hydrated films of proteins, we demonstrate here that the brilliance of tunable quantum cascade lasers is superior to perform ns time-resolved experiments even in aqueous solution (H 2 O). [ABSTRACT FROM AUTHOR]

Infrared spectroscopy has been used in the past to probe the dynamics of internal proton transfer reactions taking place during the functional mechanism of proteins but has remained mostly silent to protonation changes in the aqueous medium. Here, by selectively monitoring vibrational changes of buffer molecules with a temporal resolution of 6 μs, we have traced proton release and uptake events in the light-driven proton-pump bacteriorhodopsin and correlate these to other molecular processes within the protein. We demonstrate that two distinct chemical entities contribute to the temporal evolution and spectral shape of the continuum band, an unusually broad band extending from 2,300 to well below 1,700 cm-1. The first contribution corresponds to deprotonation of the proton release complex (PRC), a complex in the extracellular domain of bacteriorhodopsin where an excess proton is shared by a cluster of internal water molecules and/or ionic E194/E204 carboxylic groups. We assign the second component of the continuum band to the proton uptake complex, a cluster with an excess proton reminiscent to the PRC but located in the cytoplasmic domain and possibly stabilized by D38. Our findings refine the current interpretation of the continuum band and call for a reevaluation of the last proton transfer steps in bacteriorhodopsin. [ABSTRACT FROM AUTHOR]

Time-resolved femtosecond-stimulated Raman spectroscopy (FSRS) provides valuable information on the structural dynamics of biomolecules. However, FSRS has been applied mainly up to the nanoseconds regime and above 700 cm−1, which covers only part of the spectrum of biologically relevant time scales and Raman shifts. Here we report on a broadband (~200–2200 cm−1) dual transient visible absorption (visTA)/FSRS set-up that can accommodate time delays from a few femtoseconds to several hundreds of microseconds after illumination with an actinic pump. The extended time scale and wavenumber range allowed us to monitor the complete excited-state dynamics of the biological chromophore flavin mononucleotide (FMN), both free in solution and embedded in two variants of the bacterial light-oxygen-voltage (LOV) photoreceptor EL222. The observed lifetimes and intermediate states (singlet, triplet, and adduct) are in agreement with previous time-resolved infrared spectroscopy experiments. Importantly, we found evidence for additional dynamical events, particularly upon analysis of the low-frequency Raman region below 1000 cm−1. We show that fs-to-sub-ms visTA/FSRS with a broad wavenumber range is a useful tool to characterize short-lived conformationally excited states in flavoproteins and potentially other light-responsive proteins. [ABSTRACT FROM AUTHOR]