Rapid-scan Fourier Transform Infrared Research Articles

Microsecond-Resolved Infrared Spectroscopy on Nonrepetitive Protein Reactions by Applying Caged Compounds and Quantum Cascade Laser Frequency Combs.

Infrared spectroscopy is ideally suited for the investigation of protein reactions at the atomic level. Many systems were investigated successfully by applying Fourier transform infrared (FTIR) spectroscopy. While rapid-scan FTIR spectroscopy is limited by time resolution (about 10 ms with 16 cm-1 resolution), step-scan FTIR spectroscopy reaches a time resolution of about 10 ns but is limited to cyclic reactions that can be repeated hundreds of times under identical conditions. Consequently, FTIR with high time resolution was only possible with photoactivable proteins that undergo a photocycle. The huge number of nonrepetitive reactions, e.g., induced by caged compounds, were limited to the millisecond time domain. The advent of dual-comb quantum cascade laser now allows for a rapid reaction monitoring in the microsecond time domain. Here, we investigate the potential to apply such an instrument to the huge class of G-proteins. We compare caged-compound-induced reactions monitored by FTIR and dual-comb spectroscopy by applying the new technique to the α subunit of the inhibiting Gi protein and to the larger protein-protein complex of Gαi with its cognate regulator of G-protein signaling (RGS). We observe good data quality with a 4 μs time resolution with a wavelength resolution comparable to FTIR. This is more than three orders of magnitude faster than any FTIR measurement on G-proteins in the literature. This study paves the way for infrared spectroscopic studies in the so far unresolvable microsecond time regime for nonrepetitive biological systems including all GTPases and ATPases.

Coupling a Rapid-Scan FT-IR Spectrometer with Quantum Cascade Lasers within a Single Setup: An Easy Way to Reach Microsecond Time Resolution without Losing Spectral Information.

For the first time, a standard rapid-scan Fourier-transform infrared (FT-IR) spectrometer was coupled with quantum cascade lasers (QCLs) tunable within the 1876-905 cm-1 spectral range, within one single setup, by keeping one single sample compartment. The aim was to extend the time resolution of absorption measurements by several orders of magnitude thanks to the fast pulsed QCL technology without losing the spectral information provided by standard FT-IR spectroscopy, both probing the same sample. By slightly modifying the optical bench arrangement, the spectrometer now enables a fast and easy switch between the standard FT-IR mode, used for classical broadband scans from 6000 to 650 cm-1, and the new QCL-irradiation mode, used for ultrafast recording at specific wavenumbers (the two diagnostics have superimposed beam paths). So, one can study a sample (in condensed or gaseous state) during a physical or chemical transformation first as a whole in a broadband configuration and then immediately switch to the QCL mode to monitor a selected absorption feature (associated with an intermediate, a structural change, a diffusing substance, etc., for example) versus time. The QCL mode then drastically boosts the time resolution from tens of milliseconds (in rapid-scan FT-IR) to a few microseconds, as demonstrated here in the case of ammonia diffusion into a commercial zeolite ZSM-5.

Proton Transfers in a Channelrhodopsin-1 Studied by Fourier Transform Infrared (FTIR) Difference Spectroscopy and Site-directed Mutagenesis

Channelrhodopsin-1 from the alga Chlamydomonas augustae (CaChR1) is a low-efficiency light-activated cation channel that exhibits properties useful for optogenetic applications such as a slow light inactivation and a red-shifted visible absorption maximum as compared with the more extensively studied channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2). Previously, both resonance Raman and low-temperature FTIR difference spectroscopy revealed that unlike CrChR2, CaChR1 under our conditions exhibits an almost pure all-trans retinal composition in the unphotolyzed ground state and undergoes an all-trans to 13-cis isomerization during the primary phototransition typical of other microbial rhodopsins such as bacteriorhodopsin (BR). Here, we apply static and rapid-scan FTIR difference spectroscopy along with site-directed mutagenesis to characterize the proton transfer events occurring upon the formation of the long-lived conducting P2 (380) state of CaChR1. Assignment of carboxylic C=O stretch bands indicates that Asp-299 (homolog to Asp-212 in BR) becomes protonated and Asp-169 (homolog to Asp-85 in BR) undergoes a net change in hydrogen bonding relative to the unphotolyzed ground state of CaChR1. These data along with earlier FTIR measurements on the CaChR1 → P1 transition are consistent with a two-step proton relay mechanism that transfers a proton from Glu-169 to Asp-299 during the primary phototransition and from the Schiff base to Glu-169 during P2 (380) formation. The unusual charge neutrality of both Schiff base counterions in the P2 (380) conducting state suggests that these residues may function as part of a cation selective filter in the open channel state of CaChR1 as well as other low-efficiency ChRs.

Kinetic studies of the infrared-induced reaction between atomic chlorine and solid parahydrogen

We present Fourier-transform infrared (FTIR) spectroscopic studies of the IR-induced Cl+H2(v=1)→HCl+H reaction in a parahydrogen (pH2) matrix aimed at distinguishing between two proposed reactions mechanisms; direct-IR and vibron-mediated. The Cl atom reactants are produced via 355nm in situ photolysis of a Cl2 doped pH2 matrix. After photolysis is complete, a long-pass IR filter in the FTIR beam is removed and we measure the ensuing IR-induced reaction kinetics using rapid scan FTIR spectroscopy. We follow both the decay of the Cl atom reactant and growth of the HCl product using the Cl spin–orbit (SO)+Q1(0) and HCl R1(0) transitions, respectively. We show the IR-induced reaction mechanism depends on the spectral profile of the IR radiation; for IR spectral profiles that have significant IR intensities between 4000 and 5000cm−1 we observe first-order kinetics that are assigned to a vibron-mediated mechanism and for spectral profiles that have significant IR intensities that include the Cl SO+Q1(0) transition near 5094cm−1 we observe bi-exponential kinetics that are dominated by the direct-IR mechanism at early reaction times. We can distinguish between the two mechanisms using the observed kinetics. We investigate the reaction kinetics for different FTIR optical setups, for a range of sample conditions, and start and stop the IR-induced reaction to investigate the importance of secondary H atom reactions. We also study the IR-induced reaction in Br/Cl co-doped pH2 samples and show the presence of the Br atom quenches the vibron-mediated reaction kinetics presumably because the Br-atoms serve as efficient vibron traps. This paper indicates that in a highly enriched pH2 matrix the H atoms that are produced by the IR-induced Cl atom reaction likely do not play a significant role in the measured reaction kinetics which implies these secondary H atom reactions are highly selective.

Characterizing the Structure and Photocycle of PR 2D Crystals with CD and FTIR Spectroscopy†

We present here a study on proteorhodopsin (PR) 2D crystals with analytical ultracentrifugation, circular dichroism and Fourier transform infrared (FTIR) spectroscopy. The aim of our experiments was to test the activity of 2D crystal sample preparations and to gain further insight in PR structure, stability and function with these techniques. Our results demonstrate higher stability compared to detergent-solubilized or reconstituted samples. For different pH values, low pH 2D crystals tend to form bigger aggregates and are less stable than at basic pH. The pH 9 sample shows a sharp phase transition during heat denaturation and there is also evidence for protein-protein interaction due to the close proximity of the proteins in the 2D crystals. In the FTIR measurements at cryogenic temperatures (77 K), we characterized the first step in the PR photocycle. At pH 9, the K intermediate could be observed and the samples showed no orientation effects. At pH 5, we could trap the K/L intermediate, characterized by its negative IR signal at 1741 cm(-1). In rapid-scan FTIR experiments, we could also identify the M intermediate of the photocycle at basic pH. We conclude that the PR 2D crystals exhibit a fully functional photocycle and are therefore well suited for further studies on the proton transport mechanism of PR.

An ATR-FTIR spectroscopic approach for measuring rapid kinetics at the mineral/water interface

This study presents a methodology for studying rapid kinetic reactions for IR active compounds. In soils, sediments, and groundwater systems a rapid initial chemical reaction can comprise a substantial portion of the total reaction process at the mineral/water interface. Rapid-scan attenuated total reflectance (ATR) Fourier transform infrared (FTIR) spectroscopy is presented here as a new method for collecting rapid in situ kinetic data. As an example of its application, the initial oxidation of arsenite (As III) via Mn-oxides is examined. Using a rapid-scan technique, IR spectra were collected with a time resolution of up to 2.55 s (24 scans, 8 cm −1 resolution). Through observation and analysis of IR bands corresponding to arsenate (As V), rapid chemically-controlled As III oxidation is observed (initial pH 6–9) with 50% of the reaction occurring within the first one min. The oxidation of As III is followed by rapid binding of As V to HMO, at least in part, through surface bound Mn II. The experimental data indicate that rapid-scan FTIR is an effective technique for acquisition of kinetic data, providing molecular scale information for rapid reactions at the solid/liquid interface.

Thermochemical Properties, Thermal Behavior and Decomposition Mechanism of 1,1‐Diamino‐2,2‐dinitroethylene (DADE)

AbstractThe constant‐volume combustion energy, ΔcU (DADE, s, 298.15 K), the thermal behavior, and kinetics and mechanism of the exothermic decomposition reaction of 1,1‐diamino‐2,2‐dinitroethylene (DADE) have been investigated by a precise rotating bomb calorimeter, TG‐DTG, DSC, rapid‐scan fourier transform infrared (RSFT‐IR) spectroscopy and T‐jump/FTIR, respectively. The value of ΔcH⊖m (DADE, s, 298.15 K) was determined as (−8518.09± 4.59) J·g−1. Its standard enthalpy of combustion, ΔcU (DADE, s, 298.15 K), and standard enthalpy of formation, ΔfH⊖m (DADE, s, 298.15 K) were calculated to be (−1254.00±0.68) and (−103.98±0.73) kJ·mol−1, respectively. The kinetic parameters (the apparent activation energy Ea and pre‐exponential factor A) of the first exothermic decomposition reaction in a temperature‐programmed mode obtained by Kissinger′s method and Ozawa′s method, were Ek=344.35 kJ·mol−1, Ak=1034.50 s−1 and Eo=335.32 kJ·mol−1, respectively. The critical temperatures of thermal explosion of DADE were 206.98 and 207.08 °C by different methods. Information was obtained on its thermolysis detected by RSFT‐IR and T‐jump/FTIR.

Rapid-scan Fourier transform infrared spectroscopy shows coupling of GLu-L212 protonation and electron transfer to Q B in Rhodobacter sphaeroides reaction centers

Rapid-scan Fourier transform infrared (FTIR) difference spectroscopy was used to investigate the electron transfer reaction Q A −Q B→Q AQ B − ( k AB (1)) in mutant reaction centers of Rhodobacter sphaeroides, where Asp-L210 and/or Asp-M17 have been replaced with Asn. Mutation of both residues decreases drastically k AB (1), attributed to slow proton transfer to Glu-L212, which becomes rate limiting for electron transfer to Q B [M.L. Paddock et al., Biochemistry 40 (2001) 6893]. In the double mutant, the FTIR difference spectrum recorded during the time window 4–29 ms following a flash showed peaks at 1670 (−), 1601 (−) and 1467 (+) cm −1, characteristic of Q A reduction. The time evolution of the spectra shows reoxidation of Q A − and concomitant reduction of Q B with a kinetics of about 40 ms. In native reaction centers and in both single mutants, formation of Q B − occurs much faster than in the double mutant. Within the time resolution of the technique, protonation of Glu-L212, as characterized by an absorption increase at 1728 cm −1 [E. Nabedryk et al., Biochemistry 34 (1995) 14722], was found to proceed with the same kinetics as reduction of Q B in all samples. These rapid-scan FTIR results support the model of proton uptake being rate limiting for the first electron transfer from Q A − to Q B and the identification of Glu-L212 as the main proton acceptor in the state Q AQ B −.

Noise Sources in Step-Scan FT-IR Spectrometry

Step-scan Fourier transform infrared (FT-IR) spectrometry has been accepted as a useful tool for obtaining vibrational spectra of a variety of time-dependent systems. Unfortunately, a significant signal-to-noise ratio (SNR) disadvantage has been associated with the step-scan mode of data collection relative to the same data collection time with conventional rapid-scan FT-IR spectrometry. The key difference between the two methods is the average mirror velocity, which alters the dynamic range of the detector signal, as well as the frequencies of its components. The SNR disadvantage is shown to be related to low-frequency multiplicative fluctuations, caused in part by temperature variations, which convolve noise with measured spectra. Refractive index variations of air or purge gas in the paths of the infrared- and reference-laser radiation can be a particularly serious temperature-induced problem. The various noise sources are described, and experiments confirming that some are related to temperature variations are reported.

Applications of Time-Resolved Step-Scan and Rapid-Scan FT-IR Spectroscopy: Dynamics from Ten Seconds to Ten Nanoseconds

The step-scan technique in Fourier transform infrared (FT-IR) spectroscopy is employed in new applications of time resolved spectroscopy (TRS). Results are demonstrated on time-resolved laser emissions and photolytically generated chemical reactions using both emission and absorption modes. New achievements in FT-IR temporal resolution are demonstrated, as well as the complementary nature of step-scan and rapid-scan time-resolved spectroscopy.

Fourier Transform Infrared Microscopy—a Tool for Speciation of Impactor-Sampled Single Particles or Particle Clusters

With the recent advent of Fourier transform infrared (FTIR) microscopy, a powerful new tool has been added to the methods available for integrated dust analysis (IDA). While the intrinsic molecular specificity of infrared spectroscopy was applied in the 1970s with the IDA strategy of impactor-sampled aerosol analysis, at that time only average information from at least two impactor spots (1–10 μg range) could be obtained after sample preparation by grinding with KBr. This preparation step for heavily loaded samples, which may induce secondary reactions within the particles and destroy part of the reactive (acidic) species originally present, is no longer required with the new technique. FTIR microscopy allows the direct observation of isolated particles (>5 μm diameter) or particle clusters (<5 μm diameter) impacted on KBr or ZnSe (by the transmission technique) after a typical sampling duration of 5 minutes in an urban environment. This technique not only shows significant superiority in the quality of the spectra obtained but also is the key to preservation of original particle acidity. By this technique inhomogeneities within one impactor spot could be shown clearly (compound mapping). Several source, city, and background aerosols have been analyzed in the micro domain for the first time for NH4 + compounds, nitrates, sulfates, silicates, carbonates, and organics (aliphatic esters and aromatics). Acidic sulfates have been found in garage samples but not in city aerosols so far. By comparing samples at room temperature and heated up to 150°C, further information on the NH4 + compounds and the volatility of the species could be obtained. The adaptation of millisecond-time resolution of the new rapid-scan FTIR spectrometer in our laboratory for molecular specific thermal analysis research (MOSTAR) has been started, extending the capabilities of previous work.