Time-resolved Step-scan FTIR Spectroscopy Research Articles

Coupling Pulse Radiolysis with Nanosecond Time-Resolved Step-Scan Fourier Transform Infrared Spectroscopy: Broadband Mid-Infrared Detection of Radiolytically Generated Transients.

We describe the first implementation of broadband, nanosecond time-resolved step-scan Fourier transform infrared (S2-FT-IR) spectroscopy at a pulse radiolysis facility. This new technique allows the rapid acquisition of nano- to microsecond time-resolved infrared (TRIR) spectra of transient species generated by pulse radiolysis of liquid samples at a pulsed electron accelerator. Wide regions of the mid-infrared can be probed in a single experiment, which often takes < 20-30 min to complete. It is therefore a powerful method for rapidly locating the IR absorptions of short-lived, radiation-induced species in solution, and for directly monitoring their subsequent reactions. Time-resolved step-scan FT-IR detection for pulse radiolysis thus complements our existing narrowband quantum cascade laser-based pulse radiolysis-TRIR detection system, which is more suitable for acquiring single-shot kinetics and narrowband TRIR spectra on small-volume samples and in strongly absorbing solvents, such as water. We have demonstrated the application of time-resolved step-scan FT-IR spectroscopy to pulse radiolysis by probing the metal carbonyl and organic carbonyl vibrations of the one-electron-reduced forms of two Re-based CO2 reduction catalysts in acetonitrile solution. Transient IR absorption bands with amplitudes on the order of 1 × 10-3 are easily detected on the sub-microsecond timescale using electron pulses as short as 250 ns.

Time-resolved spectroscopic and electrophysiological data reveal insights in the gating mechanism of anion channelrhodopsin

Channelrhodopsins are widely used in optogenetic applications. High photocurrents and low current inactivation levels are desirable. Two parallel photocycles evoked by different retinal conformations cause cation-conducting channelrhodopsin-2 (CrChR2) inactivation: one with efficient conductivity; one with low conductivity. Given the longer half-life of the low conducting photocycle intermediates, which accumulate under continuous illumination, resulting in a largely reduced photocurrent. Here, we demonstrate that for channelrhodopsin-1 of the cryptophyte Guillardia theta (GtACR1), the highly conducting C = N-anti-photocycle was the sole operating cycle using time-resolved step-scan FTIR spectroscopy. The correlation between our spectroscopic measurements and previously reported electrophysiological data provides insights into molecular gating mechanisms and their role in the characteristic high photocurrents. The mechanistic importance of the central constriction site amino acid Glu-68 is also shown. We propose that canceling out the poorly conducting photocycle avoids the inactivation observed in CrChR2, and anticipate that this discovery will advance the development of optimized optogenetic tools.

Lipid-induced dynamics of photoreceptors monitored by time-resolved step-scan FTIR spectroscopy

The lipid environment plays a crucial role for the function of membrane proteins. We analyzed the influence of various lipid properties on the photoreceptors Bacteriorhodopsin (BR) and Proteorhodopsin (PR) by time-resolved step-scan FTIR spectroscopy. The photoreceptors were reconstituted into liposomes thereby providing an uniform biomimetic membrane, and the physical properties of the lipids were systematically varied. The lipid phase alters significantly the protonation dynamics of both, BR and PR. We also analyzed the conformational changes of the photoreceptors during the proton pumping mechanism and could demonstrate that a lipid-induced altered protonation dynamics is correlated to an altered conformational dynamics. Our results indicate that the fluidity of the membrane environment directly influences the structural-functional correlation.

Protein Dynamics of the Sensor Protein HemAT as Probed by Time-Resolved Step-Scan FTIR Spectroscopy

The heme-based aerotactic transducer (HemAT) is an oxygen-sensor protein consisting of a sensor and a signaling domain in the N- and C-terminal regions, respectively. Time-resolved step-scan FTIR spectroscopy was employed to characterize protein intermediate states obtained by photolysis of the carbon monoxide complexes of sensor-domain, full-length HemAT, and the Y70F (B-helix), L92A (E-helix), T95A (E-helix), and Y133F (G-helix) HemAT mutants. We assign the spectral components to discrete substructures, which originate from a helical structure that is solvated (1638 cm−1) and a native helix that is protected from solvation by interhelix tertiary interactions (1654 cm−1). The full-length protein is characterized by an additional amide I absorbance at 1661 cm−1, which is attributed to disordered structure suggesting that further protein conformational changes occur in the presence of the signaling domain in the full-length protein. The kinetics monitored within the amide I absorbance of the polypeptide backbone in the sensor domain exhibit two distinct relaxation phases (t1 = 24 and t2 = 694 μs), whereas that of the full-length protein exhibits monophasic behavior for all substructures in a time range of t = 1253–2090 μs. These observations can be instrumental in monitoring helix motion and the role of specific mutants in controlling the dynamics in the communication pathway from the sensor to the signaling domain. The kinetics observed for the amide I relaxation for the full-length protein indicate that the discrete substructures within full-length HemAT, unlike those of the sensor domain, relax independently.

Solid-State Step-Scan FTIR Spectroscopy of Binuclear Copper(I) Complexes.

The structure in the ground and excited electronic state of two binuclear CuI N-heterocyclic phosphine complexes that are promising for implementation in organic light-emitting diodes is investigated by a combination of the time-resolved step-scan FTIR technique and quantum chemical calculations at the DFT level of theory. In contrast to the usual application of step-scan FTIR spectroscopy in solution, the herein-presented analyses are performed in a solid phase, that is, the CuI complexes are embedded in a KBr matrix (KBr pellet). The application of solid-state time-resolved step-scan FTIR spectroscopy is of great importance for transition metal complexes, since their photophysical properties often change on moving from solid to dissolved samples. The efficient applicability of the solid-state step-scan technique in a KBr matrix is demonstrated on the chosen CuI reference systems on nano- and microsecond timescales with an excitation wavelength of 355 nm. By comparison with theoretical results, the structure of the complexes in the electronic ground and lowest-lying electronically excited state can be determined.

Probing the Role of the Heme Distal and Proximal Environment in Ligand Dynamics in the Signal Transducer Protein HemAT by Time-Resolved Step-Scan FTIR and Resonance Raman Spectroscopy.

HemAT is a heme-containing oxygen sensor protein that controls aerotaxis. Time-resolved step-scan FTIR studies were performed on the isolated sensor domain and full-length HemAT proteins as well as on the Y70F (B-helix), L92A (E-helix), T95A (E-helix), and Y133F (G-helix) mutants to elucidate the effect of the site-specific mutations on the ligand dynamics subsequent to CO photolysis. The mutations aimed to perturb H-bonding and electrostatic interactions near the heme Fe-bound gaseous ligand (CO) and the heme proximal environment. Rebinding of CO to the heme Fe is biphasic in the sensor domain and full-length HemAT as well as in the mutants, with the exception of the Y133F mutant protein. The monophasic rebinding of CO in Y133F suggests that in the absence of the H-bond between Y133 and the heme proximal H123 residue the ligand rebinding process is significantly affected. The role of the proximal environment is also probed by resonance Raman photodissociation experiments, in which the Fe-His mode of the photoproduct of sensor domain HemAT-CO is detected at a frequency higher than that of the deoxy form in the difference resonance Raman spectra. The role of the conformational changes of Y133 (G-helix) and the role of the distal L92 and T95 residues (E-helix) in regulating ligand dynamics in the heme pocket are discussed.

Ns-μs Time-Resolved Step-Scan FTIR of ba₃ Oxidoreductase from Thermus thermophilus: Protonic Connectivity of w941-w946-w927.

Time-resolved step-scan FTIR spectroscopy has been employed to probe the dynamics of the ba3 oxidoreductase from Thermus thermophilus in the ns-μs time range and in the pH/pD 6–9 range. The data revealed a pH/pD sensitivity of the D372 residue and of the ring-A propionate of heme a3. Based on the observed transient changes a model in which the protonic connectivity of w941-w946-927 to the D372 and the ring-A propionate of heme a3 is described.

Nanosecond ligand migration and functional protein relaxation in ba3 oxidoreductase: Structures of the B0, B1 and B2 intermediate states

Nanosecond time-resolved step-scan FTIR spectroscopy (nTRS 2 -FTIR) has been applied to literally probe the active site of the carbon monoxide (CO)-bound thermophilic ba3 heme-copper oxidoreductase as it executes its function. The nTRS 2 - snapshots of the photolysed heme a3 Fe-CO/CuB species captured a “transition state” whose side chains prevent the photolysed CO to enter the docking cavity. There are three sets of ba3 photoproduct bands of docked CO with different orientation exhibiting different kinetics. The trajectories of the “docked” CO at 2122, 2129 and 2137cm−1 is referred to in the literature as B2, B1 and B0 intermediate states, respectively. The present data provided direct evidence for the role of water in controlling ligand orientation in an intracavity protein environment.

Impact of the lipid environment on the protonation dynamics of bacteriorhodopsin studied with time-resolved step-scan FTIR spectroscopy

The membrane plays an important role in the structure and function of membrane proteins. We studied the influence of the lipid environment on the photocycle of the membrane protein bacteriorhodopsin (BR) with time-resolved step-scan FTIR spectroscopy. Proton transfer dynamics was monitored with mic rosecond time resolution for BR embedded in the native purple membrane as well as reconstituted into DOPC liposomes. We observed altered protonation dynamics of the Schiff base and the primary proton acceptor Asp85, revealing a faster rise as well as decay of the M state for BR surrounded by DOPC lipids. The purple membrane consists of a lipid composition that adapts better to the protein shape resulting in a stronger protein-membrane interaction as compared to the uniform DOPC lipid environment. Conformational dynamics and the correlated protonation dynamics are affected by the altered protein-membrane interaction explaining the faster photoreaction of BR in DOPC liposomes. Here we demonstrate the high sensitivity of the proton transfer dynamics to the lipid environment of BR.

Microsecond Deprotonation of Aspartic Acid and Response of the α/β Subdomain Precede C-Terminal Signaling in the Blue Light Sensor Plant Cryptochrome.

Plant cryptochromes are photosensory receptors that regulate various central aspects of plant growth and development. These receptors consist of a photolyase homology region (PHR) carrying the oxidized flavin adenine dinucleotide (FAD) cofactor, and a cryptochrome C-terminal extension (CCT), which is essential for signaling. Absorption of blue/UVA light leads to formation of the FAD neutral radical as the likely signaling state, and ultimately activates the CCT. Little is known about the signal transfer from the flavin to the CCT. Here, we investigated the photoreaction of the PHR by time-resolved step-scan FT-IR spectroscopy complemented by UV-vis spectroscopy. The first spectrum at 500 ns shows major contributions from the FAD anion radical, which is demonstrated to then be protonated by aspartic acid 396 to the neutral radical within 3.5 μs. The analysis revealed the existence of three intermediates characterized by changes in secondary structure. A marked loss of β-sheet structure is observed in the second intermediate evolving with a time constant of 500 μs. This change is accompanied by a conversion of a tyrosine residue, which is identified as the formation of a tyrosine radical in the UV-vis. The only β-sheet in the PHR is located within the α/β subdomain, ∼25 Å away from the flavin. This subdomain has been previously attributed a role as a putative antenna binding site, but is now suggested to have evolved to a component in the signaling of plant cryptochromes by mediating the interaction with the CCT.

Probing the ligand recognition and discrimination environment of the globin-coupled oxygen sensor protein YddV by FTIR and time-resolved step-scan FTIR spectroscopy.

YddV is a newly discovered signal transducer heme protein that recognizes O2 and CO. Structural differences in the ligand-bound heme complex in YddV reflect variations in catalytic regulation by O2 and CO. Time-resolved step-scan (TRS(2)) FTIR studies of the wild type and of the important in oxygen recognition and stability of the heme Fe(II)-O2 complex L65M, L65T, Y43A, Y43F and Y43W mutants were performed to determine the site-specific protein dynamics following carbon monoxide (CO) photodissociation. These mutations were designed to perturb the electrostatic field near the iron-bound gaseous ligand (CO) and also to allow us to investigate the communication pathway between the distal residues of the protein and heme. TRS(2)-FTIR spectra of YddV-heme-CO show that the heme propionates are in protonated and deprotonated states. Moreover, the rate of decay of the vibrations of amide I is on a time scale that coincides with the rate of rebinding of CO, which suggests that there is coupling between ligation dynamics in the distal heme environment and (i) relaxation of the protein backbone and (ii) the environment sensed by the heme propionates. The fast recombination rates in L65M, L65T and Y43W imply a significant role of L65 and Y43 in controlling the ligand dynamics. The implications of these results with respect to the role of the heme propionates and the charged or proton-donating residues in the distal pocket, which are crucial for stabilizing bound gaseous ligands, are discussed.

Structural Dynamics of the Signal Transducer Protein HemAT as Revealed by Time-Resolved Step Scan FTIR Spectroscopy

Aerotaxis is an important biological process for bacteria, as they utilize a signal transduction system to rapidly sense and respond to fluctuating O2 levels and retain their homeostasis. HemAT isa heme-based O2 sensor protein that acts as a signal transducer for aerotaxis in Bacillus subtilis, controlling the organisms' movement towards increasing O2 gradients. The molecular mechanisms for intra- and inter-molecular signal transduction processes are largely unknown. In our work, we have employed time-resolved step-scan Fourier transform infrared (TRS2-FTIR) spectroscopy to investigate the protein structural changes induced by ligand (CO) photodissociation and rebinding that are crucial for understanding the initial events of the intramolecular signal transduction mechanism in HemAT. We have studied the truncated sensor domain and full length HemAT-CO adducts as well as the Y70F, Y133F, L92A, and T95A mutants. Monitoring the kinetics of CO rebinding to the heme-Fe2+ (td=6μs-6ms) reveals biphasic kinetics for both the full length and truncated sensor domain HemAT. The TRS2-FTIR experiments have additionally revealed that Y70 predominantly controls the conformational changes that are induced to the protein matrix by CO photodissociation. Moreover, L92 appears to operate as the conformational gate in the migration pathway of photodissociated CO.

Ligand-Localized Triplet-State Photophysics in a Platinum(II) Terpyridyl Perylenediimideacetylide

The synthesis, electrochemistry, and photophysical behavior of a Pt(II) terpyridyl perylenediimide (PDI) acetylide (1) charge-transfer complex is reported. The title compound exhibits strong (ε ≈ 5 × 10(4) M(-1)cm(-1)) low-energy PDI acetylide-based π-π* absorption bands in the visible range extending to 600 nm, producing highly quenched singlet fluorescence (Φ = 0.014 ± 0.001, τ = 109 ps) with respect to a nonmetalated PDI model chromophore. Nanosecond transient absorption spectroscopy revealed the presence of a long excited-state lifetime (372 ns in 2-methyltetrahydrofuran) with transient features consistent with the PDI-acetylide triplet state, ascertained by direct comparison to a model Pt(II) PDI-acetylide complex lacking low-energy charge-transfer transitions. For the first time, time-resolved step-scan FT-IR spectroscopy was used to characterize the triplet excited state of the PDI-acetylide sensitized in the title compound and its associated model complex. The observed red shifts (∼30-50 cm(-1)) in the C═O and C≡C vibrations of the two Pt(II) complexes in the long-lived excited state are consistent with formation of the (3)PDI acetylide state and found to be in excellent agreement with the expected change in the relevant DFT-calculated IR frequencies in the nonmetalated PDI model chromophore (ground singlet state and lowest triplet excited state). Formation of the PDI triplet excited state in the title chromophore was also supported by sensitization of the singlet oxygen photoluminescence centered at ∼1275 nm in air-saturated acetonitrile solution, Φ((1)O(2)) = 0.52. In terms of light emission, only residual PDI-based red fluorescence could be detected and no corresponding PDI-based phosphorescence was observed in the visible or NIR region at 298 or 77 K in the Pt(II) terpyridyl perylenediimideacetylide.

Time-resolved step scan FTIR spectroscopy and DFT investigation on triplet formation in Peridinin-Chlorophyll-a-Protein from Amphidinium carterae at low temperature

We have used time-resolved step-scan FTIR spectroscopy to investigate the process of triplet formation in the peridinin–chlorophyll-a–protein (PCP) at 100 K. Results have led to the identification of possible marker bands for peridinin (Per) and 3Per. Band assignment has been carried out performing a DFT investigation on Per, 3Per and on model molecules. The main spectral feature is a couple of bands at 1746 (negative) and 1719 (positive) cm–1. The assignment of these two bands to the lactonic C═O of Per in (respectively) its fundamental S0 and triplet T1 state is based on: (1) comparison with FTIR spectra of isolated Per in literature; (2) good agreement with DFT calculations for Per and 3Per; (3) the downshift of the band upon triplet formation as predicted by DFT calculations in Per and similar lactonic systems; (4) the kinetic of band disappearance, in agreement with literature data. Another possible band for Per in its fundamental state has been identified at 1523 cm–1.

Time-resolved step scan FTIR spectroscopy and DFT investigation on triplet formation in peridinin–chlorophyll-a–protein from Amphidinium carterae at low temperature

We have used time-resolved step-scan FTIR spectroscopy to investigate the process of triplet formation in the peridinin–chlorophyll-a–protein (PCP) at 100 K. Results have led to the identification of possible marker bands for peridinin (Per) and3Per. Band assignment has been carried out performing a DFT investigation on Per,3Per and on model molecules. The main spectral feature is a couple of bands at 1746 (negative) and 1719 (positive) cm–1. The assignment of these two bands to the lactonic C═O of Per in (respectively) its fundamental S0 and tripletT1state is based on: (1) comparison with FTIR spectra of isolated Per in literature; (2) good agreement with DFT calculations for Per and3Per; (3) the downshift of the band upon triplet formation as predicted by DFT calculations in Per and similar lactonic systems; (4) the kinetic of band disappearance, in agreement with literature data. Another possible band for Per in its fundamental state has been identified at 1523 cm–1.The results are in overall agreement with a recent step-scan FTIR study on PCP at 298 K (Alexandre et al.,Biophys. J. 93 (2007), 2118–2128) even though some small discrepancies emerge, probably related to the different temperature at which experiments were carried out. Further DFT calculations have been performed to rationalise these spectral discrepancies. From a methodological point of view, the work demonstrates the potential of a combined step-scan FTIR/DFT approach in the study of photophysical processes in proteins. Furthermore, it has been shown that, when small differential IR signals are concerned, nagging photothermal effects can interfere and can possibly lead to misleading interpretations.

Direct Measurements of Rate Constants and Activation Volumes for the Binding of H2, D2, N2, C2H4, and CH3CN to W(CO)3(PCy3)2: Theoretical and Experimental Studies with Time-Resolved Step-Scan FTIR and UV−Vis Spectroscopy

Pulsed 355 nm laser excitation of toluene or hexane solutions containing W-L (W = mer,trans-W(CO)3(PCy3)2; PCy3 = tricyclohexylphosphine; L = H2, D2, N2, C2H4, or CH3CN) resulted in the photoejection of ligand L and the formation of W. A combination of nanosecond UV-vis flash photolysis and time-resolved step-scan FTIR (s2-FTIR) spectroscopy was used to spectroscopically characterize the photoproduct, W, and directly measure the rate constants for binding of the ligands L to W to reform W-L under pseudo-first-order conditions. From these data, equilibrium constants for the binding of L to W were estimated. The UV-vis flash photolysis experiments were also performed as a function of pressure in order to determine the activation volumes, DeltaV thermodynamic, for the reaction of W with L. Small activation volumes ranging from -7 to +3 cm3 mol(-1) were obtained, suggesting that despite the crowded W center an interchange mechanism between L and the agostic W...H-C interaction of one of the PCy3 ligands (or a weak interaction with a solvent molecule) at the W center takes place in the transition state. Density functional theory (DFT) calculations were performed at the B3LYP level of theory on W with/without the agostic C-H interaction of the PCy3 ligand and also on the series of model complexes, mer,trans-W(CO)3(PH3)2L (W'-L, where L = H2, N2, C2H4, CO, or n-hexane) in an effort to confirm the infrared spectroscopic assignment of the W-L complexes, to simulate and assign the electronic transitions in the UV-vis spectra, to determine the nature of the HOMO and LUMO of W-L, and to understand the agostic C-H interaction of the ligand vs solvent interaction. Our DFT calculations indicate an entropy effect that favors agostic W...H-C interaction over a solvent sigma C-H interaction by 8-10 kcal mol(-1).

Dynamics of CO in Mesoporous Silica Monitored by Time-Resolved Step-Scan and Rapid-Scan FT-IR Spectroscopy

Carbon monoxide molecules generated in the channels of mesoporous MCM-41 silica sieve from a precursor (diphenyl cyclopropenone) by photodissociation with a nanosecond laser pulse were monitored by time-resolved Fourier transform infrared (FTIR) spectroscopy using the step-scan and rapid-scan methods. A very broad absorption of CO is observed in the region 2200-2080 cm(-1) at room temperature that decays in a biphasic mode. Two-thirds of the band intensity decays on the hundreds of microsecond scale (lifetime 344 +/- 70 micros). The process represents the escape of the molecules through the mesopores into the surrounding gas phase, and a diffusion constant of 1.5 x 10(-9) m(2)/s is derived (assuming control by intra-MCM-41 particle diffusion). The broad profile of the absorption is attributed to contact of the random hopping CO with siloxane and silanol groups of the pore surface. Measurements using MCM-41 with the silanols partially capped by trimethyl silyl groups gave further insight into the nature of the IR band profile. These are the first observations on the diffusion behavior of carbon monoxide in a mesoporous material at room temperature. The residual carbon monoxide remains much longer in the pores and features distinct peaks at 2167 and 2105 cm(-1) characteristic for CO adsorbed on SiOH groups C end on and O end on, respectively. The bands decrease with time constants of 113 +/- 3 ms (2167 cm(-1)) and 155 +/- 15 ms (2105 cm(-1)) suggesting that CO in these sites is additionally trapped by surrounding diphenyl acetylene coproduct and/or precursor molecules.

Matrix merging arrangements for the study protein dynamics by time-resolved step-scan Fourier transform infrared spectroscopy and multivariate curve resolution

In this paper, we propose the construction of merging arrangements for combining the information from various runs as a powerful approach to improve the resolution. The bacteriorhodopsin (bR) photocycle has been chosen in this study as an example dealing with the protein dynamics monitored by means of time-resolved step-scan FT-IR spectroscopy. The possibilities of matrix merging are evaluated and results are compared with those from the analysis of individual and augmented matrices. As a conclusion, this strategy provides excellent results for the analysis of this type of time-resolved FT-IR data.

Consequences of counterion mutation in sensory rhodopsin II of Natronobacterium pharaonis for photoreaction and receptor activation: an FTIR study.

In many retinal proteins the proton transfer from the Schiff base to the counterion represents a functionally important step of the photoreaction. In the signaling state of sensory rhodopsin II from Natronobacterium pharaonis this transfer has already occurred, but in the counterion mutant Asp75Asn it is blocked during all steps of the photocycle. Therefore, the study of the molecular changes during the photoreaction of this mutant should provide a deeper understanding of the activation mechanism, and for this, we have applied time-resolved step-scan FTIR spectroscopy. The photoreaction is drastically altered; only red-shifted intermediates are formed with a chromophore strongly twisted around the 14-15 single bond. In addition, the photocycle is shortened by 2 orders of magnitude. Nevertheless, a transition involving only protein changes similar to that of the wild type is observed, which has been correlated with the formation of the signaling state. However, whereas in the wild type this transition occurs in the millisecond range, it is shortened to 200 micros in the mutant. The results are discussed with respect to the altered electrostatic interactions, role of proton transfer, the published 3D structure, and physiological activity.

Time-resolved step-scan FT-IR spectroscopy: focus on multivariate curve resolution.

The present paper describes the application of step-scan FT-IR spectroscopy in combination with chemometric analysis of the spectral data for the study of the photocycle of bacteriorhodopsin. The focus is on the performance of this instrumentation for time-resolved experiments. Three-dimensional data-spectra recorded over time-are studied using various factor analysis techniques, e.g., singular values decomposition, evolving factor analysis, and multivariate curve resolution based on alternating least squares. Transient intermediates formed in the time domain ranging from 1 micros to 6.6 ms are clearly detected through reliable pure time evolving profiles. At the same time, pure difference absorbance spectra are provided. As a result, valuable information about transitions and dynamics of the protein can be extracted. We conclude first that step-scan FT-IR spectroscopy is a useful technique for the direct study of difficult photochemical systems. Second, and this is the essential motivation of this paper, chemometrics provide a step forward in the description of the photointermediates.