Adding a second AgGaS2 stage to Ti:sapphire/BBO/AgGaS2 setups increases mid-infrared power twofold.

We present a method for increasing the power of mid-infrared laser pulses generated by a conventional beta-barium borate (BBO) optical parametric amplifier (OPA) and AgGaS2 difference frequency generation (DFG) pumped by a Ti:sapphire amplifier. The method involves an additional stage of parametric amplification with a second AgGaS2 crystal pumped by selected outputs of the conventional DFG stage. This method does not require additional pump power from the Ti:sapphire laser source and improves the overall photon conversion efficiency for generating mid-infrared light. It merely requires an additional AgGaS2 crystal and dichroic mirrors. Following difference frequency generation, the method reuses near-infrared light (∼1.9 µm), typically discarded, to pump the additional AgGaS2 stage and amplifies the mid-infrared light twofold. We demonstrate and characterize the power, spectrum, duration, and noise of the mid-IR pulses before and after the second AgGaS2 stage. We observe small changes in center frequencies, bandwidth, and pulse duration for ∼150-fs pulses between 4 and 5 µm.

Increasing Pump-Probe Signal toward Asymptotic Limits.

Optimization of pump-probe signal requires a complete understanding of how signal scales with experimental factors. In simple systems, signal scales quadratically with molar absorptivity, and linearly with fluence, concentration, and path length. In practice, scaling factors weaken beyond certain thresholds (e.g., OD > 0.1) due to asymptotic limits related to optical density, fluence and path length. While computational models can accurately account for subdued scaling, quantitative explanations often appear quite technical in the literature. This Perspective aims to present a simpler understanding of the subject with concise formulas for estimating absolute magnitudes of signal under both ordinary and asymptotic scaling conditions. This formulation may be more appealing for spectroscopists seeking rough estimates of signal or relative comparisons. We identify scaling dependencies of signal with respect to experimental parameters and discuss applications for improving signal under broad conditions. We also review other signal enhancement methods, such as local-oscillator attenuation and plasmonic enhancement, and discuss respective benefits and challenges regarding asymptotic limits that signal cannot exceed.

Evolution of Optimized Hydride Transfer Reaction and Overall Enzyme Turnover in Human Dihydrofolate Reductase.

Evolution of dihydrofolate reductase (DHFR) has been studied using the enzyme from Escherichia coli DHFR (ecDHFR) as a model, but less studies have used the enzyme from Homo sapiens DHFR (hsDHFR). Each enzyme maintains a short and narrow distribution of hydride donor-acceptor distances (DAD) at the tunneling ready state (TRS). Evolution of the enzyme was previously studied in ecDHFR where three key sites were identified as important to the catalyzed reaction. The corresponding sites in hsDHFR are F28, 62-PEKN, and 26-PPLR. Each of these sites was studied here through the creation of mutant variants of the enzyme and measurements of the temperature dependence of the intrinsic kinetic isotope effects (KIEs) on the reaction. F28 is mutated first to M (F28M) and then to the L of the bacterial enzyme (F28L). The KIEs of the F28M variant are larger and more temperature-dependent than wild-type (WT), suggesting a broader and longer average DAD at the TRS. To more fully mimic ecDHFR, we also study a triple mutant of the human enzyme (F32L-PP26N-PEKN62G). Remarkably, the intrinsic KIEs, while larger in magnitude, are temperature-independent like the WT enzymes. We also construct deletion mutations of hsDHFR removing both the 62-PEKN and 26-PPLR sequences. The results mirror those described previously for insertion mutants of ecDHFR. Taken together, these results suggest a balancing act during DHFR evolution between achieving an optimal TRS for hydride transfer and preventing product inhibition arising from the different intercellular pools of NADPH and NADP+ in prokaryotic and eukaryotic cells.

Least-Squares Fitting of Multidimensional Spectra to Kubo Line-Shape Models.

We report a comprehensive study of the efficacy of least-squares fitting of multidimensional spectra to generalized Kubo line-shape models and introduce a novel least-squares fitting metric, termed the scale invariant gradient norm (SIGN), that enables a highly reliable and versatile algorithm. The precision of dephasing parameters is between 8× and 50× better for nonlinear model fitting compared to that for the centerline-slope (CLS) method, which effectively increases data acquisition efficiency by 1-2 orders of magnitude. Whereas the CLS method requires sequential fitting of both the nonlinear and linear spectra, our model fitting algorithm only requires nonlinear spectra but accurately predicts the linear spectrum. We show an experimental example in which the CLS time constants differ by 60% for independent measurements of the same system, while the Kubo time constants differ by only 10% for model fitting. This suggests that model fitting is a far more robust method of measuring spectral diffusion than the CLS method, which is more susceptible to structured residual signals that are not removable by pure solvent subtraction. Statistical analysis of the CLS method reveals a fundamental oversight in accounting for the propagation of uncertainty by Kubo time constants in the process of fitting to the linear absorption spectrum. A standalone desktop app and source code for the least-squares fitting algorithm are freely available, with example line-shape models and data. We have written the MATLAB source code in a generic framework where users may supply custom line-shape models. Using this application, a standard desktop fits a 12-parameter generalized Kubo model to a 106 data-point spectrum in a few minutes.

Selectivity for water isotopologues within metal organic nanotubes.

Through a combination of many analytical approaches, we show that a metal organic nanotube (UMON) displays selectivity for H2O over all types of heavy water (D2O, HDO, HTO). Water adsorption experiments combined with vibrational and radiochemical analyses reveal significant differences in uptake and suggest that surface adsorption processes may be a key driver in water uptake for this material.

Low-Frequency Protein Motions Coupled to Catalytic Sites.

This review examines low-frequency vibrational modes of proteins and their coupling to enzyme catalytic sites. That protein motions are critical to enzyme function is clear, but the kinds of motions present in proteins and how they are involved in function remain unclear. Several models of enzyme-catalyzed reaction suggest that protein dynamics may be involved in the chemical step of the catalyzed reaction, but the evidence in support of such models is indirect. Spectroscopic studies of low-frequency protein vibrations consistently show that there are underdamped modes of the protein with frequencies in the tens of wavenumbers where overdamped behavior would be expected. Recent studies even show that such underdamped vibrations modulate enzyme active sites. These observations suggest that increasingly sophisticated spectroscopic methods will be able to unravel the link between low-frequency protein vibrations and enzyme function.

Edge-pixel referencing suppresses correlated baseline noise in heterodyned spectroscopies.

Referencing schemes are commonly used in heterodyned spectroscopies to mitigate correlated baseline noise arising from shot-to-shot fluctuations of the local oscillator. Although successful, these methods rely on careful pixel-to-pixel matching between the two spectrographs. A recent scheme introduced by Feng et al. [Opt. Express 27(15), 20323-20346 (2019)] employed a correlation matrix to allow free mapping between dissimilar spectrographs, leading to the first demonstration of floor noise limited detection on a multichannel array used in heterodyned spectroscopy. In addition to their primary results using a second reference spectrometer, Feng et al. briefly demonstrated the flexibility of their method by referencing to same-array pixels at the two spectral edges (i.e., edge-pixel referencing). We present a comprehensive study of this approach, which we term edge-pixel referencing, including optimization of the approach, assessment of the performance, and determination of the effects of background responses. We show that, within some limitations, the distortions due to background signals will not affect the 2D IR line shape or amplitude and can be mitigated by band narrowing of the pump beams. We also show that the performance of edge-pixel referencing is comparable to that of referencing to a second spectrometer in terms of noise suppression and that the line shapes and amplitudes of the spectral features are, within the measurement error, identical. Altogether, these results demonstrate that edge-pixel referencing is a powerful approach for noise suppression in heterodyned spectroscopies, which requires no new hardware and, so, can be implemented as a software solution for anyone performing heterodyned spectroscopy with multichannel array detectors already.

Isotopic Labeling of Formate Dehydrogenase Perturbs the Protein Dynamics.

Isotope substitution of enzymes has become a means of addressing the participation of protein motions in enzyme-catalyzed reactions. The idea is that only the enzyme mass will be altered and not the electrostatics, so that the protein dynamics are essentially the same but at lower frequencies because of the mass change. In this study, we variably label all carbon atoms in formate dehydrogenase (FDH) with 13C, all nitrogen atoms with 15N, and all nonexchangeable hydrogen atoms with deuterium and investigate the impact that isotopic substitution has on the dynamics at the active site by two-dimensional infrared spectroscopy and compare with the measurements of the temperature dependence of the intrinsic kinetic isotope effects (KIEs). We show that 15N labeling of FDH has the largest effect and makes the active site more heterogeneous, whereas the addition of nonexchangeable deuterium appears to have the opposite effect of 15N on active-site dynamics, resulting in a behavior similar to that of native FDH. Nevertheless, the temperature dependence of the KIEs shows a monotonic trend with protein mass that does not correspond with the changes in dynamics. These results suggest that isotope labeling has more than just a mass effect on enzyme dynamics and may influence electrostatics in ways that complicate the interpretation of the protein isotope effect.

Evolution Conserves the Network of Coupled Residues in Dihydrofolate Reductase.

Understanding protein motions and their role in enzymatic reactions is an important and timely topic in enzymology. Protein motions that are involved in the chemical step of catalysis are particularly intriguing but difficult to identify. A global network of coupled residues in Escherichia coli dihydrofolate reductase (E. coli DHFR), which assists in catalyzing the chemical step, has previously been demonstrated through quantum mechanical/molecular mechanical and molecular dynamics simulations as well as bioinformatic analyses. A few specific residues (M42, G121, F125, and I14) were shown to function synergistically with measurements of single-turnover rates and the temperature dependence of intrinsic kinetic isotope effects (KIEsint) of site-directed mutants. This study hypothesizes that the global network of residues involved in the chemical step is evolutionarily conserved and probes homologous residues of the potential global network in human DHFR through measurements of the temperature dependence of KIEsint and computer simulations based on the empirical valence bond method. We study mutants M53W and S145V. Both of these remote residues are homologous to network residues in E. coli DHFR. Non-additive isotope effects on activation energy are observed between M53 and S145, indicating their synergistic effect on the chemical step in human DHFR, which suggests that both of these residues are part of a network affecting the chemical step in enzyme catalysis. This finding supports the hypothesis that human and E. coli DHFR share similar networks, consistent with evolutionary preservation of such networks.

Optimized reconstructions of compressively sampled two-dimensional infrared spectra.

Compressive sampling has the potential to dramatically accelerate the pace of data collection in two-dimensional infrared (2D IR) spectroscopy. We have previously introduced the Generic Iteratively Reweighted Annihilating Filter (GIRAF) reconstruction algorithm to solve the reconstruction in 2D IR compressive sampling. Here, we report a thorough assessment of this method and comparison to our earlier efforts using the Total Variation (TV) algorithm. We show that the GIRAF algorithm has some distinct advantages over TV. Although it is no better or worse in terms of ameliorating the impacts of compressive sampling on the measured 2D IR line shape, we find that the nature of those effects is different for GIRAF than they were for TV. In addition to assessing the impacts on the line shape of a single oscillator, we also test the ability of the algorithm to reconstruct spectra that have transitions from more than one oscillator, such as the coupled carbonyl oscillators in rhodium dicarbonyl. Finally, and perhaps most importantly, we show that the GIRAF algorithm has a distinct denoising effect on the signal-to-noise ratio (SNR) of the 2D IR spectra that can increase the SNR by as much as 4× without any additional signal averaging and collecting fewer data points, which should further enhance the acceleration of data collection that can be achieved using compressive sampling and enable even more challenging experimental measurements.

Oscillatory Active-site Motions Correlate with Kinetic Isotope Effects in Formate Dehydrogenase.

Thermal motions of enzymes have been invoked to explain the temperature dependence of kinetic isotope effects (KIE) in enzyme-catalyzed hydride transfers. Formate dehydrogenase (FDH) from Candida boidinii exhibits a temperature independent KIE that becomes temperature dependent upon mutation of hydrophobic residues in the active site. Ternary complexes of FDH that mimic the transition state structure allow investigation of how these mutations influence active-site dynamics. A combination of X-ray crystallography, two-dimensional infrared (2D IR) spectroscopy, and molecular dynamic simulations characterize the structure and dynamics of the active site. FDH exhibits oscillatory frequency fluctuations on the picosecond timescale, and the amplitude of these fluctuations correlates with the temperature dependence of the KIE. Both the kinetic and dynamic phenomena can be reproduced computationally. These results provide experimental evidence for a connection between the temperature dependence of KIEs and motions of the active site in an enzyme-catalyzed reaction consistent with activated tunneling models of the hydride transfer reaction.

Evolutionary Effects on Bound Substrate p Ka in Dihydrofolate Reductase.

In the present study, we address the effect of active site structure and dynamics of different dihydrofolate reductase (DHFR) isoforms on the p Ka of the bound substrate 7,8-dihydrofolate, in an attempt to understand possible evolutionary trends. We apply a hybrid QM/MM free energy perturbation method to estimate the p Ka of the N5 position of the bound substrate. We observe a gradual increase in N5 basicity as we move from primitive to more evolved DHFR isoforms. Structural analysis of these isoforms reveals a gradual sequestering of water molecules from the active site in the more evolved enzymes, thereby modulating the local dielectric environment near the substrate. Furthermore, the present study reveals a clear correlation between active site hydration and the N5 p Ka of the substrate. We emphasize the role of the M20 loop in controlling the active site hydration level, via a preorganized active site with a more hydrophobic environment and reduced loop flexibility as evolution progresses from bacterial to the human enzyme.

Effect of Asp122 Mutation on the Hydride Transfer in E. coli DHFR Demonstrates the Goldilocks of Enzyme Flexibility.

Dihydrofolate reductase (DHFR) catalyzes the reduction of dihydrofolate (DHF) to tetrahydrofolate (THF) in the presence of NADPH. The key hydride transfer step in the reaction is facilitated by a combination of enzyme active site preorganization and correlated protein motions in the Michaelis-Menten (E:NADPH:DHF) complex. The present theoretical study employs mutagenesis to examine the relation between structural and functional properties of the enzyme. We mutate Asp122 in Escherichia coli DHFR, which is a conserved amino acid in the DHFR family. The consequent effect of the mutation on enzyme catalysis is examined from an energetic, structural and short-time dynamic perspective. Our investigations suggest that the structural and short-time dynamic perturbations caused by Asp122X mutations (X = Asn, Ser, Ala) are along the reaction coordinate and lower the rate of hydride transfer. Importantly, analysis of the correlated and principle component motions in the enzyme suggest that the mutation alters the coupled motions that are present in the wild-type enzyme. In the case of D122N and D122S, the mutations inhibit coupled motion, whereas in the case of D122A, the mutation enhances coupled motion, although all mutations result in similar rate reduction. These results emphasize a Goldilocks principle of enzyme flexibility, that is, enzymes should neither be too rigid nor too flexible.

Accelerating two-dimensional infrared spectroscopy while preserving lineshapes using GIRAF.

We introduce a computationally efficient structured low-rank algorithm for the reconstruction of two-dimensional infrared (2D IR) spectroscopic data from few measurements. The signal is modeled as a combination of exponential lineshapes that are annihilated by appropriately chosen filters. The annihilation relations result in a low-rank constraint on a Toeplitz matrix constructed from signal samples, which is exploited to recover the unknown signal samples. Quantitative and qualitative studies on simulated and experimental data demonstrate that the algorithm outperforms the discrete compressed sensing algorithm, both in uniform and non-uniform sampling settings.

Protein Mass Effects on Formate Dehydrogenase.

Isotopically labeled enzymes (denoted as "heavy" or "Born-Oppenheimer" enzymes) have been used to test the role of protein dynamics in catalysis. The original idea was that the protein's higher mass would reduce the frequency of its normal-modes without altering its electrostatics. Heavy enzymes have been used to test if the vibrations in the native enzyme are coupled to the chemistry it catalyzes, and different studies have resulted in ambiguous findings. Here the temperature-dependence of intrinsic kinetic isotope effects of the enzyme formate dehydrogenase is used to examine the distribution of H-donor to H-acceptor distance as a function of the protein's mass. The protein dynamics are altered in the heavy enzyme to diminish motions that determine the transition state sampling in the native enzyme, in accordance with a Born-Oppenheimer-like effect on bond activation. Findings of this work suggest components related to fast frequencies that can be explained by Born-Oppenheimer enzyme hypothesis (vibrational) and also slower time scale events that are non-Born-Oppenheimer in nature (electrostatic), based on evaluations of protein mass dependence of donor-acceptor distance and forward commitment to catalysis along with steady state and single turnover measurements. Together, the findings suggest that the mass modulation affected both local, fast, protein vibrations associated with the catalyzed chemistry and the protein's macromolecular electrostatics at slower time scales; that is, both Born-Oppenheimer and non-Born-Oppenheimer effects are observed. Comparison to previous studies leads to the conclusion that isotopic labeling of the protein may have different effects on different systems, however, making heavy enzyme studies a very exciting technique for exploring the dynamics link to catalysis in proteins.

Compressively Sampled Two-Dimensional Infrared Spectroscopy That Preserves Line Shape Information.

Two-dimensional infrared (2D IR) spectroscopy is a powerful tool to investigate molecular structures and dynamics on femtosecond to picosecond time scales and is applied to diverse systems. Current technologies allow for the acquisition of a single 2D IR spectrum in a few tens of milliseconds using a pulse shaper and an array detector, but demanding applications require spectra for many waiting times and involve considerable signal averaging, resulting in data acquisition times that can be many days or weeks of laboratory measurement time. Using compressive sampling, we show that we can reduce the time for collection of a 2D IR data set in a particularly demanding application from 8 to 2 days, a factor of 4×, without changing the apparatus and while accurately reproducing the line-shape information that is most relevant to this application. This result is a potent example of the potential of compressive sampling to enable challenging new applications of 2D IR.

Oscillatory Enzyme Dynamics Revealed by Two-Dimensional Infrared Spectroscopy.

Enzymes move on a variety of length and time scales. While much is known about large structural fluctuations that impact binding of the substrates and release of products, little is known about faster motions of enzymes and how these motions may influence enzyme-catalyzed reactions. This Letter reports frequency fluctuations of the azide anion bound to the active site of formate dehydrogenase measured via 2D IR spectroscopy. These measurements reveal an underdamped oscillatory component to the frequency-frequency correlation function when the azide is bound to the NAD(+) ternary complex. This oscillation disappears when the reduced cofactor is added, indicating that the oscillating contributions most likely come from the charged nicotinamide ring. These oscillatory motions may be relevant to donor-acceptor distance sampling of the catalyzed hydride transfer and therefore may give future insights into the dynamic behavior involved in enzyme catalysis.

Structural and Kinetic Studies of Formate Dehydrogenase from Candida boidinii.

The structure of formate dehydrogenase from Candida boidinii (CbFDH) is of both academic and practical interests. First, this enzyme represents a unique model system for studies on the role of protein dynamics in catalysis, but so far these studies have been limited by the availability of structural information. Second, CbFDH and its mutants can be used in various industrial applications (e.g., CO2 fixation or nicotinamide recycling systems), and the lack of structural information has been a limiting factor in commercial development. Here, we report the crystallization and structural determination of both holo- and apo-CbFDH. The free-energy barrier for the catalyzed reaction was computed and indicates that this structure indeed represents a catalytically competent form of the enzyme. Complementing kinetic examinations demonstrate that the recombinant CbFDH has a well-organized reactive state. Finally, a fortuitous observation has been made: the apoenzyme crystal was obtained under cocrystallization conditions with a saturating concentration of both the cofactor (NAD(+)) and inhibitor (azide), which has a nanomolar dissociation constant. It was found that the fraction of the apoenzyme present in the solution is less than 1.7 × 10(-7) (i.e., the solution is 99.9999% holoenzyme). This is an extreme case where the crystal structure represents an insignificant fraction of the enzyme in solution, and a mechanism rationalizing this phenomenon is presented.

Line shape analysis of two-dimensional infrared spectra.

Ultrafast two-dimensional infrared (2D IR) spectroscopy probes femtosecond to picosecond time scale dynamics ranging from solvation to protein motions. The frequency-frequency correlation function (FFCF) is the quantitative measure of the spectral diffusion that reports those dynamics and, within certain approximations, can be extracted directly from 2D IR line shapes. A variety of methods have been developed to extract the FFCF from 2D IR spectra, which, in principle, should give the same FFCF parameters, but the complexity of real experimental systems will affect the results of these analyses differently. Here, we compare five common analysis methods using both simulated and experimental 2D IR spectra to understand the effects of apodization, anharmonicity, phasing errors, and finite signal-to-noise ratios on the results of each of these analyses. Our results show that although all of the methods can, in principle, yield the FFCF under idealized circumstances, under more realistic experimental conditions they behave quite differently, and we find that the centerline slope analysis yields the best compromise between the effects we test and is most robust to the distortions that they cause.

2D IR spectroscopy using four-wave mixing, pulse shaping, and IR upconversion: a quantitative comparison.

Recent technological advances have led to major changes in the apparatuses used to collect 2D IR spectra. Pulse shaping offers several advantages including rapid data collection, inherent phase stability, and phase-cycling capabilities. Visible array detection via upconversion allows the use of visible detectors that are cheaper, faster, more sensitive, and less noisy than IR detectors. However, despite these advantages, many researchers are reluctant to implement these technologies. Here we present a quantitative study of the S/N of 2D IR spectra collected with a traditional four-wave mixing (FWM) apparatus, with a pulse shaping apparatus, and with visible detection via upconversion to address the question of whether weak chromophores at low concentrations are still accessible with such an apparatus. We find that the enhanced averaging capability of the pulse shaping apparatus enables the detection of small signals that would be challenging to measure even with the traditional FWM apparatus, and we demonstrate this ability on a sample of cyanylated dihydrofolate reductase.