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.

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.

Functionally relevant femtosecond to picosecond dynamics in enzyme active sites can be difficult to measure because of a lack of spectroscopic probes that can be located in the active site without altering the behavior of the enzyme. We have developed a new NAD+ analog 3-Picolyl Azide Adenine Dinucleotide (PAAD+), which has the potential to be a general spectroscopic probe for NAD-dependent enzymes. This analog is stable and binds in the active site of a typical NAD-dependent enzyme formate dehydrogenase (FDH) with characteristics similar to those of natural NAD+. It has an isolated infrared transition with high molar absorptivity that makes it suitable for observing enzyme dynamics using 2D IR spectroscopy. 2D IR experiments show that in aqueous solution, the analog undergoes complete spectral diffusion within hundreds of femtoseconds consistent with the water hydrogen bonding dynamics that would be expected. When bound to FDH in a binary complex, it shows picosecond fluctuations and a large static offset, consistent with previous studies of the binary complexes of this enzyme. These results show that PAAD+ is an excellent probe of local dynamics and that it should be a general tool for probing the dynamics of a wide range of NAD-dependent enzymes.

We report transient grating and 2D IR spectra of the C–D stretching vibration of deuterated formic acid dimer. The C–D stretching transition is perturbed by an accidental Fermi resonance interaction that gives rise to a second transition. The transient grating results show that the population lifetime of these states, which are in rapid equilibrium, is 11 ps. 2D IR spectroscopy reveals the energies of the eigenstates in the regions of one quantum and two quanta of C–D stretching excitation. Using these eigenstate energies, we construct a simplified model for the zeroth-order states that we then use to simulate the 2D IR spectrum. The results of this simulation suggest that the model captures the essential features of the vibrational spectroscopy in the region of the C–D stretching transition and compares well with previous gas-phase spectroscopy of the C–D stretch of deuterated formic acid dimer.

Enzyme active-site dynamics at femtosecond to picosecond time scales are of great biochemical importance, but remain relatively unexplored due to the lack of appropriate analytical methods. Two-dimensional infrared (2D IR) spectroscopy is one of the few methods that can examine chemical biological motions at this time scale, but all the IR probes used so far were specific to a few unique enzymes. The lack of IR probes of broader specificity is a major limitation to further 2D IR studies of enzyme dynamics. Here we describe the synthesis of a general IR probe for nicotinamide-dependent enzymes. This azido analog of the ubiquitous cofactor nicotinamide adenine dinucleotide is found to be stable and bind to several dehydrogenases with dissociation constants similar to that for the native cofactor. The infrared absorption spectra of this probe bound to several enzymes indicate that it has significant potential as a 2D IR probe to investigate femtosecond dynamics of nicotinamide-dependent enzymes.

The potential for femtosecond to picosecond time-scale motions to influence the rate of the intrinsic chemical step in enzyme-catalyzed reactions is a source of significant controversy. Among the central challenges in resolving this controversy is the difficulty of experimentally characterizing thermally activated motions at this time scale in functionally relevant enzyme complexes. We report a series of measurements to address this problem using two-dimensional infrared spectroscopy to characterize the time scales of active-site motions in complexes of formate dehydrogenase with the transition-state-analog inhibitor azide (). We observe that the frequency–frequency time correlation functions (FFCF) for the ternary complexes with NAD+ and NADH decay completely with slow time constants of 3.2 ps and 4.6 ps, respectively. This result suggests that in the vicinity of the transition state, the active-site enzyme structure samples a narrow and relatively rigid conformational distribution indicating that the transition-state structure is well organized for the reaction. In contrast, for the binary complex, we observe a significant static contribution to the FFCF similar to what is seen in other enzymes, indicating the presence of the slow motions that occur on time scales longer than our measurement window.

The spectral position of C−D stretching absorptions in the so-called “transparent window” of protein absorption (1800−2300 cm−1) makes them well suited as probes of protein dynamics with high temporal and structural resolution. We have previously incorporated single deuterated amino acids into proteins to site-selectively follow protein folding and ligand binding by steady-state FT IR spectroscopy. Ultimately, our goal is to use C−D bonds as probes in time-resolved IR spectroscopy to study dynamics and intramolecular vibrational energy redistribution (IVR) in proteins. As a step toward this goal, we now present the first time-resolved experiments characterizing the population and dephasing dynamics of selectively excited C−D bonds in a deuterated amino acid. Three differently deuterated, Boc-protected leucines were selected to systematically alter the number of additional C−D bonds that may mediate IVR out of the initially populated bright C−D stretching mode. Three-pulse photon echo experiments show that the steady-state C−D absorption linewidths are broadened by both homogeneous and inhomogeneous effects, and transient grating experiments reveal that IVR occurs on a subpicosecond time scale and is nonstatistical. The results have important implications for the interpretation of steady-state C−D spectra and demonstrate the potential utility of C−D bonds as probes of dynamics and IVR within a protein.

We present three-pulse vibrational echo measurements of azide ion bound to the active site Zn of human carbonic anhydrase II (HCA II) and of two separate active-site mutants Thr199 → Ala () and Leu198 → Phe (). Because structural motions of the protein active site influence the frequency of bound ligands, the differences in the time scales of the frequency-frequency correlation functions (FFCFs) obtained from global fits to each set of data allow us to make inferences about the time scales of the active site dynamics of HCA II. Surprisingly, the deletion of a potential electrostatic interaction in results in very little change in the FFCF, but the insertion of the bulky phenylalanine ring in causes much faster dynamics. We conclude that the fast, sub-picosecond time scale in the correlation function is attributable to hydrogen bond dynamics, and the slow, apparently static contribution is due to the conformational flexibility of Zn-bound azide in the active site.

We report transient grating and 2D IR spectra of the C–D stretching vibration of deuterated formic acid dimer. The C–D stretching transition is perturbed by an accidental Fermi resonance interaction that gives rise to a second transition. The transient grating results show that the population lifetime of these states, which are in rapid equilibrium, is 11 ps. 2D IR spectroscopy reveals the energies of the eigenstates in the regions of one quantum and two quanta of C–D stretching excitation. Using these eigenstate energies, we construct a simplified model for the zeroth-order states that we then use to simulate the 2D IR spectrum. The results of this simulation suggest that the model captures the essential features of the vibrational spectroscopy in the region of the C–D stretching transition and compares well with previous gas-phase spectroscopy of the C–D stretch of deuterated formic acid dimer.