Evaluation of p-(13C,15N-Cyano)phenylalanine as an Extended Time

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Evaluation of p-(13C,15N-cyano)phenylalanine as an extended timescale 2D IR probe of proteins Amanda L. Le Sueur, Sashary Ramos, Jonathan D. Ellefsen, Silas P. Cook, and Megan C. Thielges Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b04650 • Publication Date (Web): 13 Apr 2017 Downloaded from http://pubs.acs.org on April 14, 2017

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Analytical Chemistry

Evaluation of p-(13C,15N-cyano)phenylalanine as an Extended Timescale 2D IR Probe of Proteins Amanda L. Le Sueur, Sashary Ramos, Jonathan D. Ellefsen, Silas Cook, Megan C. Thielges* Department of Chemistry, Indiana University, 800 East Kirkwood Avenue, Bloomington, IN 47405, United States Two-dimensional infrared (2D IR) spectroscopy provides a powerful approach for the direct study of molecular dynamics with high spatial and temporal resolution. Its application for investigating specific locations in proteins requires the incorporation of IR probe groups with spectrally isolated absorptions to avoid the congestion inherent to protein spectra. This has motivated extensive efforts toward the development of new IR probes, but there remains a need for those that can extend the experimental time range, which is limited by their vibrational lifetimes. Toward this goal, isotopically labeled p-(13C15N-cyano)phenylalanine was synthesized, siteselectively incorporated into the protein plastocyanin, and evaluated for its potential as a 2D IR probe. The isotopic labeling increases the vibrational lifetime about two-fold, which results in larger signals at longer timescales. However, isotopic labeling simultaneously shifts the absorption to a spectral region with greater water absorbance, which results in greater heating-induced signals in the background that overlap those of the nitrile probe. The study demonstrates the use of a new 2D IR probe to measure the side chain dynamics in a protein and also illustrates the multiple factors to consider in development of 2D IR probes for studying proteins.

Multidimensional infrared (IR) spectroscopy has become a powerful approach for studying molecular structure and dynamics. Two-dimensional (2D) IR spectroscopy makes possible, for example, direct measurement of coupling between vibrational modes, frequency heterogeneity, and the temporal evolution of the frequencies.1-3 Application of 2D IR spectroscopy to a variety of both small molecule and complex systems has been demonstrated to investigate a wide range of questions, from understanding hydrogen bonding of water to the dynamics of protein folding.1,2,4-9 The characterization of proteins by IR spectroscopy however is challenged by the massive spectral congestion and complexity inherent to biological samples that hinder accurate discrimination and analysis of specific absorption bands. To alleviate this issue, significant effort has focused on development of with IR probe groups that have frequency-resolved absorptions. For example, 13C and/or 18O labeling of the amide backbone can be used to shift the absorptions of specific residues.10-12 Characterization of proteins at side chains is possible by incorporation of unnatural amino acids with functional groups that absorb within a relatively transparent frequency window of protein IR spectra (~1800-2500 cm-1).4,6,13,14 Here we evaluate the utility of isotopically labeled p-(13C,15N-cyano)phenylalanine (13C15NPhe) as an IR probe of proteins and its potential for increasing the experimental timescale for 2D IR studies. A variety of IR probe groups with suitable vibrational frequencies have been introduced as unnatural amino acids and demonstrated as 2D IR probes of proteins or peptides.6-10,14-17 As a probe of side chain dynamics, the cyano group is the smallest and hence least potentially perturbative, yet has a sufficiently strong transition dipole strength to be feasible as a probe for 2D IR studies of proteins. Of the available cyanosubstituted amino acids, the aromatic nitrile of p-

cyanophenylalanine (CNPhe) provides the most intense signal.18,19 Furthermore, CNPhe can reasonably substitute for Tyr or Phe residues and is easily site-selectively incorporated into proteins via a variety of biosynthetic approaches. Although the combination of 2D IR spectroscopy and siteselective incorporation of CNPhe or other IR probe groups provides a route for measurement of protein dynamics with high spatial and temporal resolution, the approach has a limited experimental timescale. 2D IR signals decay with the vibrational lifetime of the IR probe, and so the lifetime ultimately determines the duration of protein dynamics accessible with an IR probe. Unfortunately, vibrational lifetimes are often short. For instance, that for CNPhe is ~ 4 ps. This hinders the application of 2D IR spectroscopy to measure protein motions on longer timescales that also contribute to their function. This issue has motivated effort to IR probes with longer lifetimes, including thiocyano-, selenocyano-, and isocyanofunctionalized amino acids.1,20-23 The Fayer group recently pointed out that isotopic labeling of the nitrile leads to an increase in its vibrational lifetime.24 This effect was first noticed for the vibration corresponding to the naturally abundant 13C of the cyano group of the ionic liquid 4-cyano-4’pentylbiphenyl, which showed a doubled lifetime. A longer vibrational lifetime was likewise observed for 13C-labeled benzonitrile. The Gai group more recently reported pumpprobe spectroscopy studies that found a longer vibrational lifetime for the isotopically labeled amino acid 13C15NPhe than CNPhe in aqueous solution.25 To explore the potential for 13 15 C NPhe as a 2D IR probe for the measurement of dynamics in proteins, we report its incorporation into plastocyanin (Pc) and evaluation of its utility for characterizing the dynamics via 2D IR spectroscopy.

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Sample Preparation. Synthesis of 13C15NPhe was performed using palladium-catalyzed cyanation of BOC-protected 4bromophenylalanine as reported in literature,26 but with minor modifications. Complete details about synthesis and characterization by NMR and mass spectrometry are provided in Supporting Information. To express Pc, the leader sequence of the petE gene encoding Pc in Nostoc PCC7119 was removed to achieve cytoplasmic expression according to literature procedures.27 The amber codon suppressor approach was used to site-specifically incorporate CNPhe or 13C15NPhe at residue 36 of Pc by coexpression of the Pc gene containing a TAG at codon 36 with the tRNA/aminoacyl-tRNA synthetase pair from plasmid pULTRA-CNF in media supplemented with 1 mM CNPhe (or 13C15Phe).28 The plasmid construction, expression, purification and characterization of all proteins are detailed in the Supporting Information. For all spectroscopic measurements, solutions of the amino acids at were prepared at 50 mM in 200 mM sodium phosphate, pH 7. Samples of Pc, CNPhe36Pc, and 13C15NPhe36Pc were dialyzed into 1 mM sodium phosphate, pH 6.8. Samples for 13C15NPhe36Pc were at 5.3 mM (1.9 mM in 13C15N probe considering incomplete labeling), while those for CNPhe36Pc were at 2.6 or 8.5 mM (1.9 or 3.8 mM in CN probe). For FT IR spectroscopy, all samples were sealed between two CaF2 windows separated by a 38 µm or 76 µm Teflon spacer for linear or 2D IR spectroscopy, respectively. FT IR spectroscopy. FT IR spectroscopy was performed with an Agilent Cary 670 FT IR spectrometer using a liquid nitrogen cooled mercury-cadmium-telluride detector at 4 cm-1 resolution. IR absorption spectra of CNPhe36Pc (13C15NPhe36Pc) were generated using transmission spectra of unlabeled Pc and CNPhe36Pc (13C15NPhe36Pc) acquired under identical conditions. After subtraction of a polynomial fit to correcting for the background, the absorption bands were fit to a Gaussian function to determine the center frequency and full width at half maximum (FWHM) line width. Additional details are provided in the Supporting Information. Pump-probe and 2D IR spectroscopy. Pump-probe and 2D IR spectroscopy were performed as previously reported and detailed in Supporting Information.29,30 Experiments utilized ~170 fs pulses at 1 kHz repetition rate and centered at 2225 and 2142 cm-1 generated with a home-built optical parametric amplifier pumped by a Ti:sapphire oscillator/regenerative amplifier (Spectra Physics). During all experiments, the sample cell was rotated continuously. To exclusively measure population dynamics, the probe beam was set at magic angle (54.7°) relative to the horizontally polarized pump beam by placement of a waveplate/polarizer before and a polarizer after the sample.31 The transmitted probe beam was directed into a 0.32 m spectrograph (300 gr/mm grating) onto a 32-element MCT array (~1.5 cm-1/pixel resolution). The pump-induced change in the probe beam transmission was measured as a function of the pump-probe delay. Experiments were performed in triplicate. 2D IR spectroscopy was performed in the “conventional” geogetry,3,32 where the mid-IR beam is split into three, separately delayed excitation beams that are directed in a BOXCARS geometry and focused at the sample. To generate a single 2D

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IR spectrum, the time between the first two pulses was scanned (τ), while keeping the time between the second and third pulses, the waiting time (Tw), constant. The third-order signal emitted from the sample in the phase-matched direction was heterodyne-detected with a fourth local oscillator beam. The heterodyned signal was dispersed with a spectrograph onto a MCT array to generate the ωm (vertical) axis of the 2D spectrum. Interferograms along τ at each pixel of the array were Fourier transformed to produce the ωτ (horizontal) axis. To investigate time-dependent changes of the system, 2D spectra were obtained at increasing Tw. For each amino acid and protein sample at each Tw, 3-8 independent 2D spectra were acquired and analyzed; standard errors are reported in the figures and tables. Additional experimental details are provided in the Supporting Information. Data Analysis. The time-dependent change in the pumpinduced difference absorption at the 0-1 frequencies was fit to an exponential decay with an offset to determine the vibrational lifetimes of the CNPhe and 13C15NPhe. 2D IR spectra of the aqueous solutions of amino acids were corrected for phase errors guided by matching the 2D projection along ωm to the pump-probe spectra, as well as corrected for inner filter effects, as previously described.29,33 Because we are unable to acquire pump-probe spectra for the low concentration Pc samples, simulated pump-probe spectra were used instead. For phasing the 2D spectra of the Pc samples at long Tws (8-16 ps), where high overlap of the lower frequency regions occurred with features associated with solvent, only the higher frequency spectral region encompassing the 0-1 diagonal band was considered during initial phase correction. Slight adjustment was then made to optimize the appearance of the expected band at lower frequency along ωm associated with the 1-2 CN transition. The Tw-dependent changes in the lineshape of the 2D spectra were analyzed via the center-line-slope (CLS) method.34,35 The CLS decays in combination with the linear spectra were used to determine the frequency-frequency correlation functions (FFCFs), which connects the spectroscopic observables to the underlying dynamics of the protein system. The FFCF was modeled according to the Kubo model36 as ߜ(‫)ݐ‬ ‫∗ = ܨܥܨܨ‬ + ∆ଵଶ ݁ ି௧/ఛభ + ∆ଶ௦ ܶଶ + 2ܶଵ The latter two terms describe the dynamics among the inhomogeneous distribution of frequencies, where ∆ଵ is the frequency fluctuation amplitude sampled on timescale ߬ଵ , and the static term ∆s, reflects the frequency fluctuation amplitude sampled more slowly than the experimental time window. The ߜ(‫)ݐ‬/(ܶଶ∗ + 2ܶଵ ) term accounts for the homogeneous contribution to the FFCF. ܶଶ∗ = (∆ଶ ߬)ିଵ is the pure dephasing time that describes very fast fluctuations in the motionally narrowed limit where the frequency amplitude and time scale cannot be separated (∆߬ ≪ 1). The homogeneous dynamics lead to a Lorentzian contribution to the line shape, Γ ∗ = 1/ߨܶଶ∗ . ܶଵ is the vibrational lifetime, which was measured independently for the amino acid samples via IR pump-probe spectroscopy and determined to be 4.2 ± 0.3 ps and 10.7 ± 0.1 ps for the CNPhe and 13C15NPhe probes, respectively. Results and Discussion.

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Table 1. FFCF parameters, frequencies and linewidths for nitrile labeled amino acid and Pc samples.

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Frequency (cm-1) 13

C15NPhe36Pc

Linewidth (cm-1)

T2* (ps)

Γ*(cm-1)

∆1 (cm-1)

τ1 (ps)

∆2 (cm-1)

2154.6 ± 0.06

11.7 ± 0.5

1.5 ± 0.04

7.3 ± 0.2

2.8 ± 0.4

2.9 ± 0.9

1.7 ± 0.7

2155.9 ± 0.01

11.9 ± 0.1

1.2 ± 0.02

9.0 ± 0.2

2.5 ± 0.1

1.8 ± 0.2

1.4 ± 0.1

CNPhe36Pc

2235.2 ± 0.07

12.6 ± 0.3

1.4 ± 0.1

7.5 ± 0.3

3.0 ± 0.1

1.1 ± 0.2

2.2 ± 0.2

CNPhe

2236.7 ± 0.02

12.6 ± 0.1

1.4 ± 0.03

7.9 ± 0.2

2.7 ± 0.2

1.2 ± 0.2

1.5 ± 0.1

13

15

C NPhe

We prepared isotopically labeled 13C15NPhe using palladium catalyzed cyanation of BOC-protected 4bromophenylalanine.26 Linear FT IR spectra of the aqueous solutions of CNPhe and 13C15NPhe show absorption bands assigned to the nitrile stretches at 2236.7 and 2155.9 cm-1, respectively (Table 1). The vibrational lifetimes of the nitrile stretch for CNPhe and 13C15NPhe amino acids in aqueous solution were measured using pump-probe spectroscopy. The pump-induced changes in the transmission at the fundamental 0-1 frequencies were well fit to an exponential decay with an offset (Figure 1). The offset reflects a long-lived signal due to pump-induced heating of the sample (see discussion below). The fit to the data gives 4.2 ± 0.3 ps and 10.7 ± 0.1 ps for the vibrational lifetimes of CNPhe and 13C15NPhe, respectively. Both values generally agree with the observations of others,7,8,25 although the lifetime that we determined for 13 15 C NPhe is slightly longer than previously reported (7.9 ps).25 Thus, 13C15N-labeling roughly doubles the lifetime of the CNPhe IR probe.

Figure 2. Structural model of Pc (PDB 1TU2) showing location of introduced CNPhe36. The main chain is depicted in ribbon representation, and side chains are shown for CNPhe36 and Cu site of Pc.

duce the overall signal from the CN probe from that expected from the total protein concentration. The linear FT IR spectra acquired for CNPhe36Pc and 13 15 C NPhe36Pc show nitrile stretching absorptions at 2235.2 or 2154.6 cm-1, respectively, which are shifted to lower frequencies by 1.3-1.5 cm-1 from those of the amino acids in aqueous solution (Figure 3, Table 1). The variation in frequency indicates that the CN experiences different average environments in aqueous solution and the protein, with the shift to lower frequency upon incorporation into Pc suggesting that the CN is in a slightly less polar environment, less optimally hydrogen bonded, or less tightly packed when incorporated into the protein.37-40 However, relative to the frequency range observed experimentally for the absorption of CNPhe,7,8,18,37,38,41-43 the vibrational frequency of CNPhe36Pc

Figure 1. Pump-induced change in transmission at CN probe frequency for CNPhe (black points) and 13C15NPhe (green points) in aqueous solution. Lines show fits to exponential decays with a constant offset to account for sample heating.

We then substituted CNPhe or 13C15NPhe for Val36 of Pc (CNPhe36Pc and 13C15NPhe36Pc), a small, highly soluble blue copper protein that serves as an electron shuttle in photosynthesis (Figure 2). Selective labeling was accomplished using evolved tRNA/tRNA synthetase pairs for incorporation via the amber codon suppressor method.28 The integrity of the labeled proteins was assayed by mass spectrometry (MS) (Figure S1). Analysis of the MS peak amplitudes indicates ~40% efficiency for CNPhe or 13C15NPhe incorporation at position 36, with a Phe found otherwise. Phe incorporation does not introduce spectral interference in the IR region of interest, but does re-

Figure 3. FT IR spectra of aqueous solutions of the amino acids CNPhe (black) and 13C15NPhe (green), and of CNPhe36Pc (red) and 13C15NPhe36Pc (blue).

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is consistent with the CN probe’s incorporation at a solventexposed position of the protein. The dynamics reported by the CNPhe and 13C15NPhe probes for aqueous solutions of the free amino acids and the labeled protein were then measured by 2D IR spectroscopy (Figure 4). 2D IR spectroscopy provides correlation spectra that associate initial (ωτ) and final (ωm) frequencies of a vibrational probe measured with a time separation (Tw). The experiment involves application of three temporally controlled laser pulses to the sample and the heterodyned detection of an emitted third order signal. The temporal evolution of the signal between the first two pulses, the coherence time, τ, effectively labels the initial frequencies of the ensemble, giving the horizontal axis of the 2D spectrum. During the time between the second and third pulses, Tw, this information is stored in population states, consisting of excess excited state and depleted ground state population. After application of the third pulse, the frequency-resolved detection of the third order signal emitted from the sample interrogates the final frequencies, giving the vertical axis of the 2D spectrum. The dynamics of the probe during Tw among its microstates in a protein or solvent lead to the temporal evolution of its frequencies, which manifest as time-dependent changes in the lineshapes of the 2D bands. All 2D spectra for CNPhe and 13C15NPhe show bands on the diagonal at the fundamental 0-1 frequency (red contours, positive) along both the ωτ and ωm axes (Figure 5). The appearance of this band reflects excitation of the initial ground state population, followed by a combination of ground state bleaching and stimulated emission. A second strong band (blue con-

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tours, negative) occurs off the diagonal at ωτ of the 1-0 frequency and ωm of the 1-2 frequency, displaced along ωm by the anharmonicity (25 cm-1), which arises from excited state absorption. With increasing Tw, the 2D lineshapes show changes that reflect spectral diffusion due to motions among the probe’s environments. At the shortest Tw measured (0.25 or 0.35 ps, Figure 4), the spectra for all samples are elongated along the diagonal, reflecting that in each case much of the system is in the same state when probed after the third pulse as it initially was during τ, i.e. the spectra show high correlation between the two frequency axes. As Tw is increased, the spectra appear less elongated because a greater proportion of the ensemble change states within Tw, leading to different initial and final frequencies. Analysis of the Tw-dependent lineshapes was performed using the CLS method, which provides an approximation of the normalized FFCF (Figure 5).34 The timescales of the inhomogeneous contributions to the FFCFs were taken from exponential fits to the CLS decays. The CLS decay in combination with the linear absorption spectra were then fit to determine the full FFCFs, including the homogeneous contribution and absolute inhomogeneous frequency fluctuation amplitudes associated each timescale (Table 1).29 The FFCF reports how the inhomogeneous distribution of frequencies of the CN probe is sampled over time, and so reflects the dynamics of the CNPhe among its ensemble of microenvironments in aqueous solution or Pc. The CLS decays determined from line shape analysis of the 2D IR spectra in all cases were well modeled as the sum of three components: a homogeneous contribution, an exponential decay for ps dynamics, and a constant offset indicative of dynamics on timescales longer than experimentally accessible.

Figure 4. Tw-dependent 2D IR spectra of (a) 13C15NPhe, (b) CNPhe, (c) 13C15NPhe36Pc, (d) CNPhe36Pc. For ease in visualizing background signals panels (a) and (b) are plotted with 60 total contours, while panels (c) and (d) show 40 contours

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The CLS decays for the concentrated aqueous solutions of amino acids well overlap (Figure 5A). Likewise, the FFCFs determined for the amino acids are similar (Table 1), although small differences were obtained for τ1 and T2*, which is likely due to the uncertainty of the CLS method in deconvoluting the homogeneous and fast inhomogeneous contributions (as much as 15% potential error is estimated).34 For the amino acids, the largest component of the inhomogeneous distribution of frequencies (∆1 ൎ 2.6 cm-1) is sampled on a ~1.5 ps timescale, similar to previous reports.7,8 Our data also finds a smaller contribution (∆s ൎ 1.5 cm-1) from dynamics slower than the timescale of the experiment. The CLS decays for the Pc samples are also similar; however, the standard deviations for determination of the CLS values from the 2D spectra were greater for 13C15NPhe36Pc (Figure 5B). Correspondingly, the FFCF parameters obtained from combined fitting of the CLS decays with the linear spectra for 13C15NPhe36Pc showed greater uncertainty. The results for 13C15NPhe36Pc also show slightly greater τ1, which is unexpected. The inhomogeneous contributions to the FFCFs for the probes primarily reflect the fluctuating interaction of the side chain and its surrounding protein/solvent environment, which should not depend on isotopic labeling. We attribute the poorer data quality and fits for 13 15 C NPhe36Pc to the greater persistent background features in the 2D spectra, which both overlap the 2D bands of the CN as well as complicate phasing. For CNPhe36Pc, the FFCF shows similar timescales for τ1 and

Figure 5. CLS decays determined for (A) aqueous solutions of CNPhe (black) and 13C15NPhe (green) amino acids, and for (B) CNPhe36Pc (red) and 13C15NPhe36Pc (blue). The error bars reflect standard deviations obtained from analysis of three independent sets of 2D spectra. T2* as observed for the amino acids in solution. This suggests that that water and/or other protein groups to which the CNPhe is sensitive are highly mobile at the protein surface, similar to bulk water. The major difference between the FFCFs for the protein

and aqueous solution was the greater frequency fluctuation amplitude associated with the slowest timescale dynamics (∆s). This

observation implies that the CN probe experiences greater heterogeneity in slowly interconverting environments at the protein surface than in aqueous solution, which is likely due to the greater chemical diversity present within the context of a protein than in a pure water environment.

Figure 6. Tw-dependent decay of the diagonal 2D band amplitudes for CNPhe (black points) and 13C15NPhe (green points). Exponential decays fit to the data (black and green lines) give time constants of 4.4 and 9.3 ps, respectively. The results illustrate the utility of the CNPhe probe to measure the dynamics at their local environments in proteins. However, the FFCFs show long-time offsets, reflective of dynamics on longer timescales than measured. Unfortunately, the experimental timescale is limited by the decay of the 2D bands with increasing Tw, which occurs at a rate equal to the vibrational lifetime of the IR probe. The information about system’s frequencies during the first time interval τ is stored during Tw in depleted ground state and excess excited state populations. As vibrational relaxation during Tw abolishes the population difference, the intensity of the emitted third order signal and consequently the 2D band amplitude decrease. This is illustrated in Figure 6, where the normalized amplitudes of the 2D bands at the 0-1 frequencies for the aqueous solutions of CNPhe and 13 15 C NPhe are plotted as function of Tw. Fitting these curves to exponential decays yields time constants of 4.4 and 9.3 ps for the naturally abundant and isotopically labeled CNPhe, respectively. These correspond to the vibrational lifetimes measured via pump-probe spectroscopy. The longer vibrational lifetime measured for 13C15NPhe suggests that it should provide an extended-timescale probe of protein dynamics. However, we encountered another challenge – additional, obscuring features that appear in the 2D spectra, particularly in the lower ωm frequency regions (Figure 4). Similar features also appear for samples of pure water but not for samples of DMSO solvent (Supporting Information), so we attribute them to water modes of the aqueous samples. Despite that the CN absorptions occur in a relatively transparent frequency window of protein IR spectra, they sit atop a broad absorption centered at 2130 cm-1 associated with a bend-libration combination mode of water (Figure 7).44

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Figure 7. Normalized FT IR spectra of aqueous solution of 13 15 C NPhe and CNPhe showing absorptions at 2156 cm-1 and 2237 cm-1, respectively, atop the broad absorption from a bend-libration combination mode of water. To help illustrate these background features, 1D slices of the 2D spectra were extracted (Figure 8). The slices along the ωm axis at those ωτ frequencies not obscured by the CN bands (e.g. black lines A and B, top panels, Figure 8) show a complex band pattern consisting of a number of induced bleach, absorption, and/or shifted water bands (Figure 8 A and B). These appear at the shortest Tws (0.35 ps) and show little decay at the longest measured Tws (10 ps). The slow decay of these features indicates that they likely arise from heating of the sample, in accord with numerous previous studies of the excitation of the O-H stretch vibration of water that find intraand inter-molecular vibrational relaxation to occur on ultrafast timescales, while the dissipation of the excess thermal energy by density fluctuations occurs on a slower, ns timescale.44-48 An analogous band pattern has been previously reported by Bian, et al. in 2D spectra of the O-D stretch of D2O/H2O solutions of potassium selenocyanate.49 To assess whether the perturbed water combination modes are correlated to those of the CN probes, we inspected 1D slices (green lines C and D, top panels, Figure 8) along the pump ωτ axis at the ωm frequencies of the 0-1 CN transitions. These show low intensity water band bleach and/or induced absorptions at lower frequencies from the strong CN band. Their intensities do not scale with CN probe concentration, suggesting that they are not associated with the CN mode, but rather reflect correlation with water modes at frequencies that overlap the 0-1 CN band. Because the solvent band pattern persists as the CN bands decay, discernment of the latter are increasingly challenging for longer Tws. Moreover, the protein samples are necessarily at low concentration, so the solvent bands become commensurate or even greater than those from the CN probes. This is problematic because heat-induced solvent features directly overlap with the 0-1 band, and so consequently can directly affect lineshape analysis to determine the FFCFs. In addition, the high overlap of the solvent-associated cross bands at lower frequency and the band associated with the 1-2 transition of CN complicates the phase-correction during spectral processing, where standard practice involves comparison of the 1D projection along ωτ with the pump-probe spectrum. The CN absorption for 13C15NPhe (~2156 cm-1) occurs nearer than the band for CNPhe (~2237 cm-1) to the maximum of the absorbance of a water combination mode (~2130 cm-1), and so the background absorbance is greater for the l3C15NPhe probe

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(Figure 7). Because of this, we found the 2D spectra for the l3 15 C NPhe probe more challenging to phase correctly, as well as obtained greater uncertainty in CLS determination by 2D lineshape analysis. Although decreasing excitation energy reduces the water band pattern, it simultaneously decreases the emitted third-order signal from the CN probe. The phasing problems could be eliminated by alternately implementing 2D IR spectroscopy with the pump-probe geometry with pulse shaping, instead of the conventional BOXCARS geometry employed here for its high sensitivity. Even higher absorbance at the frequencies of these probes is found for D2O (Supporting Information), but it could be a better solvent with probes with lower frequency absorptions (< 2130 cm-1). Further effort is warranted to optimize detection of the CN while minimizing interference from water heating.

Figure 8. 1D slices of 2D spectra for 13C15NPhe (A,C) and for CNPhe (B,D). Shown are 1D slices along ωm at (A) ωτ = 2132 cm1 , (B) ωτ = 2210 cm-1. 1D slices along ωτ are shown at (C) ωm = 2156 cm-1 and (D) ωm = 2237 cm-1. Slices were taken with Tw of 0.5 ps (red), 1.0 ps (black), 2.0 ps (cyan), 4.0 ps (magenta), 6.0 ps (blue) and 10 ps (green). 2D spectra are provided in the top panel to illustrate the locations of the 1D slices.

Conclusion We evaluated isotopically labeled 13C15NPhe for its potential to serve as an extended timescale 2D IR probe for protein studies. 13C,15N labeling does increase the vibrational lifetime by about two-fold, suggesting it could extend the experimental time scale of 2D IR studies. However, the isotopic labeling also shifts the CN frequency to a more greatly absorbing region of an overlapping combination water mode, which generates an obscuring heat-induced band pattern that persists as the signals from the CN probes rapidly decay. Whereas the background features are relatively innocuous for highly concentrated solutions of the amino acids, they become problematic for protein samples that are necessarily at low concentration. Overall, the benefit of the increased lifetime of 13C15NPhe is mitigated by the higher background absorbance at the frequency of its absorption. On the other hand, the difference in fre-

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quencies of the CNPhe and 13C15NPhe makes possible their use as probes in combination, enabling simultaneous measurement of dynamics at two sites in a protein. In addition, their coupling to each other or to other incorporated probes could also lead to cross bands that would inform on their distance and orientation. Similar to previous studies employing backbone labeling,11,12 such information would provide insight into protein structure. Overall, this study demonstrates the utility of both probes for characterizing local sites in proteins, but illustrates the challenges and multiple factors to consider when developing better 2D IR probes for site-selective characterization of proteins. ASSOCIATED CONTENT

Supporting Information Experimental details of protein expression, spectroscopic measurement, and additional multidimensional spectra. The Supporting Information is available free of charge on the ACS Publications website.

AUTHOR INFORMATION Corresponding Author *Email: [email protected]

ACKNOWLEDGMENT A.L.L., S.R., and M.C.T. thank Indiana University and the Department of Energy (Grant DE-FOA-0000751) for funding.

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Evaluation of p-(13C,15N-Cyano)phenylalanine as an Extended Time

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