Correction : Vibrational dynamics and solvatochromism of the label SCN in various solvents and hemoglobin by time dependent IR and 2 DIR spectroscopy

~ FOnsager 1⁄4 m0 ! 4pe0a 2 e 1 ð Þ n þ 2 3 2eþ n2 ð Þ In accordance with the previous literature, a was erroneously set equal to the volume V of the Onsager spherical cavity. Because a represents the Onsager spherical cavity radius, a = V 3/(4p) has to be used instead. This has recently been pointed out by Bagchi and coworkers and was mentioned before in the supplementary information of a previous publication. For MeSCN, the Onsager spherical cavity volume is VMeSCN = MW/(r NA) = 1.19 10 28 m and the cavity radius cubed becomes aMeSCN 3 = 2.8 10 29 m. Correspondingly, the estimated Onsager field FOnsager is a factor of 4p/3 too low in our work as well as in the previous work of others. In Fig. 8 and Fig. S5 of the ESI, the Onsager solvent field on the X-axis therefore has to be multiplied by 4p/3. The corrected figures are shown below. The slope of the line fitting the aprotic solvents in Fig. 8 and Fig. S5 (ESI) produces the Stark tuning rate, which accordingly was a factor of 4p/3 too high in the previous publication and now becomes |D~m| = 1.7(4) 10 9 cm /(V/m). Note that the references in the captions to Fig. 8 and Fig. S5 (ESI) refer to the references in the original publication.


Introduction
][3][4][5][6][7][8][9][10][11][12] These labels are incorporated at specific target sites and provide local information on a molecule's structure, polarity, hydrogen bonding and electric field.Ideally, these labels are small, do not perturb the protein fold and, more importantly, do not affect the protein's function.Suitable candidates should furthermore have large oscillator strengths, and exhibit isolated infrared absorption frequencies (also separated from solvent absorption), that facilitate their tracking, and sensitively respond to their surroundings.For 2D-IR spectroscopy, they should in particular have long vibrational lifetimes, as the lifetime determines the time interval during which the oscillator frequency can be tracked and report on the dynamics of the local environment.A large anharmonicity of the label is beneficial to minimize cancelation between the 0→1 and 1→2 transition.The label we focus on is thiocyanate (SCN) which can be incorporated into proteins by post-translational chemical modification of a cysteine residue that can be placed by sitedirected mutagenesis.We insert SCN in hemoglobin by cysteine cyanylation and show that it possesses the aforementioned desired properties.We show steady-state FTIR and time-dependent 2D-IR data which indicate the presence of a nonhomogenous structural distribution in the surroundings of the S 13 C 15 N label.We analyze the central wavenumber, lineshape, anharmonicity and the lifetimes of the thiocyanate vibration in different environments.The results are interpreted in terms of 50 local electric fields and solvent properties (polar vs apolar and protic vs aprotic).We demonstrate that the use of the SCN label in 2D-IR spectroscopy of a protein gives rich in-situ dynamic information.This makes it promising for applications in proteins and other systems which do not possess intrinsic infrared 55 structural markers and for which local information is not accessible otherwise.We have chosen bovine hemoglobin (Hb) to incorporate the SCN label and determine its (ultrafast) infrared properties.Hb is a transporter protein that can bind a small ligand to a covalently-60 bound heme group (see Figure 1), and is found in all vertebrates.Hb consists of four subunits (two α and two β monomers ) that are non-covalently associated. 13Bovine Hb contains one heme per subunit, only one cystein in each β subunit 14 and no cysteine in the α subunits.Here we substitute the cysteine's SH for SCN 65 via cyanylation that is similar to the one performed in refs 15,16 .Because of significant H 2 O absorption in the region where the cyanide stretch frequency appears, the 2D-IR spectra are collected on the isotope S 13 C 15 N-labeled protein in deuterated aqueous buffer.The protein preparation protocol described here Panel A shows the secondary structure of Hb through its transparent surface (adapted from pdb-file 2QSS 17 , carbon monoxide not shown).For clarity the two α-and β-subunits are colored green and grey, respectively.Each subunit contains one 5 heme (blue), but only the β-monomers contain one cysteine each.B Zoom on one of the two cysteines that are to be cyanylated.C Structure of MeSCN. 18The images are generated by Chimera. 19n be easily used to built in S 13 C 15 N instead of S 12 C 14 N, 10 highlighting the simplicity of the described cyanylation procedure.
Ignited by the current large interest, there are already several examples of proteins with inserted thiocyanate labels: into the active site of ketosteroid isomerase (KSI), 20 in a bacterial reaction 15 center, 15 modification of S-peptide bound to the ribonuclease Sprotein (RNase S), 15 human aldose reductase (hALR2), 15

Methylthiocyanate Samples
Methyl thiocyanate and all other solvents and chemicals were purchased from Sigma Aldrich and used without further purification unless stated otherwise.D 2 O was purchased from Deutero.For the measurements in buffer, 100 mM sodium 35 phosphate buffer with pH 7.9 was used.Other used solvents are tetrahydrofuran (THF), tetrachloroethylene (PCE) and 2,2,2trifluoroethanol (TFE).For the measurements in dimethyl sulfoxide (DMSO) the purchased solvent was dried over night using molecular sieve. 40

Protein sample preparation
In order to incorporate the SCN label into a protein, bovine hemoglobin (Sigma Aldrich) was used.Its tetrameric structure was verified by ultracentrifugation.SCN was introduced via 45 cysteine cyanylation. 15,16The protocols in the literature involve protein purification by using columns, which we replaced by dialysis.The lyophilized protein was dissolved in 100 mM sodium phosphate buffer (pH 7.9) without further purification.Then, 5,5'-dithiobis(2-nitrobenzoicacid) (DTNB) was added in 50 two-fold excess and the mixture was filled into a membrane (cutoff at 14 kDa) -covered micro dialysis capsule QuixSep (Carl Roth), and dialyzed against 1000 ml of 100 mM sodium phosphate buffer (pH 7.9) for approximately 8 hours.The dialysis solvent exhibits a faint yellow color.In the next step, an 55 approximately 100 fold excess of potassium cyanide was added, 27 which resulted in a change of color of the protein solution from brown to red due to complexation of the heme iron.The mixture was subsequently dialyzed (overnight) against 200 ml of 100 mM Sodium phosphate buffer (pH 7.9).The dialysis solvent turns 60 bright yellow if the protein is successfully labeled.Finally, a second dialysis step of 8 hours against 800 ml 100 mM sodium phosphate buffer (pH 7.9) is required to obtain pure protein (approximately 8 mM of labeled protein with no significant absorption of free cyanide (2094 cm -1 ) 28,29 and no traces of 65 yellow, which would hint at DTNB or TNB (2-nitro-5thiobenzoate) in the dialysis solution.Cyanylation at pH > 8 can lead to cleavage of the protein at the cysteine 30,31 .The sample used for further measurements is therefore investigated by MALDI MS to rule out the presence of protein fragments.

70
Isotope-labeled protein was prepared in the same way, the only differences being that K 13 C 15 N (Sigma Aldrich; 99% 13 C and 98% 15 N) was used as label, and the final dialysis step (3-4 hours) was done in 100 ml pD-adjusted D 2 O-buffer.Above protocol resulted in SCN-labeled hemoglobin that was 75 stable under lab conditions for more than a week (evident by very minor intensity changes in the FTIR spectrum).The stability and labeling efficiency was determined by mass-spectra (MS-MALDI) to be >95% after two days and ~85% after 7 days, based on the relative intensity of the β-subunit peak with/without (i.e.80 native) label (see Figure S4).

Steady-State Measurements
The FTIR spectra were measured with a Bruker Tensor 27 equipped with an MCT detector using a flow cell 32 with a sample 85 Fig. 2 Normalized FTIR absorption spectra of bovine hemoglobin (in aqueous buffer solution) with CN bound to the heme (blue), labeled with SCN (red), and S 13 C 15 N (black, in D2O buffer).All spectra are scaled to the low frequency peak and have the water background subtracted.Note that lower panel is shifted by 80 cm -1 due to isotope labeling.5 thickness of 100 µm.A long-pass filter, which blocked light with wavenumbers over 2500 cm -1 , was used.For the protein measurements baseline correction was necessary since the thiocyanate signal was on top of a water absorption band.This 10 band was fitted with a 5th order polynomial and subtracted (raw data in Figure S1).For concentration determination, UV-Vis absorption measurements were carried out with a JASCO V-670 spectrophotometer using a 1 mm quartz cuvette. 15

Extinction coefficient determination
For the determination of the IR extinction coefficient concentration series from 5 mM to 30 mM in 5 mM steps were done for MeSCN in buffer, THF and DMSO.In PCE the concentration was varied from 10 mM to 40 mM in 5 mM steps The literature UV-Vis extinction coefficient for methemoglobin with cyanide bound to the heme at 540 nm is 11 mM -1 cm -1 according to [33][34][35][36] .With this we calculate an extinction coefficient of 41 ± 5 M -1 cm -1 for the SCN absorption (see Supporting Information).

Ultrafast measurements
For the ultrafast measurements an experimental setup was used that consisted of a Ti:Sapph regenerative amplifier (3.5

Results
In this section we show by steady-state FTIR spectroscopy that we have successfully incorporated the SCN label in Hb.Because it has previously been shown that the central frequency of the CN 70 vibration is modulated by solvent properties and CN-solvent interaction, 2,3,38,39 we have collected steady-state and timeresolved infrared spectra of the model compound MeSCN.We have investigated the effect of the solvent on thiocyanate vibration's vibrational relaxation, anharmonicity, central frequency and lineshape.To mimic the effect of different protein environments on the above properties of the thiocyanate group, we used protic and aprotic solvents with different polarities, i.e. dielectric constants.Finally, we present time dependent 2D-IR data on S 13 C 15 N-Hb.Hemoglobin (see the Supplementary Information for details on sample characterization) does not exhibit absorption bands in the 2100-2180 cm -1 region. 40Upon binding of cyanide (CN -) to the 15 heme a single peak appears at 2122 cm -1 (Figure 2; blue spectrum), consistent with results of McCoy et al., 28 who showed that the reaction of potassium cyanide with hemoglobin leads to absorption at this wavenumber.Upon cyanylation (see Materials and Methods for our protocol, which is simplified with respect to 20 previously published ones) an additional peak appears at 2156 cm -1 (Figure 2; red spectrum, see Discussion for the used background subtraction procedure).Therefore, the second peak is assigned to the absorption of the thiocyanate label, and not to free CN -which absorbs at lower frequencies (2080 cm -1 ). 41,42These 25 assignments are supported by the FTIR spectra of the doubly isotope labeled S 13 C 15 N (the black spectrum), which shows a downshift of almost 80 cm -1 for both bands: the heme-bound cyanide shifts by 77 cm -1 to 2045 cm -1 , the thiocyanate absorption by 79 cm -1 to 2077 cm -1 .Previous work on S 13 C 15 N-labels in 30 ketosteroid isomerase mutants 43 reports on thiocyanate absorption frequencies in a similar spectral region, i.e. 2088 cm -1 -2090 cm -1 .
The downshift has the advantage that the isotope label now absorbs in a region with less water background absorption, and even less in D 2 O.This is beneficial in particular for 2D-IR 35 spectroscopy, where the excitation that generates our signal competes with direct solvent excitation.
As is known from previous work on MeSCN and ethylthiocyanate in solution the SCN label can be used as probe of the local environment, 2,3,38,39 because its central frequency is 40 sensitive to its polarity, hydrogen bonding and temperature.Therefore, we measured the model compound MeSCN in

Ultrafast measurements
In the previous section we have seen that the central wavenumber 65 changes if the polarity and hydrogen bonding properties of the solvent are changed.This is one requirement of a useful vibrational label.Its applicability for 2D-IR spectroscopy dramatically increases if the vibrational lifetime is long (in the order of tens of picoseconds) so that time-dependent changes in 70 the local environment (i.e.solvent or protein dynamics that alter the local electric field) can be tracked as long as possible.The lifetimes of MeSCN and the label SCN inside the protein are therefore determined using ultrafast infrared pump-infrared probe spectroscopy (see Figures 4 and 5).We focus on the spectral 75 regions where we observe the solvent-dependent bleach of the absorption of the thiocyanate stretch vibration and its corresponding vibrationally excited state absorption (ESA).The time traces of the ESA and the bleach are depicted at the maximum and minimum signal frequencies (Figure 4A and B, 80 respectively).For unscaled traces the reader is referred to Figure S2.The complete measured spectral window (as shown in Figure 5) is also subjected to global analysis which, after application of a sequential model, allows to extract species-associated difference spectra and their associated time constants, 37 which are shown in  than those of the model compound (the traces in Figure 4 are normalized at 1 ps, but the real amplitude is better seen in Figure S2 where the traces have been multiplied by 40 for better visibility).We find that the vibrational lifetime of S 13 C 15 N in the protein is similar to MeSCN in buffer, but shorter than MeSCN in the other used solvents.Nevertheless, the lifetime in the protein is 37 ps, making it a relatively long-lived infrared marker band (vibrational lifetimes are typically only a few picoseconds 45 ) that could report on changes in local environment.The anharmonicity of the thiocyanate stretch frequency is obtained by fitting Lorentzians to the spectra of Figures 5 and shown in Table 2.The anharmonicity in the protein is a bit smaller than that of MeSCN in solution but large enough for the bleach and the ESA not to significantly overlap and cancel the signal.

Spectral diffusion in haemoglobin
The protein label is followed by 2D-IR spectroscopy as function of delay time (Figure 6).The wavenumber of the narrow-band infrared pump pulse is scanned and the resulting probe spectrum is collected for each time delay.The 2D-IR spectra reveal more details than can be observed in time-dependent IR spectra with only the band maximum excited (Figure 5F).To better visualize 35 the differences at different pump frequencies, cuts through the 2D-IR spectra at different times are shown (Figure 7).The cuts clearly show spectral diffusion: with increasing waiting time the central bleach wavenumber in each cut converges to the band maximum at 2077 cm -1 .These results are discussed in terms of 40 Fig. 6 2D-IR spectra of Hb-S 13 C 15 N at different delays between the narrow-band IR pump (FWHM = 10.3 cm -1 ; 1 ps duration) and probe pulses.All panels share the same color scale, and the contours are equidistant (2.9 µOD steps).The measurement at -20 ps is subtracted as background, and in addition the average over all pixels in each 2D-IR-5 spectrum is subtracted as well to correct for a transient heating signal (evident in the blue spectrum of Figure 5F).The black line represents the diagonal, and the colored arrows at 2 ps correspond to the pump wavenumbers 2070 cm -1 , 2074 cm -1 and 2078 cm -1 of the spectral cuts through the data depicted in Figure 7.

the extinction coefficient and central frequency of SCN
In this section we discuss the steady-state spectroscopic properties of MeSCN in different solvents and examine the influence of the solvent's polarity and hydrogen-bonding properties (i.e.solvatochromism).First we focus on the extinction 20 coefficient, then on the linewidth and the central frequencies.
A quick look at Table 1 that summarizes the extinction coefficients leads to the conclusion that they are not or only mildly dependent on the solvent's polarity and hydrogen-bonding capacity.A similar observation has been made by Maienschein- Cline et al. for a different set of solvents. 38The extinction coefficient of MeSCN in aqueous buffer is consistent with the value reported previously, 38 but our value for TFE is slightly different (142 M -1 cm -1 instead of 163.6 M -1 cm -1 ).The results for the absorption in THF, often used as a model system for the 30 protein interior because of its similar dielectric constant, agree with those obtained by McMahon et al.. 39 To further check if there is a strong relationship between the extinction coefficient of MeSCN and the solvents polarity and hydrogen-bonding capabilities we also measured in DMSO, which is a polar solvent 35 that cannot form hydrogen bonds.The extinction coefficient obtained for DMSO is similar as for the apolar, aprotic THF and the only slightly less polar, protic TFE.It is lower than for highly polar and protic aqueous buffer.The extinction coefficient for apolar PCE is remarkably low.An even lower value has been 40 reported for CCl 4 (79 M -1 cm -1 ). 38Fig. 7 Spectral diffusion of the S 13 C 15 N label in Hb.Spectral slices at selected time points of the 2D-IR spectra that are shown in Figure 6.All 45 panels share the same legend, and both the data (circles) and its corresponding fit (continuous line; resulting from a simultaneous fit with 2 Lorentzians) is shown at different pump wavelengths and delay times.
The average of each spectrum is set as baseline.The vertical dashed colored lines represent guides to the eye.

50
In order to examine the measured spectra a baseline correction needs to be applied, because the spectra of the SCN absorption band are superimposed on a water band which is fitted by a 5th order polynomial function and subtracted as baseline.Although 55 the center frequency is hardly affected by this correction, this procedure may affect the observed lineshape, which is the reason why we refrain from an extensive lineshape analysis.Referring to the ultrafast data, which do not require a potentially distorting background correction, we can however conclude that the 60 linewidth is broader in H-bonding solvents than in aprotic solvents (see the values for FWHM in Table 2).Both steady-state and ultrafast data (see Tables 1 and 2) show TFE to have the broadest linewidth.The comparison between aqueous buffer and PCE, both having almost the same central frequency, shows the 65 absorption in the aprotic PCE to be significantly narrower.
The shifts in central frequency reported in Table 1 cannot be explained by solvent polarity alone: for example, MeSCN in aqueous buffer (ε = 80.1; all ε values com from ref. 44 ) and PCE 70 (ε = 2.268) absorb almost at the same wavenumber.Hydrogen bonding has to be taken into account as an additional factor.This can be achieved using empirical solvatochromic scales such as the Kamlet-Taft solvatochromic relationship (depending on three parameters α, β, π* characterizing the solvent´s hydrogen bond 75 donor acidities, hydrogen bond acceptor basicities and solvent dipolarity/polarizability, 46,47 which we successfully used to fit the center wavenumber of MeSCN in both protic and aprotic solvents (see Figure S6).However, the empirical Kamlet-Taft relationship which has been derived for bulk solvents cannot directly be 80 transferred to a protein environment and a strict molecular interpretation is not possible.We therefore discuss the solvatochromic shift in terms of the vibrational Stark effect, 50,3 keeping in mind that hydrogen bonding will cause additional effects.The shift in central wavenumber of a solute with the  38 with the exception of DMF (dimethylformamide), which has been taken from 22 .Right: Central 10 wavenumber of thiocyanate incorporated into proteins for which the orientation is known from X-Ray data (data points 1, 2, 5, 11 and 12 are taken from 48 , 3, 4, 6, 7 and 8 from 21 , and 9 and 10 from 49 ).An additional figure with SCN central frequencies in proteins summarized from the literature is shown in Figure S5.
15 theory. 51,52This approach models the electric field of the solute's dipole and its interaction with the solvent.The solute-induced polarization of the solvent generates an electric field that is sensed by the solute and causes a solvatochromic wavenumber shift ∆߭ ൌ െ ߂ߤ റ ݄ܿ ⁄ • ‫ܨ∆‬ റ through the vibrational Stark effect.∆ߤ റ 20 is the change of the dipole moment of the oscillator caused by vibrational excitation (often expressed as the Stark tuning rate |߂ߤ റ| in cm -1 /(V/m)) and ‫ܨ∆‬ റ is the change of the field acting on the oscillator.In the Onsager theory this field (in V m ⁄ ) is given by: 25 where ߝ is the dielectric constant of the solvent, n is the refractive index of the solute, ߝ the permittivity (in F m ⁄ ), ߤ ሬሬሬሬറ is the permanent dipole moment of the solute in vacuo (ߤ റ ୣୗେ ൌ 30  typically is small (at least two orders of magnitude smaller than in the case of electronic transitions, where changes of several Debye are typical) 52 , the treatment of the solvatochromic shift of vibrational transitions used here assumes that the reaction field is not changed by vibrational excitation. 53We apply a linear fit (see .88 cm -1 /(MV cm -1 nm 3 ) for MeSCN vs 5.14 cm -1 /(MV cm -1 nm 3 ) for EtSCN 3 , respectively, which corresponds reasonably.These ratios are in the same range as those for several (amino-)benzonitriles: 4.12-7.65 cm -1 /(MV cm -1 nm 3 ). 52he central wavenumber in DMSO is to our knowledge the  54 However, this approach has previously given good results for the modeling of HOD in D 2 O. 55 For CN it is proposed by Cho and coworkers that additional electric field components (caused by additional water 60 molecules placed in vicinity of the CN bond) need to be included which could cause a blue-shift of the CN stretch frequency (which we also observe experimentally in the polar buffer and TFE solvents, see Figure 3).They show that the angle of the formed hydrogen bond determines the direction of the frequency 65 shift (i.e. a linear geometry leads to a blue shift, a distorted one to a red shift) with respect to the non-hydrogen bonded case.In our analysis (see Figure 8) we always observe a blue shift for hydrogen bonding solvents with respect to the value predicted by Onsager theory.A linear geometry (the C and N atoms of 70 MeSCN and the H atom of the solvent lie on a line) seems therefore likely.A red shift with respect to the Onsager value in H-bonding solvents so far has not been reported.Such a red shift with respect to the non-H-bonding situation might occur in a protein environment, where the geometry of the protein enforces 75 the formation of a distorted H-bond.However, in the solvents analyzed here we generally observe two opposing trends: higher polarity as reflected by higher  causes a solvatochromic shift to lower wavenumbers and the presence of H-Bonds, which is usually correlated with higher  causes a shift to higher 80 wavenumbers.In protic solvents, we therefore expect partial cancelation of the two influences, as reflected e.g. by the approximately equal wavenumber of MeSCN in aqueous buffer and PCE. 85

Protein label: steady state spectroscopic properties and local environment
The absorption spectra of Hb show that the SCN label appears about 35 cm -1 upshifted with respect to the heme-bound cyanide (Figure 2).Upon cyanylation, the absorption band of the heme-90 bound cyanide does not change at all, indicating that the label does not cause structural changes at the binding site.The intensity of the two bands is similar in the 13 C 15 N sample, but a bit lower for the thiocyanate in the 12 C 14 N sample, which we attribute to lower labeling yield.The aforementioned baseline 95 subtraction may also induce artificial peak intensity differences, in particular as the isotopologues lie on different parts of the water background.Because the observed heme-bound peak does not increase with higher KCN concentration, the heme groups are taken to be saturated with CN -(see Supporting Information).The SCN peak does not alter its relative intensity with higher KCN concentration during the labeling reaction either, which could be 5 explained by a maximum labeling efficiency reached, or by a fully labeled protein.In order to determine the labeling efficiency mass spectra are collected on the native and labeled protein (see Figure S4), which show a negligible amount of unlabeled protein in the labeled samples, and we are therefore confident that we 10 have obtained a completely labeled protein.In our best samples the bands of heme-bound CN and thiocyanate are approximately equally intense, implying that the oscillator strength of the label is about twice that of the heme bound cyanide (there are four heme groups and two cyanylated cysteines). 15 The absorption of SCN in the protein is significantly broadened (compared to MeSCN in THF), but because the protein absorption is superimposed on a water background it is difficult to reliably determine the linewidth from FTIR spectroscopy.The broader lineshape of SCN in the protein is caused by 20 inhomogeneous broadening, i.e.SCN interacting with the local protein environment, as can be seen in the 2D-IR spectra (Figure 6, see discussion on spectral diffusion below).Interestingly, the estimated extinction coefficient at the band maximum of SCN in the protein is about two times smaller than that for MeSCN in 25 CCl 4 .The extinction coefficient of a thiocyanate group in its present protein environment seems therefore to be significantly smaller than that of MeSCN in the investigated solvents and the one reported by Fafarman et al. (120 M -1 cm -1 ) for the S-Peptide bound to RNAseS at 80 K.A closer look at the left part of Figure 8 shows that the frequency of the SCN label in Hb (i.e. the nitrile vibration) is similar to that of MeSCN in a solvent that has a high solvent field (i.e.where the green line intersects the black line) and therefore a high dielectric constant.In addition, because the recorded central 35 wavenumber lies below or on the Stark-tuning rate line of the apolar solvents (the black line), it is likely to assume that the label is not hydrogen bonded in the protein (all hydrogen-bonding solvents lie above the black line), although the calculations by  S5, reviewing a large number of examples, 15,2,39,57,21,49,48,22 the 65 wavenumber of the current label is among the lowest reported so far.The fact that central frequency of the label in the protein is rather low due to the possible absence of a hydrogen bond has previously been observed as well. 48,49,57The central frequency in the RNase S complex (2156.2cm -1 ) (Numbers 9 and 10 in Figure 70 8) is explained by prevention of a thiocyanate-water H-bond upon complex formation. 49The low wavenumber in the M150C-CN mutant of ketosteroid isomerase (2153.2-2154.0cm -1 , depending on mutant and ligand) (Numbers 11 and 12 in Figure 8) is according to the authors also caused by a not hydrogen-bonded 75 thiocyanate group. 48

Polarity of the surrounding and hydrogen bonding determine the lifetime of the CN vibration
We determined the vibrational lifetime of the CN vibration in 80 different solvents as well as in hemoglobin by global analysis (Figure 5) to range from 32 ps to 158 ps.These long lifetimes are comparable to different proline-SCN derivatives that have been reported to have lifetimes of 45-75 ps in D 2 O and CHCl 3 , with the slower lifetimes in the latter solvent. 58The spectral features 85 show large similarities for all solvents, although differences in baseline dynamics are seen, which cause fast components in THF and buffer solvents (a consequence of minor direct solvent excitation).Similarly, the 4.8-ps component in PCE and the 1.2ps component in DMSO are attributed to solvent dynamics and 90 are therefore not discussed further (the spectral changes are very minor with respect to the slower components).As a trend observed in the data less polar solvents lead to longer vibrational lifetimes (τ DMSO < τ THF < τ PCE , Table 2).The latter correlation is consistent with previous reports 59,60 that vibrational relaxation is 95 faster in more polar solvents, which has been attributed to stronger modulation of the vibrational energy levels. 59,61,62urthermore, vibrational relaxation in protic TFE is faster than in aprotic DMSO, although the latter has a higher dielectric constant.This suggests an additional speed-up of vibrational 100 relaxation by hydrogen bonding.Accordingly, we observe the fastest vibrational relaxation in aqueous buffer.Our observations are consistent with previous studies on smaller solutes (SCN -, N 3 and [N(CN) 2 ] -), where a speed up of a factor 2-3 has been observed when the solvent is changed from polar, non-hydrogen 105 bonding to polar, hydrogen bonding. 63,64Broader linewidths are observed in hydrogen-bonding solvents, which in addition cause shorter lifetimes. 64Such a correlation between lifetimes and linewidths has been reported by Ohta and Tominaga, who attributed it to the fact that enhanced modulation of the 110 vibrational energy levels caused by interactions between the oscillator and its surrounding lead to both, broader linewidth and shorter lifetime.The lifetime and linewidth of our protein label (Figure 5D) is very similar to that in buffer.A sub-picosecond signal as seen in buffer in Figure 5B (due to direct solvent much lower excitation level of the solvent when using narrowband excitation (excitation at the main bleach with FWHM = 11 cm -1 , corresponding to pulse duration of 1 ps).As discussed in the context of the low center wavenumber, we exclude the existence of a hydrogen bond.However, the label seems to experience a high field whose fluctuations could also cause modulation of the vibrational energy levels and thus rapid vibrational relaxation as observed for MeSCN in buffer.Attachment of the label to the protein backbone and van der Waals contacts to the protein surrounding are likely to open 10 additional relaxation channels.The observed anharmonicities (the wavenumber difference of about 25 cm -1 between the excited state and the bleach) are identical within experimental error for all solvents.Only the protein's anharmonicity is slightly lower (21 cm -1 , or 22 cm -1 when corrected for isotopic shift), which 15 might be caused by the local environment of the label inside the protein.We can however not exclude a difference caused by the protein backbone to which the label is connected to as opposed to a single methyl group for MeSCN. 20

Spectral diffusion of SCN in hemoglobin
The collected time dependent 2D-IR spectra in Figure 6 show that the shape of the bleach signal changes as function of time.To highlight the changes spectral cuts are made at the colored positions (Figure 7).In the case of a homogenously broadened 25 band the signal shape does not change as a function of time.
Regardless of the pump wavenumber, the signal would look the same in all cuts and only decrease in size with time. 65In contrast, inhomogeneously broadened spectra lead to varying spectral shapes as function of delay time and pump wavenumber (i.e. 30 spectral diffusion).The 2D-IR signal would change from a line shape that extends along the diagonal to a signal where only the amplitude but not the line shape varies with pump wavenumber if spectral diffusion is completed.The selected time slices for different pump wavenumbers show that initially different bleach 35 positions are observed (at 2 ps).However, a single bleach position remains at 5 ps, irrespective of the used pump wavenumber (i.e.spectral diffusion has occurred within 5 ps).To show that spectral diffusion occurs at all pump frequencies, more pump frequencies are plotted in Figure S3.There is no clear 40 evidence of spectral diffusion in the excited state (a similar effect is also observed for 13 C 15 N -in D 2 O 66 ).The available S/N ratio does not allow a center line slope analysis (a measure of the frequency fluctuation correlation function 45 ).A more powerful pump pulse 67 than our current few 100 nJ will certainly lead to 45 higher S/N ratios and enable a more detailed analysis of the frequency fluctuations.Note that the observed dynamics is an intrinsic protein property (which may depend on temperature and solvent properties).Therefore, our measurements show that hemoglobin in buffer exhibits ultrafast rearrangements at room 50 temperature that are reflected by the observed spectral diffusion The protein obviously is not a static molecular framework, but it dynamically interacts with its surroundings and continuously reorganizes its structure, which can be sensed by the here employed CN vibration.In some special cases it may even be 55 possible to probe the protein dynamics without the need of cyanylation: it has been demonstrated by Hamm and coworkers that the (very polarizable) S-H stretch vibration of a cysteine significantly gains in oscillator strength if it is involved in a strong (protected) hydrogen bond. 68Without this interaction, 60 however, the oscillator strength is too weak to be observed.In contrast to our results they observe very slow spectral diffusion (diffusion time of 12 ps in porcine hemoglobin) because of the hydrogen-bond induced structural rigidity within the helix in which the cysteine is located, ultimately providing a different 65 type of information that is dominated by the fluctuations of the hydrogen bond.

Conclusions
We have shown that the incorporation of a SCN label inside a protein can give rich dynamic information about the direct 70 surroundings of the unnatural amino acid.Our results on hemoglobin, which contains a cyanylated cystein with S 13 C 15 N in each of its two β-subunits, show that the label experiences a strong field in the proximity of the charged, solvent exposed surface and does not possess a hydrogen bond.Evidence for this 75 comes from the fact that the central frequency of the thiocyanate stretch vibration is rather low.In addition, the observed linewidth is relatively broad and the lifetime rather short (both compared to apolar solvents) albeit still long for a vibrational label (37 ps).Depending on the positioning of the SCN label, even longer To investigate the cause of spectral diffusion of a label more closely, further studies would be required.9][70][71] Future experiments with higher pump power and photon echo implementation of 2D-IR will allow for a higher signal to noise ratio.At longer delays, the low extinction coefficient of SCN is 100 overcompensated by the long vibrational lifetime.The combination of a long vibrational lifetime and its high sensitivity for local electric fields make the SCN label therefore a useful and versatile probe for 2D-IR spectroscopy of protein dynamics.
measurements, and Sabrina Oesteritz and Georg Wille for helping with the development of the protocol for protein preparation.J. B. thanks the Alexander von Humboldt Foundation for a Sofja

Fig. 1
Fig. 1 Structures of bovine hemoglobin and methylthiocyanate.Panel A shows the secondary structure of Hb through its transparent surface (adapted from pdb-file 2QSS 17 , carbon monoxide not shown).For clarity the two α-and β-subunits are colored green and grey, respectively.Each subunit contains one

20 and
in TFE from 50 mM to 200 mM in 25 mM steps.For the steady-state measurements shown in Figure 3 the MeSCN concentrations were 25 mM in buffer and 20 mM in PCE, THF and TFE and 15 mM for DMSO and for the ultrafast measurements shown in Figures 4 and 5 90 mM in THF, PCE, 25 TFE and DMSO and 450 mM in buffer.

Fig. 4
Fig. 4 Vibrational lifetimes of MeSCN and the isotope labeled protein Hb-S 13 C 15 N. Panel A/B show the ESA and bleach amplitudes, respectively (normalized to the maximum absorption in the raw data at 1 ps; the bleach minimum is scaled with the same normalization factor) of the MeSCN samples in different solvents after broadband infrared 5

Figures 5 .
Figures 5. Note that the protein signals are significantly smaller

Fig. 5
Fig. 5 Species-associated difference spectra of MeSCN and the isotope labeled protein Hb-S 13 C 15 N.The continuous lines result from a global fit after applying a sequential model (its species-associated difference spectra -with error bars-and associated time constants are shown.The long-lived components represent the spectra that do not decay on the 5

Fig. 8
Fig. 8 Central wavenumber of the nitrile group in MeSCN in different solvents (left) and selected proteins (right).Left: Central wavenumber of MeSCN's nitrile group in protic (open symbols) and aprotic (solid symbols) solvents as function of the calculated solvent field ‫ܨ‬ ை௦ .For5

50 lowest
reported for MeSCN yet.For all protic, hydrogen bonding solvents we observe a blue shift with respect to the value predicted by Onsager theory.Recent DFT calculations by Cho and coworkers for CN in a hydrogen bonding environment have shown that if only the field component along the cyanide bond is 55 taken into account, the observed solvatochromic shifts (for MeSCN with H 2 O clusters) cannot be explained.

30
80lifetimes might occur and allow extended observation of protein dynamics, as we observed a great variability of MeSCN vibrational lifetimes ranging from 32 ps in aqueous buffer to 158 ps in PCE.We have demonstrated the use of the isotope label S 13 C 15 N in the protein that functions as local probe inside a 85 protein to report on local structural dynamics.The use of the isotope facilitates 2D-IR spectroscopy of the CN stretch vibration in D 2 O because of the higher solvent transmission at the wavenumber of the isotopologue.Time resolved 2D-IR spectra show spectral diffusion on a few picosecond timescale.The initial 90 inhomogeneity (at delays < 5 ps) reflects the dynamic heterogeneous local environment of the label inside the protein.

Table 1 .
Central wavenumber, FWHM and extinction coefficients of the nitrile vibration in thiocyanate in different environments.The value between brackets corresponds to the standard deviation of the last 45 decimal, and are a result of a Lorentzian fit to the data shown in Figure3.
38The lineshape is not a simple Lorentzian (evident by the relative large error).†Calculatedbyaveraging5concentrationmeasurements,see the supplementary information for more details.††This is the value for pure water.†differentsolvents,mimickingapolar,polarand hydrogen bonding environments in proteins (Figure3).For comparison also the protein absorption is depicted.Fits to the absorption bands and determined extinction coefficients (see the Materials and 55 Methods section for details) are displayed in Table1.To the best of our knowledge, the values for the apolar solvent (PCE) and for the polar, but aprotic solvent (DMSO) were not reported yet.PCE's central wavenumber is similar to MeSCN in aqueous buffer, which is remarkable because the buffer is highly 60 polar.This is in contrast to the previously reported trend that non-H-bonding solvents exhibit the lowest frequencies.38

Table 2 .
Analysis of the time dependent spectra.Wavenumbers of the bleach and excited state signal result from a least-squares fit of two Lorentzians to the spectrum associated with the lifetime shown in Figure305.The anharmonicity is the difference between the central frequencies of the (simultaneous) fit to the bleach and excited state.A linear baseline connecting the lowest and highest frequencies is subtracted.
Cho et al.suggest that this could also be caused by a distorted H-It is striking to see that our central frequency for SCN in Hb lies in the same region as the 60 thiocyanate vibrations for which the absence of a hydrogen bond is confirmed by X-ray crystallography.The low frequency indicates a high field at the thiocyanate, similar to the field for MeSCN in highly polar solvents.As shown in Figure