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Year: 2008

Two-dimensional infrared spectroscopy of photoswitchablepeptides

Hamm, P; Helbing , J; Bredenbeck, J

Hamm, P; Helbing , J; Bredenbeck, J (2008). Two-dimensional infrared spectroscopy of photoswitchable peptides.Annual Review of Physical Chemistry, 59:291-317.Postprint available at:http://www.zora.uzh.ch

Posted at the Zurich Open Repository and Archive, University of Zurich.http://www.zora.uzh.ch

Originally published at:Annual Review of Physical Chemistry 2008, 59:291-317.

Hamm, P; Helbing , J; Bredenbeck, J (2008). Two-dimensional infrared spectroscopy of photoswitchable peptides.Annual Review of Physical Chemistry, 59:291-317.Postprint available at:http://www.zora.uzh.ch

Posted at the Zurich Open Repository and Archive, University of Zurich.http://www.zora.uzh.ch

Originally published at:Annual Review of Physical Chemistry 2008, 59:291-317.

Two-dimensional infrared spectroscopy of photoswitchablepeptides

Abstract

We present a detailed discussion of the complimentary fields of the application of two-dimensionalinfrared (2D-IR) spectroscopy in comparison with two-dimensional nuclear magnetic resonance(2D-NMR) spectroscopy. Transient 2D-IR (T2D-IR) spectroscopy of nonequilibrium ensembles isprobably one of the most promising strengths of 2D-IR spectroscopy, as the possibilities of 2D-NMRspectroscopy are limited in this regime. T2D-IR spectroscopy uniquely combines ultrafast timeresolution with microscopic structural resolution. In this article we summarize our recent efforts toinvestigate the ultrafast structural dynamics of small peptides, such as the unfolding of peptidesecondary structure motifs. The work requires two ingredients: 2D-IR spectroscopy and the possibilityof triggering a structural transition of a peptide on an ultrafast timescale using embedded or intrinsicphotoswitches. Several photoswitches have been tested, and we discuss our progress in merging thesetwo pathways of research.

2D-IR Spectroscopy of Photoswitchable Peptides

Peter Hamm, Jan Helbing, Jens BredenbeckPhysikalisch–Chemisches Institut, Universität Zürich,Winterthurerstrasse 190, 8057 Zürich, Switzerland

We present a detailed discussion of the complimentary fields of application of 2D-IR spec-troscopy in comparison with 2D-NMR spectroscopy. Transient 2D-IR spectroscopy of non-equilibrium ensembles is probably one of the most promising strengths of 2D-IR spectroscopy,as the as the possibilities of 2D-NMR spectroscopy are very limited in this regime. Transient2D-IR spectroscopy uniquely combines ultrafast time-resolution with microscopic structural res-olution. Our recent efforts to investigate ultrafast structural dynamics of small peptides, suchas the unfolding of peptide secondary structure motifs, are summarized. The work requires twoingredients: 2D-IR spectroscopy and the possibility to trigger a structural transition of a peptideon an ultrafast timescale using embedded or intrinsic photo-switches. Several photo-switcheshave been tested, and our progress in merging these two pathways of research is discussed.

I. INTRODUCTION

The importance of structural dynamics of biomoleculesfor their function is widely appreciated. 2D-IR spec-troscopy is a novel spectroscopic tool that uniquely com-bines ultrafast time resolution with appreciable structureresolution. The transferability of the concepts of multi-dimensional NMR spectroscopy [1] to IR spectroscopyhas been postulated already in the earliest publicationson two-dimensional NMR [2], however, the first two-dimensional IR spectrum was measured only relativelyrecently [3]. In analogy to 2D-NMR spectroscopy, theconnectivity of various vibrational states can be relatedto local contacts of the corresponding molecular groups,which is the basic principle of structure determination inboth cases. Equivalents of all fundamental NMR experi-ments (COSY [4, 5], NOESY [6], and EXSY [7–9]) havebeen demonstrated in the IR range in the meanwhile [10].

By nature of the close analogy, it might seem that thebiggest competitor of 2D-IR spectroscopy will always be2D-NMR spectroscopy. That would put the bar high!Both 2D-NMR and 2D-IR spectroscopy are capable ofresolving certain functional groups in a molecule, work(mostly) in the solution phase and aim to elucidate struc-ture as well as dynamics of solution phase systems. Overthe course of the last 20-30 years NMR spectroscopy hasproven to be so much more versatile that it has largelysuperseded conventional (1D) IR spectroscopy for stan-dard analytic purposes. This has essentially two reasons:(i) NMR spectra are more structured and allow one toresolve individual resonances from much larger moleculesthan IR spectroscopy. (ii) The interpretation and assign-ment of NMR spectra works on the basis of simple em-

pirical rules, whereas the assignment of infrared spectrais currently still a challenge to even the most powerfulquantum-chemistry codes for only a mid-size moleculewith, say, 30 atoms.

When we started out to develop 2D-IR spectroscopyas a tool to determine molecular structure, we concen-trated on a small peptide, trialanine [6, 11–13]. Much toour surprise, the conformation of that molecule, or bet-ter the distribution of conformations, was not known atthe time although it has important model character: The’backbone’ of trialanine contains only one central (φ, ψ)-pair of dihedral angles and hence reflects the intrinsicstructure propensity of the peptide bond itself withoutthe additional complication of intermolecular hydrogenbonds in larger peptides that form secondary or tertiarystructure motifs. In a series of papers [6, 11–13], we de-termined the preferred conformation, the relative popula-tions of conformations, as well as the flexibility of triala-nine. We found that trialanine populates predominantlythe so-called poly-prolin II structure (PII) with about80%. Other structure elements like α-helical and β-sheetmotifs seemed to contribute only little. This result wasunexpected since the PII structure is not common inlarger proteins, and since standard molecular dynamics(MD) force fields predicted different structure distribu-tions [14]. Our results (among others) lead to suggestionsfor modifications of the AMBER force field [15].

Very recently, our structure prediction of trialanine hasessentially been confirmed in a combined NMR and MDsimulation study, going just a little bit beyond the mostelementary methods of the rich toolbox of NMR spec-troscopy [16]. This is good and bad news: On the onehand, it validates that 2D-IR spectroscopy can indeed be

2

EXSYspectral diffusion

T1, T2, HetNOE

ps ns µs ms s

T1ρ, T2 EXSY

pump-probeRDC

a

b

fs

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ps ns µs ms s

pump-probe

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‘real time’

FIG. 1: Time arrow from femtoseconds to seconds togetherwith time regimes that can be covered by various techniquesof (a) 2D-NMR and (b) 2D-IR spectroscopy. Panel (a) isadapted from Ref. [17].

used to determine the unknown structure of small pep-tides, even when the experimenter cannot put any biasedexpectation into the structure analysis. On the otherhand, it says that if 2D-NMR spectroscopy tries hardenough it almost always will beat 2D-IR spectroscopy interms of pure structure resolution capabilities.

It has often been argued that the big potential of 2D-IR spectroscopy, as compared to 2D-NMR spectroscopy,is the by many orders of magnitudes higher intrinsic timeresolution of the former. However, one must very care-fully specify what exactly is meant by ’time resolution’.Fig. 1a shows the time regimes that can be covered byvarious variants of NMR spectroscopy, which do in factinclude the ultrafast picosecond and even sub-picosecondrange [17]. For example the orientational diffusion timeof bulk water has been deduced already in the mid-70’sby NMR spectroscopy [18], revealing a value of 2.3 psfor D2O. Only about 20 years later [19], IR spectroscopycame into the position to repeat the measurement, re-vealing that the process is more complex. The gener-ally accepted picture now is that the anisotropy decayis biphasic with a sub-100 fs librational component priorto a 2.5-3 ps diffusive component [20, 21]. Hence, whileNMR spectroscopy is essentially right for to the diffusivepart, it completely misses the fast component and cannotdifferentiate the contributions in a heterogeneous process– a subtle but important difference.

The way how the ultrafast time regime is accessed bythe intrinsically very slow NMR spectroscopy is indirectthrough relaxation methods (T1, T2, HetNOE, T1ρ andresidual dipolar couplings (RDC) in Fig. 1a). For ex-ample, in the case of orientational diffusion of water dis-cussed above, a simple theory exist which relates the T1relaxation time of the proton spin transition, which iswhat is actually measured in the experiment and whichis quite slow (0.45 s), to the fast orientational correlation

time τc of the molecule [18]. Both quantities are inverselyproportional, hence the faster the molecular structuralprocess, the slower the spin relaxation. The importantpoint is that this theory relies on a model assumption –free orientational diffusion – which is not extremely welljustified in the case of water [22]. NMR relaxation tech-niques cannot validate the model assumption made tointerpret the data, and hence tend to oversimplify com-plex systems such as liquid water. For a truly diffusiveprocess, for example the reorientation of a larger, closeto spherical molecule in solution, the results from NMRspectroscopy are usually perfectly correct.

The important difference between IR and NMR spec-troscopy is the time range that can be accessed in ’realtime’. That is, the experimenter varies a time variablesomewhere in the measuring instrument – e.g. the tim-ing of one part of a NMR pulse sequence with respect tothe rest, or the sledge of an optical delay line – and thattime is related directly to the time a particular struc-tural process takes in a one-to-one manner, i.e. the timefor interconversion of two molecular species. (For thepurpose of this review, we distinguish between structuraland energy relaxation processes, the latter of which canbe investigated as well by 2D-IR spectroscopy.) In thecontext of this definition, this is exchange spectroscopy(EXSY) and pump-probe spectroscopy in Fig. 1:

• In an EXSY experiment, an initial part of a pulsesequence (preparation phase) is used to label asub-ensemble of molecules in a certain conforma-tion by exciting a spectrally distinct spin or vibra-tional state. The sub-ensemble is then given timeto change its conformation during a waiting period,after which the conformation is finally read out. Byvarying the waiting period one observes the con-formational transition in real time. EXSY has anupper limit of the accessible time window which isdictated by the excitation lifetime of the spin or vi-brational state used as a label. In the case of 2D-IRspectroscopy, the time-window ranges from about100 fs for e.g. the frequency fluctuations of theOH vibration in water (i.e. spectral diffusion) [23–26] to typically a few 10’s of picoseconds for e.g.hydrogen bond on- and off-times in solute-solvent-systems [7–9].

• In a pump-probe experiment, a molecular systemis perturbed in some way (e.g. by triggering a pho-toreaction, or by a temperature jump) and therebybrought into a non-equilibrium state. As a re-sult, all molecules in the sample volume will un-dergo a conformational transition in a concertedway, and 2D-NMR or 2D-IR spectroscopy is thenused to elucidate a snap-shot structure of that en-semble on the fly. The lower time limit of pump-probe spectroscopy is dictated by the spectroscopicmethod (about 1 ps in the case 2D-IR spectroscopy,and 10’s of milliseconds for 2D-NMR spectroscopy),whereas no intrinsic limitation is imposed towards

3

longer timescales.

From all the methods indicated in Fig. 1, pump-probespectroscopy is the only one which allows one to accessthe non-equilibrium regime. Comparing Fig. 1a withFig. 1b, clearly the biggest potential of IR spectroscopy,as compared to NMR spectroscopy, lies in the almost un-limited time window accessible to such non-equilibriumexperiments. This is why we made a strategic decisionsome time ago to explore and develop exactly this capa-bility of 2D-IR spectroscopy: Measuring snap-shot struc-tures of fast evolving molecular systems by means oftransient 2D-IR (T2D-IR) spectroscopy. This is wherethe dynamical aspect of NMR spectroscopy almost com-pletely breaks down.

T2D-IR spectroscopy is a versatile technique that canbe applied to all sorts of problems in chemistry and bio-physics. For example, we have studied in detail the po-larization dependence of 2D-IR spectra of a metal-to-ligand charge transfer complex in its electronically ex-cited state [27], have established a technique that al-lows one to correlate vibrations in the electronic groundto those in a photo-product state (’labeling vibrationsby light’) [28], applied that technique to elucidate theconnectivity of binding sites after photo-dissociation ofCO in myoglobin [29], and studied solvation phenomenabeyond the linear response regime [30]. Very recently,T2D-IR experiments have also been reported by Tok-makoff and coworkers [31, 32] as well as Kubarych andcoworkers [33]. In this review we focus on the foldingdynamics of photoswitchable peptides [34–36]. Our ap-proach requires two ingredients that we currently developfurther in parallel: (i) 2D-IR and T2D-IR spectroscopy(Sec. IIA) and (ii) concepts of ultrafast photo-switchingof peptides (Sec. II B). In Sec. III, we report on the stagewe currently reached in merging these two pathways ofresearch.

II. METHODOLOGY

A. 2D-IR and Transient 2D-IR Spectroscopy

While the first 2D-IR experiment was a pulsed fre-quency domain experiment [3], a technique that has beenadapted and refined by various groups [4, 6, 7, 11–13, 37–41], time domain pulsed Fourier transform tech-niques have been devised as well [8, 9, 23–26, 42–57]. Variants of 2D vibrational spectroscopy are 2D-Raman spectroscopy [58–60] or DOVE spectroscopy (i.e.doubly vibrationally enhanced four wave mixing, a hy-brid frequency-time domain spectroscopy) [61, 62]. 2Dspectroscopy has also been applied to electronic transi-tions [63–68], has been addressed extensively by theoreti-cal groups [10, 69–82] and has been reviewed from variousperspectives [5, 83? –91].

The information content of the time domain and fre-quency domain implementations of 2D-IR spectroscopy is

|0〉〉〉〉

|1〉〉〉〉

|2〉〉〉〉

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|02‘〉〉〉〉

b

FIG. 2: 2D-IR spectrum of two vibrators that are (a) notcoupled or (b) coupled.

largely identical [55, 92]: The pulsed-frequency domainexperiment does a pre-selection of the pump frequencywith the help of a tunable Fabry-Perot filter, whereasin the time-domain Fourier transform techniques thepump process is achieved by two phase-locked, spectrallybroad pulses that interfere in the sample. In the lat-ter case, the spectral information is regained by Fouriertransforming the data after the measurement. With re-spect to the probe process, both techniques are identical.The time-domain Fourier transform technique has con-ceptual advantages (highest possible spectral resolution,higher sensitivity, more control over pulse parameters ofall three field interactions) which, however, are boughton the expense of a more difficult experiment (interfer-ometic stability, accurate determination of the absolutephase) [92]. Zanni and coworkers have recently proposeda hybrid method based on an acousto-optical modulatorthat seems to combine the advantages of the two ap-proaches [54, 55].

It is more intuitive to discuss the outcome of a 2D-IRexperiment in a frequency domain picture. First considertwo vibrators that are uncoupled, in which case a char-acteristic double-peak structure is obtained for each vi-brator on the diagonal of the 2D-IR spectrum (Fig. 2a).Whenever the pump-frequency is resonant with one ofthe vibrators, the latter is promoted into the first ex-cited state (|1〉 or |1′〉, respectively), and the probe beamthen probes the fundamental 0-1 transition (bleach andstimulated emission) as well as the first 1-2 overtone tran-sition (excited state absorption). Since chemical bondsare always weakly anharmonic with slightly different fre-

4

ω

pu

ωpr

ωpr

ω

pr

b ca

FIG. 3: Schematic illustration of transient 1D (top) and 2D-IR (bottom) spectroscopy. The absorption bands shift fromthe steady state a to b, each yielding a characteristic 2D-IRspectrum. In c, difference 1D and 2D-IR spectra are shown.Negative response is depicted in blue, positive response in red.Adapted from Ref. [34].

quencies of the 0-1 and the 1-2 transitions, the two peakswill separate along the probe frequency axis. Both peakshave opposite signs, and we usually color-code them asblue (negative, bleach and stimulated emission) and red(positive, excited state absorption).

When the coupling between the two vibrators isswitched on (Fig. 2b) by bringing them into close spa-cial proximity, additional cross peaks appear in the off-diagonal region of the 2D-IR spectrum. This is since(for example) the |00′〉 → |10′〉 transition –exciting thefirst vibrator while the second is in its ground state– isdifferent in frequency from the |01′〉 → |11′〉 transition,where the second vibrator is already in its first excitedstate. This so-called intermode anharmonicity is a di-rect manifestation of the coupling between the two vi-brators [86]. The coupling can be of static nature (in thesense of a level scheme as shown in Fig. 2b, right), orof dynamic nature by the flow of energy between twovibrators [6]. In any case, the intensity of the crosspeak carries information on the distance, whereas theanisotropy of the cross peak reveals the relative orien-tation of the corresponding molecular groups. Both areimportant structure indicators which can be used to de-termine the unknown structure of small peptides from2D-IR spectroscopy [4, 11, 12, 43, 49].

For reasons discussed in the introduction, we concen-trate on an extension of 2D-IR spectroscopy, transient2D-IR (T2D-IR) spectroscopy [27–30, 34–36]. For thatpurpose, we introduce an additional optical or UV pulsepreceding the 2D-IR pulse sequence, which triggers aphotochemical reaction, or a conformational transition,of the molecular system under study. The evolving non-equilibrium ensemble is then probed by the 2D-IR partof the experiment, and we may call the outcome a UV-pump-2D-IR-probe experiment.

Since the UV pump pulse will in general not con-vert 100% of the reactant species into the photo-productspecies, the 2D-IR spectrum in the presence of the UV-switch pulse always contains contributions from both. In

order to eliminate contributions of molecules that havenot absorbed any UV photon, two sets of 2D-IR spectraare recorded simultaneously - one with the UV-switchpulse on and one with the UV-switch pulse off - and sub-tracted from each other [34]. Hence, just like it is com-mon practice in conventional pump-probe spectroscopy,our T2D-IR spectra are in fact T2D-IR difference spec-tra.

To facilitate the interpretation of T2D-IR spec-troscopy, we introduce the schematic response of an ideal-ized molecule with one single homogeneously broadenedoscillator, the frequency of which shifts upon triggeringthe photo-reaction. In analogy to Fig. 2, the vibratorwould give rise to a 2D-IR spectrum with two peaks ineach of the initial and product states: a negative (blue)bleach and stimulated emission signal at the vibrator’soriginal frequency and a positive (red) excited state ab-sorption signal, which is red-shifted owing to the intrin-sic anharmonicity of the oscillator. The T2D-IR spec-trum (Fig. 3c), however, is the difference of the 2D-IRspectra of the initial (Fig. 3a) and the transient species(Fig. 3b). In a transient 1D difference spectrum, nega-tive contributions arise from the depleted initial popula-tion, while positive contributions stem from the transientphoto-product (Fig. 3c, top). Accordingly, the signs ofthe various 2D-IR peaks (color-coded in Fig. 3) of theinitial and the product state are interchanged also in theT2D-IR difference spectrum (Fig. 3c, bottom).

If the frequency shift upon the photo-reaction, as wellas the anharmonicities of reactant and product states, arelarge compared to the linewidth of a band, in total fourpeaks are expected along the diagonal for each individualresonance. Principally speaking, the same is true for eachcross peak. However, we will see that this is in generalnot the case, and the various contributions overlap andtend to strongly cancel due to their opposite signs. Inparticular for transient cross peaks, where we anticipatea change of coupling and hence a change of intermodeanharmonicity, only the two peaks related to the confor-mation with the stronger coupling tends to survive thiscancelation effect [35]. However, T2D-IR line shapes arenot yet thoroughly studied from the theoretical side.

B. Photoswitchable Peptides

The second important ingredient for studying non-equilibrium structural dynamics is the possibility to initi-ate a conformational transition of a peptide or a proteinon an ultrafast picosecond timescale. The best estab-lished approach to achieve this is a laser induced temper-ature jump to initiate, for example, folding or unfoldingof a peptide [93–101]. Temperature jumps can, in prin-ciple, be very fast and are only limited by the thermalequilibration time of the bulk solvent that is in the rangeof a few tens of picoseconds, depending on the concentra-tion of the molecule used to absorb light and to depositenergy into the solvent [102]. Nevertheless, most tem-

5

perature jumps studies so far were limited to the 10 nsregime for technical reasons: Q-switched Raman-shiftedNd-YAG lasers were used to generate the intense IR pulsewith an energy of typically 1 mJ needed to heat the sol-vent water directly by exciting an overtone of either itsOH or OD stretch vibration.

However, the intrinsic time resolution of 2D-IR spec-troscopy is significantly faster (1 ps) than the pulse du-ration of suitable Q-switched lasers (10 ns). We felt thatlimiting the overall time resolution of the experiment to10 ns would potentially miss important advantages of 2D-IR spectroscopy (although this time resolution might besufficient for most of the problems of peptide dynamicsstudied by temperature jump experiments).

Temperature jumps introduce another problem whencombining it with a 2D-IR experiment as a probe pro-cess: A significant amount of energy is deposited in arelatively small volume which causes a sudden jump ofpressure in a small sample volume. Strategies to reduceartifacts from the resulting shock waves and the result-ing index of refraction gradients have been worked outfor conventional pump-probe experiments [103], but 2D-IR spectroscopy, consisting of a sequence of IR pulses, ismore critical in this regard. The fact that the pulsed-frequency domain experiment is not a phase sensitivetechnique has proven to be a crucial advantage. Only re-cently, Tokmakoff and coworkers succeeded to cope withthe problem of phase distortions using the time-domainFourier transform technique [31, 32].

We have chosen another approach to initiate confor-mational transitions in small peptides. By incorporat-ing a photoswitch into a peptide which undergoes a pho-tochemical reaction after electronic excitation, one mayalter the conformation of the peptide in a very con-trolled way. The switching time is ultrafast in mostcases (≈1 ps). Furthermore, since the photoswitch is di-rectly incorporated into the molecular system, its actionis much more local than that of a temperature jump.As a consequence, the overall energy pumped into thesample volume is much smaller (at least a factor of 10),and hence problems with shock waves are considerablyreduced (albeit still present in some cases). We distin-guish between photoswitches, when they are reversible,or phototriggers, when it is a one-time switch. Variousphotoswitches have been proposed in the literature, ofwhich we tested a few for our purpose to combine it withT2D-IR spectroscopy: azobenzene (Fig. 4a,b) as wellas disulfide-bridges and thiopeptides as intrinsic photo-switches (Fig. 4c,d). The use of photoswitches for variousapplications is reviewed in Refs. [104–106].

a

d

c

430 nm

366 nm

260 nm

260 nm

280 nm

b430 nm

366 nm

FIG. 4: Photoswitchable peptides studied in our lab: Azoben-zene (a) embedded into peptide backbone (b) or cross-linkingamino acid side chains. Intrinsic photoswitches: (c) Photo-cleavable disulfide-bridge and (d) Thiopeptides.

III. TRANSIENT 2D-IR SPECTROSCOPY OFPHOTOSWITCHABLE PEPTIDES

A. Azobenzene

The cis-trans isomerization of azobenzene is widelyused for photo-switching [106]. It is characterized by ahigh quantum yield (50%), high photostability, ultrafastswitching speed (about 1 ps) and the fact that cis andtrans isomers have absorption bands which are well sep-arated, and which therefore allow for a selective switch-ing in both directions [107]. Azobenzene has been incor-porated directly into the backbone of peptides by vari-ous groups [108–121], switching the conformation of thelatter by photo-exciting the former. In a first example,we concentrated on a small cyclized peptide designed byMoroder and coworkers. The peptide resembles the ac-tive site of thioredoxin reductase (Fig. 4a), and was cy-clized by an azobenzene-based ω-amino acid [112]. UVspectroscopy has been applied, which reports on the re-sponse of the chromophore [122–124], as well as IR spec-troscopy, which addresses directly the dynamics of thepeptide backbone [125].

Isomerization of the photoswitch couples very directlyto a conformational transition of the peptide back-bone. In fact we find that the peptide backbone canbe stretched on an ultrafast 20 ps timescale upon cis-trans isomerization of an azo-moiety [125]. After this

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FIG. 5: (a)–(l): Time resolved pump-probe (top of eachpanel) and T2D-IR spectra (bottom of each panel) atT=20 ps, 200 ps and 1.7 ns, respectively. The left columnshows experimental results, the right column the simulation.The labelled arrows refer to features described in the text.Adapted from Ref. [34].

initial driven phase, the molecule keeps on equilibratingon a slower nanosecond timescale, but the major part ofthe conformational transition clearly occurs within thefirst 20 ps. This is concluded from the transient pump-probe signals which has built up already 20 ps after theUV pump pulse (Fig. 5a, Pos. 1) and changes onlymarginally between 20 ps and 1.7 ns (Fig. 5a,c,e). The20 ps timescale might seem surprisingly fast for a con-formational transition of a peptide. However, given thefact that a major fraction of the energy of the excitationphoton (290 kJ/mol) is used to drive the conformationaltransition, we concluded that the peptide backbone isstretched with an artificially high force

Nevertheless, such cyclized azo-peptides appear to beideal model systems to test the capability of 2D-IR spec-troscopy in combining ultrafast time-resolution with di-rect structural resolution. This is why we used exactlythis system for our first demonstration of transient 2D-IR spectroscopy [34]. Despite the fact that hardly any

time-dependence is observed in the pump-probe spectrabetween 20 ps and 1.7 ns (Fig. 5a,c,e), we find large dif-ferences in the T2D-IR spectra (Fig. 5b,d,f), illustratingthe information gain of the latter as compared to theformer. In particular, we find only a small signal fromthe photoswitched peptides in the T2D-IR spectrum af-ter 20 ps (expected at Pos. 2 in Fig. 5b) although it givesrise to a band in the 1D spectrum (Fig. 5a, Pos. 1). Onlythe T2D-IR signal of the initial cis state is observed. Af-ter 200 ps, however the band of the trans-ensemble hasgrown in (Fig. 5d, Pos. 3) and within 1.7 ns we observe atilt of this band towards the diagonal of the 2D spectrum(Fig. 5f, Pos. 4).

Interestingly, the T2D-IR spectra can be modeled al-most quantitatively by just varying the homogeneous linewidth γt of the amide I band of the photo product by 10-15% between 20 ps and 1.7 ns (Fig. 5h, j, l). In particularthe low intensity of the transient product band at 20 ps aswell as its rise at later times are perfectly matched. Tran-sient 2D-IR spectroscopy is sensitive to relatively smallchanges in the lineshape function due to strong cancela-tion effects in the double-difference spectrum.

The homogeneous linewidth γt in 2D-IR experimentsis a measure of fast (< 1 ps) fluctuations of the pep-tide backbone. The time scale relevant for homogeneousbroadening (

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FIG. 6: (a+b) Equilibrium and (c+d) transient non-equilibrium spectra 100 ps after photo-cleavage of cyclo-(Boc-Cys-Pro-Aib-Cys-OMe). The top panels (a+c) show 1D-IR spectra and the bottom panels (b+d) the corresponding2D-IR spectra. The equilibrium 2D-IR spectrum (b) is theweighted difference between parallel and perpendicular po-larization measurement (to enhance the cross-peak contribu-tion), whereas the transient 2D-IR spectrum (d) is with allthree laser beams polarized parallel. The crosspeak markedby circles in either spectrum relates to the coupling across thehydrogen bond in the β-turn structure. The example struc-tures were obtained form an accompanying MD simulation.Adapted from Ref. [35]

B. Intrinsic Photoswitches

The disulfide bridge between two cysteines, that occursin natural peptides and proteins, may be used as pre-determined breaking point that photo-dissociates uponelectronic excitation at ≈260 nm, and hence as photo-trigger. This concept was first reported in pioneeringwork of Hochstrasser and coworkers [129, 130]. A 20residue peptide was designed to fold into an α-helix af-ter photo-cleavage, while no distinct secondary struc-ture was possible in the initial bridged state. Unfor-tunately, geminate recombination of the liberated thiylradicals occurred before helix formation could be de-tected. We have recently used this approach for a muchsmaller peptide (Fig. 4c) [131] that forms a β-turn struc-ture in the disulfide-bridged configuration. Upon photo-cleavage of the disulfide bridge, the hydrogen bond inthe β-turn is destabilized and the ring structure opens.In contrast to the molecule studied by Hochstrasser andcoworkers [129, 130], the molecule is much more straineddue to the shortness of the backbone and stays open onlong timescales. In fact, only about half of the radicalsare quenched after 1 ms either intra- or intermolecular-ily [132].

The peptide features a completely resolved and easy

to assign IR spectrum of the five C=O groups withouthaving to invoke isotope labeling. This fortunate sit-uation made it the ideal test case for transient 2D-IRspectroscopy that allowed us to directly extract micro-scopic structural information on an ultrafast timescalefor the conformational transition of the molecule [35].Fig. 6b shows an equilibrium 2D-IR spectrum of themolecule which is rather congested since essentially allC=O groups are coupled to each other. Fig. 6d, in con-trast, shows a transient 2D-IR spectrum 100 ps afterphoto-cleavage of the disulfide bridge. The diagonal isdominated by signals similar to those in Fig. 5. Moreimportant for the structural resolution aspect is the off-diagonal region, where only one dominant peak remains,related to the coupling between Cys(1)-Aib(3) across theintramolecular hydrogen bond. The corresponding crosspeak is weak in the equilibrium case (Fig. 6b) and ishidden underneath the wings of the much stronger crosspeaks of Boc-Cys(1) and Cys(1)-Pro(2), both related tonearest neighbors in the sequence. In contrast, Cys(1)and Aib(3) are ’far in sequence’, but close in space in thefolded structure. Therefore, upon opening of the ringstructure, changes in coupling are expected only for thelatter in the transient 2D-IR spectrum, which is a differ-ence 2D-IR spectrum between photo-product state andreactant state. A time series showed that the transientcross-peak rises on a 100 ps timescale, in good agreementwith accompanying results from MD simulations [35].The transient cross-peak constitutes a direct and easyto interpret structural indicator.

Another potential intrinsic photoswitch is the -CO-NH- peptide unit itself, which is thermodynamically morestable in the trans state, but which can be switched to thecis conformation by electronic ππ∗ excitation [133], forc-ing the peptide to change its secondary structure [134,135]. However, the required excitation wavelength liesdeep in the UV (190 nm) which is hard too reachtechnically and which can destroy the molecule. Ex-citation at 190 nm is also non-selective and can ran-domly switch any peptide unit in a polypeptide. Bothproblems are avoided by replacing one peptide unit bya thiopeptide (i.e. -CSNH-) unit (Fig. 4d) [136–140].This red-shifts the wavelength of the ππ∗ transition ofthe trans isomer to ≈260 nm [141, 142] which is eas-ily accessible by frequency-tripling a Ti:S laser. Fur-thermore, incorporation of a thioamide linkage resultsin only minor changes of the secondary structure of apeptide [143, 144]. A combination of theoretical andtime-resolved experimental studies on the photoswitchitself (N -Methylthioacetamide) have shown that the iso-merization quantum yield is high (30-40% in the trans-cis and 60-70% in the cis-trans direction) although themolecule remains trapped in a relatively long-lived ex-cited state for 200-300 ps, where it is almost free torotate about the thioamide bond [145, 146]. Neverthe-less, thiopeptides are particularly useful as model sys-tems for T2D-IR spectroscopy, because the isomeriza-tion of the thioamide bond changes the relative orienta-

8

tion of the adjacent peptide units, which are sufficientlyclose to each other for amide I mode coupling. In anal-ogy to trialanine in 2D-IR spectroscopy the smallest testmolecule for T2D-IR is thus a tetrathiopeptide, with twoamide I oscillators separated by the thio-photoswitch.The first thiopeptide we have investigated by T2D-IRspectroscopy (Boc-Ala-Gly(=S)-Ala-Aib-OMe) [36, 147],in addition contains two protecting groups (Boc andAib/OMe, providing two more C=O oscillators), whichstabilize the trans-molecules in a looped conformation viaan intramolecular hydrogen bond. In the T2D-IR spec-tra, we observe resolution enhancement along the diago-nal as well as a transient cross peak due to the breakingof that hydrogen bond [36].

IV. DISCUSSION

Comparing the results of Ref. [34] (Fig. 5) with thoseof Refs. [35, 36] (Fig. 6), one may suggest concrete de-sign criteria to render T2D-IR spectroscopy an expressivemethod to time-resolve transient structures. In the caseof the azobenzene switched cyclic octapeptide of Ref. [34],the first example we investigated, the amide I′ band wasnot spectrally resolved, hence, we could not interpret theT2D-IR spectra in terms of structural motifs. The struc-tural information did not go any deeper than that of thecorresponding 1D spectroscopy; nevertheless, additionaldynamic information could be extracted. In contrast, incase of the disulfid-bridged β-turn [35], we deal with asomewhat smaller peptide whose amide I′ spectrum isfully resolved. As a result, specific local contacts can beidentified whose time-dependence can be interpreted interms of the breaking of certain loop structures.

In the examples of Refs. [35, 36], bands are resolvedsince the different C=O groups are sitting in differentchemical environments. For larger peptides, this will ingeneral not be the case. However, spectral resolutioncan always be regained when employing isotope label-ing [12, 46, 49, 147]. The synthesis of site-selectivelyisotope labelled peptides is not particularly demanding.For example, in our studies of the folding of a photo-switchable α-helix (Fig. 4b), we employ double 13C16Oand 13C18O isotope labeling in order to single out twospecific sites from the unstructured main amide I′ bandof unlabeled groups. 13C16O-labeling causes a frequencydownshift of the amide I′ band of about 30 cm−1, whereas13C18O shifts it by 50 cm−1. In that way, the local con-tact between a specific pair of amino acids can be iden-tified (e.g. the i → i + 4 configuration of a α-helicalloop [49]), and its time-dependence can potentially bemeasured. These experiments promise to give unprece-dented structural insights into which helix-loop is form-ing at what time to what extend during the folding pro-cess.

An important, but not yet thoroughly explored issue isthe lineshapes of T2D-IR spectra [148]. If the frequencyshifts induced by the photo-reaction are larger than the

linewidth of the band, in total four spectrally resolvedbands are expected for each diagonal and cross peak inthe T2D-IR spectrum (Fig. 3). This is the case for e.g.the metal-to-ligand charge transfer of a metal-carbonylcompound we studied extensively ([Re(CO)3(dmbpy)Cl],dmbpy=4,4’-dimethyl-2,2’bipyridine) [27]. However, inthe case of photoswitchable peptides, the transient fre-quency shifts that are induced by a conformational tran-sition are typically very small – much smaller than theline width of the amide I band. In this case the two con-tributions Fig. 3a and Fig. 3b will overlap and tend tocancel each other in Fig. 3c. If the lineshape functionsof reactant and product species are identical (which willnot be the case in general), then the T2D-IR is expectedto be symmetric, as in Fig. 3c. Due to the strong cance-lation between reactant and product contributions, how-ever, small changes in the line shape functions of eithercontribution may cause strong effects for the resultingdifference T2D-IR spectrum (see Fig. 5b, Position 2).

The theoretical modeling of these cancelation effects isquite demanding and has not yet been achieved. First,it requires very high accuracy to obtain still reasonableresults after subtracting the two contributions from re-actant and product state, and second, so far almost alllineshape calculations are based on an equilibrium as-sumption [70]. However, it is not clear after which timeone can think about an evolving structural ensemble asbeing in a quasi-equilibrium. We have shown that this as-sumption is definitely not valid on a 10 ps timescale [30],but we do not know whether it might become reasonableon a 100 ps to 1 ns timescale [34, 35]. This problem callsfor significant effort from the theoretical side.

V. CONCLUSION

NMR is a high-resolution spectroscopy, capable tospectrally isolate thousands of individual resonances.However, the narrow line width implies that direct timeresolution of NMR spectroscopy is intrinsically slow. Thetime resolution is ultimately limited by the dephasing(T2) time of the spectroscopic transition used as molecu-lar probe (typically 1 ms for spin transitions), rather thanby the measurement instrument. In contrary, typical de-phasing times of vibrational transitions in the solutionphase are in the range of 1 ps, hence IR bands are broad,and the number of resolved bands in a given spectralwindow is restricted. Nevertheless, the fast dephasingtime is responsible for the high direct time resolution of2D-IR spectroscopy. Spectral congestion can be circum-vented to a significant extent by isotope labeling. In thatsense, 2D-IR and 2D-NMR spectroscopy should not beviewed as competing methods, rather 2D-IR spectroscopyshould be further developed as a complementary method.We have focussed here on transient non-equilibrium 2D-IR spectroscopy as one of the most promising strengthsof 2D-IR spectroscopy (it certainly is not the only one).Learning to use the structure resolution capabilities of

9

2D-IR spectroscopy, also in equilibrium, is of course aninevitable prerequisite before extending it to the non-equilibrium regime, and work in this direction is still re-quired.

Since the typical length and time scales of T2D-IRspectroscopy coincides exactly with what is accessibleto MD simulations, the latter can directly be testedagainst experiment. MD simulations currently are theonly approach to understand and visualize the compli-cate structure and dynamics - and hence the function -of biomolecules on an atomic level. Yet, it is clear thatthe MD approach is based on, in part very crude, approx-imations, while experimental techniques which can pro-vide pictures of similar clarity are scarce. With Ref. [35],we made a significant step towards a ’molecular movie’of ultrafast structural dynamics extracted directly fromexperimental data.

Acknowledgement: We are thankful to numerouscontributions from our coauthors, in particular ChristophKolano, Mariusz Kozinski, Luis Moroder, Christian Ren-ner, Wolfram Sander, Gerhard Stock, Josef Wachtveitl,Sander Woutersen and Wolfgang Zinth. The work hasbeen financially supported by the University of Zurich,the Swiss Science Foundation (SNF) and the GermanScience Foundation (DFG).

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