Folding and unfolding of a photoswitchable peptide from picoseconds to microseconds

G. Andrew Woolley, and Peter Hamm Janne A. Ihalainen, Jens Bredenbeck, Rolf Pfister, Jan Helbing, Lei Chi, Ivo H. M. van Stokkum,

doi:10.1073/pnas.0607748104 published online Mar 19, 2007; PNAS

This information is current as of March 2007.

www.pnas.org#otherarticlesThis article has been cited by other articles:

E-mail Alerts. click hereat the top right corner of the article or

Receive free email alerts when new articles cite this article - sign up in the box

Rights & Permissions www.pnas.org/misc/rightperm.shtml

To reproduce this article in part (figures, tables) or in entirety, see:

Reprints www.pnas.org/misc/reprints.shtml

To order reprints, see:

Notes:

Folding and unfolding of a photoswitchable peptidefrom picoseconds to microsecondsJanne A. Ihalainen*, Jens Bredenbeck*, Rolf Pfister*, Jan Helbing*, Lei Chi†, Ivo H. M. van Stokkum‡,G. Andrew Woolley†, and Peter Hamm*§

*Physikalisch-Chemisches Institut, Universitat Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland; †Department of Chemistry,University of Toronto, 80 Saint George Street, Toronto M5S 3H6, Canada; ‡Faculty of Sciences, Department of Physics and Astronomy,Vrije Universiteit, De Boelelaan 1081, 1081 HV Amsterdam, The Netherlands

Edited by William A. Eaton, National Institutes of Health, Bethesda, MD, and approved January 2, 2007 (received for review September 6, 2006)

Using time-resolved IR spectroscopy, we monitored the kinetics offolding and unfolding processes of a photoswitchable 16-residuealanine-based �-helical peptide on a timescale from few picosec-onds to almost 40 �s and over a large temperature range (279–318K). The folding and unfolding processes were triggered by anultrafast laser pulse that isomerized the cross linker within a fewpicoseconds. The main folding and unfolding times (700 ns and 150ns, respectively, at room temperature) are in line with previousT-jump experiments obtained from similar peptides. However,both processes show complex, strongly temperature-dependentspectral kinetics that deviate clearly from a single-exponentialbehavior. Whereas in the unfolding experiment the ensemblestarts from a well defined folded state, the starting ensemble in thefolding experiment is more heterogeneous, which leads to dis-tinctly different kinetics of the experiments, because they aresensitive to different regions of the energy surface. A qualitativeagreement with the experimental data-set can be obtained by amodel where the unfolded states act as a hub connected to severalseparated ‘‘misfolded’’ states with a distribution of rates. Weconclude that a rather large spread of rates (k1 : kn � 9) is neededto explain the experimentally observed stretched exponentialresponse with stretching factor � � 0.8 at 279 K.

Protein relaxation and protein folding kinetics are in manycases extremely complex processes, mainly because of the

large distribution of the time scales of processes (1). Thiscomplexity often results in nonexponential kinetics and leads tothe kinetics depending on the monitored observable (2–5).Because of their relatively fast folding times and plainness, alarge amount of work has been concentrated on �-helix forma-tion of Ala-rich model peptides with strong helix-propensity(6–8). Because the �-helix is one of the predominant secondarystructures in many proteins and peptides, detailed understandingof its conformational dynamics can give important insight intothe conformational dynamics of naturally occurring proteins.

Helix dynamics have been studied for the most part by usinglaser induced T-jump methods to perturb the equilibrium, andthe subsequent conformational dynamics are detected either bytime-resolved IR spectroscopy (9–13), by fluorescence (14, 15),or by Raman scattering techniques (16). It has been establishedfor some time that helix folding is not a two-state process (17).The overall relaxation of an �-helical peptide after an unfoldingperturbation has been found to occur between 120 ns and 420 ns,at room temperature (9–12, 14, 15). The folding of an �-helix hasbeen found to occur with a time constant of �1.2 �s (13, 18).However, molecular dynamics simulations suggest that polypep-tides can undergo considerable structural changes within 1 ns orless (19–21). By means of a triplet-triplet-quenching technique,intramolecular contact formation between side chains and in-tramolecular chain diffusion of a polypeptide have been ob-served experimentally to take place in the order of 20 ns (22, 23).Thus, an �10-ns time resolution (the typical time-resolution ofT-jump experiments) is not necessarily sufficient to resolve these

fast processes taking place during the early phases of a foldingor an unfolding process (7).

To study both folding and unfolding for one and the samemolecule, Woolley and coworkers (24, 25) have designed pho-toswitchable peptides where a crosslinked photoisomerizableazobenzene determines the helix propensity of the peptide.When the crosslinker is attached to two cysteines spaced 11residues apart, the �-helical conformation is stabilized by thelinker in its trans conformation, while in the cis conformation,the linker considerably diminishes the helicity of the peptide, andas such, the free energy surfaces of these conformations areconsiderable different (See Fig. 1A) (24, 25). Moreover, becausethe isomerization of the linker is ultrafast (26), folding andunfolding can be triggered with high time-resolution (18, 27–29).The sequence of the peptide, Ac-AACARAAAARAAACRA-NH2 (hereafter noted as the AARA peptide), is the same as thatused in many T-jump studies (10, 14, 16) but is slightly differentfrom the one studied in our previous report (18), where addi-tional salt bridges stabilized the helical form.

In an ‘‘energy landscape picture,’’ the aim is to reduce theoverwhelmingly large coordination space, which obviously existin protein dynamics, to a few (typically one) representativereaction coordinates. Along that reaction coordinate, a freeenergy surface can be established to account for the thermody-namics, and at the same time one is hoping that kinetics can alsobe modeled by using some diffusion process on that surface(30–33). Conceptually, the first step is always possible whethera ‘‘good’’ or ‘‘bad’’ reaction coordinate has been chosen, but thesecond step is problematic and depends on the choice of thereaction coordinate (34, 35).

T-jump experiments have often been modeled by ‘‘initiation–propagation models’’ (or the kinetic zipper model) (5, 14, 36).Each residue of the polypeptide chain is considered to exist inone of two possible configurations [coil-like (c) or helical-like(h)] the latter having lower entropy because of the smallernumber of possible conformations. Although this type of analysissuccessfully describes a large number of helix relaxation data, theinitiation-propagation model has a tendency to predict com-pressed rather than stretched exponential kinetics in the foldingdirection (14, 33), which contradicts the results of our experi-ments (this study and ref. 18). Moreover, molecular dynamicsimulations of small peptides (37–41), consistently demonstratethe existence of low free energy traps in the unfolded state thatare mostly due to nonnative hydrogen bonds. This idea hasrecently been supported by UV resonance Raman experiments,

Author contributions: J.A.I., J.B., J.H., G.A.W., and P.H. designed research; J.A.I., J.B., R.P.,J.H., L.C., G.A.W., and P.H. performed research; R.P., L.C., and G.A.W. contributed newreagents/analytic tools; J.A.I., J.B., I.H.M.v.S., and P.H. analyzed data; and J.A.I., G.A.W., andP.H. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS direct submission.

§To whom correspondence should be addressed. E-mail: p.hamm@pci.unizh.ch.

© 2007 by The National Academy of Sciences of the USA

www.pnas.org�cgi�doi�10.1073�pnas.0607748104 PNAS � March 27, 2007 � vol. 104 � no. 13 � 5383–5388

BIO

PHYS

ICS

which suggested that, e.g., 310 helices might act as traps as well,in particular for low temperatures (42). Such misfolded traps arecompletely ignored in initiation–propagation models. However,if these misfolded traps turn out to be rate limiting, and neitherinitiation nor propagation of the helix itself, then the number ofnative contacts will no longer be a good reaction coordinate thatcan be used to describe the kinetics of folding.

We demonstrate in this study that detecting both folding andunfolding processes of an �-helix over a large range of temper-atures and a wide span of time (from �30 ps up to 36 �s) permitsa more detailed discussion of the energy landscapes of both thefolded and unfolded states.

ResultsSteady-State IR Spectroscopy. Throughout this article, conforma-tional changes of the peptide are followed by detecting changesof the amide I� band (CAO stretch), because this band is verysensitive to transition dipole coupling effects and to the H-bonding of the CAO-group (43). The absorption spectra of thecrosslinked AARA peptide in its trans state and in its cis stateare shown in Fig. 1B. The FTIR difference spectra between thefolded conformation (trans state) and unfolded conformation(cis state), measured under illumination either at 435 nm or 365

nm, respectively, are shown in Fig. 1C. As observed in manyother spectroscopic studies (9–13, 18, 44), the amide I� modeshifts to lower frequency in the folded conformation, andtherefore the absorption difference spectrum shows a positivesignal �1,633 cm�1 and negative signals �1,655 cm�1 and 1,680cm�1 (solid line). Indeed, the crosslinker increases considerablythe helix propensity in its trans state (24, 25), as is demonstratedby CD measurements (Table 1). The perfect mirror symmetry ofthe folding and unfolding FTIR difference spectra demonstratesthe reversibility of the processes.

Time Evolution of the IR Spectra. Overall, the spectral dynamics ofthe folding process triggered by 425 nm excitation of themolecules in the cis state closely resemble the results reported inref. 18 (Fig. 2A). The early signals up to 30 ps are omitted,because they are dominated by the response of the linker, whichis heated by absorption of the UV photon and subsequentultrafast electronic relaxation of the azobenzene moiety (18, 45).The first signal that can be unambiguously assigned to thedynamics of the peptide is seen as a broad bleach at the positionof the amide I� band �30 ps after the laser flash (Fig. 2 A, cyan

430 nm366 nm

A

1600 1650 1700

∆rosb

Atpi

no

Energy, 1/cm

Folding

Unfolding

rosbA

tpi

no

C

TransCisB

Fig. 1. Photoswitchable peptide and its steady-state CAO absorption spec-trum. (A) Schematic models of the cis (Upper) and trans (Bottom) peptides,illustrating the conformational transition induced by the photoswitchablelinker. Hydrogens and side chains are omitted for clarity. (B) FTIR absorbancespectrum of the peptide in the trans (solid line) and cis (dashed line) states. Thecis spectrum was constructed by using the trans spectrum and the cis–transdifference spectrum assuming 65% conversion to the cis form [estimationfrom the UV-visible absorbance difference (data not shown)]. (C) FTIR differ-ence spectrum of the peptide under 436-nm (trans � cis, solid line) or 365-nmirradiation (cis � trans, dashed line) at room temperature. The FTIR differencespectra of the crosslinker under the same irradiation conditions are shown asa dotted line (trans � cis) and a dash-dotted line (cis � trans).

Table 1. Helicities, folding times, and unfolding times of the photoswitchable peptide

Helicity, % Folding Unfolding

Temp., °C Trans Cis �1f , ps �2

f , ns �3f , ns �3

f Temp., °C �1u, ps �2

u, ns �3u, ns

6 66 20 80 18 1,370 0.8012 64 18 120 17 1,040 0.82 8 50 9 37019 60 13 100 15 770 0.91 19 40 5 16032 55 10 140 15 430 1.0 29 30 3 9045 46 11 140 6 270 0.98 41 20 1 50

Helicities in the trans and cis states of the linker are estimated from the CD signal at [� ]222 without irradiation (for the trans state) and �365-nm irradiation(for the cis state). A 65% conversion to the cis form under 365-nm light is used [based on the UV-visible absorbance difference (data not shown)]. The foldingand unfolding times at various temperatures are fitted with three time constants, and in the case of folding, one free stretching factor was used (�3). Thewavelength-dependent amplitudes of the components at room temperature are shown as decay-associated difference spectra [�l(� )] in Fig. 3. The estimatederror of the analysis is 10% for the CD experiment, 30% for the two first-lifetime components, 10% for �3, and 5% for �.

-0.5

0.0

0.5

1.0

1560 1600 1640 1680-0.4

0.0

0.4B

0.03 ns1 ns45 ns141 ns3600 nsFTIR

A

Energy, 1/cm

Folding

Unfolding

∆rosb

Atpi

,noD

Om

0.03 ns0.07 ns2 ns50 ns696 nsFTIR

Fig. 2. Time-resolved IR-spectra of the folding and unfolding processes atroom temperature. (A) Transient absorption signal after 425-nm excitation,which corresponds to the spectral dynamics of the folding process of the�-helix. (B) Transient absorption signal after 380-nm excitation, which corre-sponds to the spectral dynamics of the unfolding process of the �-helix. TheD2O heat signal is subtracted from the spectra. The FTIR difference spectrum(dashed lines; compare Fig. 1C) are scaled for better comparison. The arrowsindicate the direction of the spectral evolution.

5384 � www.pnas.org�cgi�doi�10.1073�pnas.0607748104 Ihalainen et al.

line). This indicates a perturbation of the peptide backbone bythe stretching of the photoswitch in the early times after theexcitation pulse. The broad bleach undergoes a blue shift anddisappears within 45 ns (red and green lines). Then, a spectrumwith a shape similar to the steady-state difference spectrum (Fig.1C) appears at �140 ns after the laser flash (Fig. 2 A, blue line)consistent with the formation of the �-helix structure. The finalspectrum is obtained after 3.6 �s (black line).

Fig. 2B shows the spectral evolution of the unfolding processtriggered by 380 nm excitation of the molecules in the trans state.The signals due to peptide dynamics are smaller overall in theunfolding experiment, because the quantum yield of the azolinked peptide for the trans 3 cis isomerization (0.08) is muchsmaller than that of the cis3 trans isomerization (0.62) (46). Thespectrum at 30 ps after the laser flash is again dominated byrelaxation of the linker, which is seen as negative signals at 1,600cm�1 and 1,670 cm�1 and a positive signal at 1,645 cm�1 (Fig. 2B,cyan line). A narrow bleach at �1630 cm�1, which reportsperturbations in the peptide, can be observed 70 ps after the laserflash (red line). The unfolding signal develops further over 2 ns(green line), and after 50 ns, the signal is more than half-waycomplete (blue line), although the spectral shapes between 1,660cm�1 and 1,680 cm�1 are still underdeveloped. The final spec-trum, which closely resembles the steady-state cis–trans differ-ence spectrum (Fig. 1C), is obtained �700 ns after excitation(Fig. 2B, black line). Thus, at room temperature, the unfoldingprocess is about five times faster than the folding process.

Temperature Dependent Amide I� Dynamics. Fig. 3 shows time tracesat various wavelengths of the amide I� band during the foldingand unfolding processes at various temperatures. Immediateobservations for the folding (Fig. 3 A–H Upper) and unfolding(Fig. 3 A–H Lower) processes are as follows: (i) a strongtemperature dependence of both processes, (ii) strong nonex-ponentiality at low temperatures in the folding experiment andat all temperatures in the unfolding experiment where a clearbiphasic behavior can be observed, and (iii) the spectroscopicresponses are different at different wavelengths, in agree-ment with earlier observations made by using T-jump experi-ments (9, 10).

The time traces observed between 1,616 cm�1 and 1,717 cm�1

were globally fit to

��t, ) � �l�1

n

�l(�)exp�� t/�l)�l]

with a common set of time constants, where �(t,) is the timeresolved spectrum which is a sum of �l(�) spectra (or decayassociated difference spectra) multiplied by exponential decayswith time constants �l and stretching factors �l. This is a typicalglobal analysis method (with stretched exponential kinetics) thattakes into account both the spectral information and the tem-poral behavior of the process in ref. 47. The fitted traces areshown as solid lines passing through the measured data points(Fig. 3 A–F). One should note that spectral shifts, which obvi-ously take place in the spectral dynamics of both processesstudied, partially obstruct this type of analysis. The rather largeerror margins associated with the first two time constants (�1

f and�2

f) in the folding process are due to such spectral shifts but alsodue to the small amplitudes of the folding signal. However, byusing a global analysis method, the time scales and the relativemagnitudes of particular spectral ‘‘events’’ become clearly ap-parent. In the unfolding experiment, the time traces showmultiphasic behavior. In the folding experiment, spectrally dis-tinct kinetics dictate the use of several components (Fig. 3).Three time constants were found to be required for a satisfactoryfit of the data for both the folding and unfolding processes. Inprinciple, one should be able to fit a stretching factor for eachcomponent, but the stretching factor could reliably be fitted onlyfor the main folding phase (�3

f ) as a free parameter, because ofits clear spectral and temporal separation from the other com-ponents. The fitting parameters are collected in Table 1, and thewavelength-dependent amplitudes of the components �l(�) atroom temperature are plotted in Fig. 3 G and H. The spectra�1(�) to �3(�) (circles) correspond to the amplitudes of thechange of the signal to the final spectrum �inf(�), which hasinfinite lifetime (blue line).

It is obvious that the spectral dynamics are entirely differentin the unfolding process (which is usually studied in T-jumpexperiments) from those of the folding process (Figs. 2 and 3).In the case of folding (Table 1 and Fig. 3G), the first componentshows the blue shift of the broad bleach, observed in the timeevolution of the IR-spectra (see Fig. 2). A further blue-shift andthe disappearance of the broad bleach take place during �2

f. Thethird lifetime corresponds to the main folding phase. Theamplitudes of the first two components are much smaller thanthat of the third component and only the third phase shows adistinct temperature dependence, both in terms of time constantand the stretching factor (�3

f, and �3f , respectively). In the

unfolding process (Table 1 and Fig. 3H), the first time constantis dominated by the dynamics of the linker, because the signalsfrom the peptide are much smaller. In contrast to the folding

-1

0

1

10-1 100 101 102 103-1

0

1

10-1 100 101 102 103 10-1 100 101 102 103

1677 cm-1

1677 cm-1

B

F

1657 cm-1

1657 cm-1

1630 cm-1

1630 cm-1

6oC45oC

A

D

C

E

Time, ns

roN

la

mi

dez

∆D

O

Unfolding

Folding

8oC41oC

1620 1650 1680

,S

DA

D.a

.u

ε2

ε3

Energy, 1/cm

εinf.

εinf.

Unfolding

ε1(*0. 5)

ε1

G

ε3

ε2

HFolding

Fig. 3. Kinetic traces of folding and unfolding processes. (A–F) Dynamic helix folding (A–C) and unfolding (D–F) signals at various temperatures (black, lowesttemperature; blue, highest temperature) observed at 1,630 cm�1, 1,657 cm�1, and at 1,677 cm�1. The traces are normalized to their maxima for better comparison.The data are shown as circles, and the fits are shown as solid lines. (G and H) Room temperature decay-associated difference spectra of the folding and theunfolding processes. The infinitely long component is shown as a blue line.

Ihalainen et al. PNAS � March 27, 2007 � vol. 104 � no. 13 � 5385

BIO

PHYS

ICS

experiment, the transient signal during the subsequent 10 ns ofthe unfolding process shows clear absorption changes, a consid-erable decay amplitude [�2(�) in Fig. 3H], and a clear temper-ature dependence. The final part of the unfolding process takesplace in the 100-ns range [�3(�) in Fig. 3H). All time componentsare strongly temperature-dependent (Table 1).

DiscussionIt has been argued that the trans state of the linker stabilizes the�-helix structure but does not force the molecule into thissecondary structure (18). In the early phase of the foldingprocess the spectral responses in the amide I� difference signalare relatively minor compared with the overall signal. The broadbleach �1,630 cm�1, observed �30 ps after initiation of thefolding process (Fig. 2A), suggests that a number of (native ornonnative) hydrogen bonds responsible for the small helicity inthe cis conformation break because of the isomerization of thelinker. However, the conformational space of the initial state inthe unfolding experiment is much narrower (see Fig. 1A),especially at low temperatures, and therefore the isomerizationof the linker can be expected to rapidly lead to large changes inthe peptide. Although the signal from the linker itself is domi-nant in the unfolding experiment, the effects of the isomerizationof the linker on the peptide are observable as a narrow bleachat 1,630 cm�1 as early as 70 ps after the laser flash, indicating animmediate breakage of almost one-third of the (native) H-bonds.The amplitude (Fig. 3D) of this phase indicates that a largernumber of hydrogen bonds are broken at lower temperatures,consistent with the larger helicity of the peptide at lowertemperatures (Table 1). However, although a considerable frac-tion of the response of the unfolded state occurs on a picosecondtimescale (up to 2 ns), further unfolding processes take place upto a few hundreds ns. The unfolding behavior of the peptideobserved during this time is similar to that observed in T-jumpexperiments on closely related peptides (9–13), suggesting thatour molecule behaves in a way similar to unlinked �-helices.Although one should keep in mind that the photoswitch mightreduce the accessible configuration space, the molecule stillshows much of the complexity of protein folding.

In agreement with earlier studies (9, 10, 13, 14, 18), we findthat the folding and unfolding of an �-helix are strongly ther-mally activated (Figs. 3 and 4). From the slopes of the logarithmicrate constant as a function of reciprocal temperature (Fig. 4),one would conclude that a large enthalpic barrier exists betweenfolded and unfolded states (31 kJ/mol for folding and 37 kJ/molfor unfolding). In a two-state picture, such a high barrier wouldlead to single-exponential kinetics, in clear contradiction to theexperimentally observed nonexponential response.

In our previous study we resolved the conflict between a highapparent folding barrier on the one hand and nonexponential

kinectics on the other hand by introducing a diffusive process ona relatively shallow one-dimensional free energy surface (18). Inthis case, the temperature dependence would be governed by asuper-Arrhenius law (48); that is, by many but much smallerbarriers on a rugged energy surface. Initiation–propagationmodels, in contrast, would provide a reasonable fit for theunfolding experiment for any given temperature.

However, without adding a strongly temperature-dependentdiffusion constant to the model (with a variation that exceedsthat expected because of the change of solvent viscosity),initiation–propagation models cannot resolve the conflict be-tween apparent high activation barrier and nonexponentialkinetics (15). This is because the free energy surface consists oftwo wells, i.e., effectively a two-state systems, except when thebarrier is in the range of kBT or smaller [which is the case whena double (31) or multiple (33) sequence approach is used].Furthermore, as discussed in detail and on quite general groundsin ref. 33, initiation–propagation models (as well as downhillfolding models) have the tendency to produce compressed ratherthan stretched exponential kinetics for the folding direction,where the ensemble is approaching the state the spectroscopicobservable is most sensitive to. In fact, compressed kinetics hasbeen predicted already quite some time ago for the foldingdirection (see figure 10 of ref. 14) but remained undiscussed.

All these models have in common that they try to reduce thekinetics onto a one-dimensional free energy surface. In this case,the total of all experimental observations (i.e., folding andunfolding kinetics of one and the same molecule including allrelevant timescales from picoseconds to microseconds and in alarge temperature range) render constraints on the nature of thefree energy surface that are difficult to resolve. If, however, onegives up the assumption of diffusion on a one-dimentionalsurface, the constraints are significantly less. Indeed, in networkanalyses of the folding of similarly small peptides, it has beenargued that the conformational ensemble of small peptides insolution is composed of relatively few clusters of states (40, 41,39, 49), comprising the native (folded) state and misfolded statestrapped by nonnative hydrogen bonds and potentially salt-bridges. The network analysis furthermore suggests that onemisfolded state can transfer into another misfolded state onlythrough a hub, whereas the direct transfer between misfoldedstates is significantly slower (40). Reducing these ideas to a

T1 T2

T3

T4T5

U

T1 T2

T3

T4T5

F

T1 T2

T3

T4T5

U

T1 T2

T3

T4T5

F

U

FT1-T5U F

HU

-TSU

HU

-TSUkf

kt

kt

kf

T1-T5

A B

C D

Fig. 5. Kinetic model. Kinetic scheme connecting the folded state (F) with afew traps (T1-T5) through an ensemble of unfolded states (U) in a star-likemanner. The thickness of the arrows connecting the states symbolize a distri-bution of rates. (A and B) Shown are the population flow in folding (A) andunfolding (B) experiments, respectively. (C and D) Energetics of the relevantstates at low (C) and high (D) temperatures.

3.2 3.4 3.6

6.0

6.5

7.0

goL

01

[k

s-1]

Folding

Unfolding

1000/ T

Fig. 4. Logarithmic folding and unfolding rates as a mean decay rate atvarious temperatures estimated from the integrated area under the normal-ized signal at 1,630 cm�1 and at 1,677 cm�1 for folding and unfolding,respectively.

5386 � www.pnas.org�cgi�doi�10.1073�pnas.0607748104 Ihalainen et al.

simple kinetic scheme, one arrives at a model where an ensembleof unfolded states U is connected to a set of misfolded traps ina star-like manner (Fig. 5). Here, we discuss to what extent sucha scenario can account for our experimental findings.

In the folding experiment (Fig. 5A), all traps are initiallypopulated and feed into the folded states (F) through theunfolded ensemble U. The response, therefore, will be a rela-tively nonspecific sum of all contributions that results instretched exponential relaxation provided the individual rates kt,are all different. Solving a system of rate equations according toFig. 5A with equally distributed barrier heights (assuming thatthe folding rate kt is not rate limiting, see below) shows that aspread of rates �9 is required to obtain a stretching factor of � �0.8 (a spread of �20 is needed for the larger stretching factor � �0.7 in ref. 18). Thus, although a stretching factor of � � 0.8appears to be a relatively small deviation from exponentialbehavior, it can be the result of dramatic effects on a microscopiclevel (50).

In the unfolding experiment (Fig. 5B), in contrast, the processstarts from the better defined folded state F. Initially, it will feedonly into kinetically favored states (Fig. 5B, T1). Only afterlonger times will thermodynamic equilibrium be achieved, whichmay even disfavor the kinetically favored states. In an unfoldingexperiment, the ensemble of trajectories will initially be muchmore focused than in the folding experiment, because it startsfrom a defined state F, rather than from a broad distribution ofstates. In other words, the ensemble of trajectories will initiallyfollow a relatively specific pathway. This is in qualitative agree-ment with a number of experimental observations: the distinctbiphasic kinetics (Fig. 3E), the large amplitude �2(�) observed inthe early phase of the unfolding experiment (Fig. 3H), and thestronger wavelength dependence in the unfolding experiment(Fig. 3 E and F versus Fig. 3 B and C). In fact, solving a rateequation system according to the model in Fig. 5B leads to twodistinct timescales for the unfolding experiment. Hence, thecounterpart to the stretched exponential response in the foldingexperiment (component �3

f in Table 1) is the biphasic responsein the unfolding experiment with two distinct timescales (com-ponents �2

u and �3u in Table 1). Component �2

f is negligibly smallin the folding experiment (Fig. 3G). Solving the same system ofrate equations furthermore suggests that the ratio of foldingversus unfolding rates is a qualitative measure of the number ofaccessible traps (if one assumes that the photoswitch modifiesthe energetics of the folded state solely, and not that of anybarrier relative to the traps). As we observe a value of �5 for thatratio experimentally, we conclude that indeed only relatively fewsuch traps exist. In part, this might be due to the photoswitch thatreduces the accessible configuration space of the molecule.

The average rate increases with temperature and at the sametime the nonexponential response disappears (Table 1). In theframework of the model discussed here, this can be understoodas follows: The unfolded ensemble U is the one with the highestentropy (because, as open structure it has the largest confor-mational space) and the highest enthalpy (because hydrogen-bonds are missing). As such, its free energy varies strongest withtemperature, such that it might effectively act as a transition stateat low temperatures (Fig. 5C). In this case, the inhomogeneousdistribution of rates between U and the trapped states arerelevant, rendering the overall kinetics nonexponential. At hightemperatures, in contrast, the unfolded state U is lowered,shifting the equilibrium toward the unfolded state U, away fromboth the folded state F and the traps Ti (Fig. 5D). The moleculesthat fold directly out of the unfolded state U will lead toessentially exponential kinetics. Furthermore, the folding barrierhas disappeared, and the individual rates from the traps willapproach the more uniform ‘‘speed limit’’ of folding on anessentially f lat free energy surface (22, 23). In fact, the fastesttime constants we observe in our experiment (�2 in Table 1)

coincide with reported values of the folding speed limit. Inagreement with experiment, the transition between nonexpo-nential and exponential kinetics will occur in the same temper-ature regime as the shift of the equilibrium constant, because thetemperature variation of the unfolded state U is responsible forboth effects.

If one were to combine all misfolded traps Ti together with theunfolded ensemble U into one thermodynamic state, that statewould be entropically lowered, and one could regain an initia-tion–propagation model. From the perspective of thermodynam-ics, this is, of course, possible. From the perspective of kinetics,however, such an unification is meaningful only if the folding-rate kt were the rate limiting step. In this case, however, atwo-state model would effectively be recovered, leaving us againwith the conflict between high apparent folding barrier andnonexponential kinetics. Similarly, if the traps were connectedamong each other directly by fast rates, and not through a hub,one could again unify them, still yielding the inconsistency of aone-dimensional reaction coordinate. Hence, within the frame-work of the star-like model Fig. 5, we can indeed provideexperimental validation to the theoretical suggestion of theexistence of a hub for folding (40). The hub would be theunfolded state U, but because that is coupled to the folded stateF in a non-rate-limiting manner, the latter would effectively bea hub as well (40).

ConclusionIn a one-dimensional diffusion model, the total of all experi-mental observations renders constraints on the nature of the freeenergy surface that are difficult to solve and tend to producecontradictions in terms. These problems disappear when givingup the one-dimensional assumption. However, it should bestressed that the problem is highly underdetermined experimen-tally, and current experiments do not allow one to uniquelyresolve it. Only all-atom molecular dynamics simulations canprovide the information content sufficient to distill out physicalpictures. The set of experimental data presented here is morecomplete than any experiment so far, and sets clear benchmarksfor comparison with computational results. To summarize ourkey observations: (i) both folding and unfolding show complexspectral kinetics at all time ranges from picoseconds up tomicroseconds, (ii) the kinetics of both folding and unfoldingprocesses show strong temperature dependence, (iii) differenttypes of kinetics are obtained when one observes folding orunfolding process at different wavelengths within the amide I�band, (iv) the spectral responses are different in the folding andunfolding experiments, and finally (v) the whole data set can beexpressed with a rather simple star-like model. All this has beenobserved for one molecule. Molecular dynamics simulations ofa photoswitchable helical peptide are helical peptide are cur-rently underway to investigate the kinetics at a atomic level ofdetail and compare them with our experimental data.

Materials and MethodsPeptide Synthesis. The 16-residue peptide was prepared by usingFmoc-based solid phase peptide synthesis methods (25) (JPTPeptide Technologies, Berlin, Germany). The two cysteine res-idues were crosslinked with the photoisomerizable linker ac-cording to refs. 24 and 25 to obtain the photoswitchable AARApeptide. For the spectroscopic experiments, TFA was removedby liquid chromatography (Bond Elut SAX; Varian, Palo Alto,CA) columns rinsed with H2O, 10 mM phosphate buffer, and 1mM HCl.

Steady-State IR and CD Measurements. The desired state of thephotoswitch was obtained by properly filtered high-power Hglight before taking IR and CD spectra in FTS 175C (Bio-Rad,Cambridge, MA) or Jasco (Gross-Umstadt, Germany) Model

Ihalainen et al. PNAS � March 27, 2007 � vol. 104 � no. 13 � 5387

BIO

PHYS

ICS

J-710 spectrometers, respectively. The helix content of bothconformations was estimated from the CD signal at 222 nm asdescribed in ref. 24.

Time-Resolved IR Spectroscopy. The dissolved sample was circulatedin a closed cycle CaF2 flow cell with a 100-�m optical pathlength(51). The closed cycle was thermostated to �1°C. The dark-adaptedAARA peptide is in the trans-azo conformation (ccis � 1%) (25).To monitor unfolding, the transition from trans (folded) to cis(unfolded) was initiated by a short (700-fs) laser pulse at 380 nm.Between the experiments at different temperatures, the sample washeated to 318 K in darkness for 30 min to relax the small amountof molecules (ccis � 10%) accumulated in the cis conformation backto trans-azo conformation. To monitor folding, the initial cis statewas prepared by using continuous UV irradiation with an Ar-IonLaser (363 nm, 50 mW; Coherent Innova 100, Santa Clara, Cali-fornia). The transition from cis to trans was initiated by a 700-fslaser pulse at 425 nm.

The evolution of the peptide after photoswitching the linkerwas monitored by time resolved IR spectroscopy. A setup

consisting of two synchronized 1-kHz Ti:sapphire-oscillator/regenerative amplifier femtosecond laser systems (Spectra Phys-ics, Mountain View, CA) was used (52). The output of system 1was frequency doubled to generate pulses at 380 nm or 425 nm.The output of system 2 was used to pump an optical parametricamplifier with a difference frequency-mixing stage to obtain IRprobe pulses (100 fs, center frequency 1,620 cm�1, bandwidth240 cm�1 FWHM) (53). The delay between the pulses of systems1 and 2 was controlled electronically. The IR output of theoptical parametric amplifier was split into a probe and a refer-ence beam, which were focused into the sample with a spot sizeof 80 �m. The probe beam was centered in the UV pump spot(120 �m) and the reference beam passed the flow cell 1 mmupstream. Probe and reference beams were frequency-dispersedin a spectrometer (Triax Series, Jobin Yvon, France) and imagedonto a 2 32 pixel HgCdTe detector (Infrared Associates,Stuart, IL) array.

We thank Amedeo Caflisch for many instructive discussions and EllenBackus for careful reading of the manuscript. The work was supportedby Swiss Science Foundation Grant 200020-107492/1.

1. Daniel RM, Dunn RV, Finney JL, Smith JC (2003) Annu Rev Biophys BiomolStruct 32:69–92.

2. Hagen SJ, Eaton WA (1996) J Chem Phys 104:3395–3398.3. Yang WY, Gruebele M (2004) J Am Chem Soc 126:7758–7759.4. Gruebele M (2005) C R Biologies 328:701–712.5. Naganathan AN, Doshi U, Fung A, Sadqi, Munoz V (2006) Biochemistry

45:8466–8475.6. Rohl CA, Baldwin RL (1998) Meth Enzymol 295:1–26.7. Ferguson N, Fersht A (2003) Curr Opin Struct Biol 13:75–81.8. Gruebele M (2005) Protein Folding Handbook, eds Buchner J, Kiefhaber T

(Wiley, New York), pp 454–490.9. Huang, C.-Y, Getahun Z, Zhu Y, Klemke JW, DeGrado WF, Gai F (2002) Proc

Natl Acad Sci 99:2788–2793.10. Williams S, Causgrove TP, Gilmanshin R, Fang KS, Callender RH, Woodruff

WH, Dyer RB (1996) Biochemistry 35:691–697.11. Huang, C-Y, Klemke JW, Getahun Z, DeGrado WF, Gai F (2001) J Am Chem

Soc 123:9235–9238.12. Petty SA, Volk M (2004) Phys Chem Chem Phys 6:1022–1030.13. Werner JH, Dyer RB, Fesinmeyer RM, Andersen NH (2002) J Phys Chem B

106:487–494.14. Thompson PA, Eaton WA, Hofrichter J (1997) Biochemistry 36:9200–9210.15. Thompson PA, Mu noz V, Jas GS, Henry ER, Eaton WA, Hofrichter J (2000)

J Phys Chem B 104:378–389.16. Lednev IK, Karnoup AS, Sparrow MC, Asher SA (1999) J Am Chem Soc

121:8074–8086.17. Schwarz G, Seelig J (1968) Biopolymers 6:1263–1277.18. Bredenbeck J, Helbing J, Kumita JR, Woolley GA, Hamm P (2005) Proc Natl

Acad Sci USA 102:2379–2384.19. Hummer G, Garcia AE, Garde S (2000) Phys Rev Lett 85:2637–2640.20. Zhou Y, Karplus M (1999) Nature 401:400–402.21. Lazaridis T, Karplus M (1997) Science 278:1928–1930.22. Bieri O, Wirz J, Hellrung B, Schutkowski M, Drewello M, Kiefhaber T (1999)

Proc Natl Acad Sci USA 96:9597–9601.23. Lapidus LJ, Eaton WA, Hofrichter J (2000) Proc Natl Acad Sci USA 97:7220–

7225.24. Kumita JR, Smart OS, Woolley GA (2000) Proc Natl Acad Sci USA 97:3803–

3808.

25. Flint DG, Kumita JR, Smart OS, Woolley GA (2002) Chem Biol 9:391–397.26. Nagele T, Hoche R, Zinth W, Wachtveitl J (1997) Chem Phys Lett 252:489–495.27. Sporlein S, Carstens H, Renner HSC, Behrendt R, Moroder L, Tavan P, Zinth

W, Wachtveitl J (2002) Proc Natl Acad Sci USA 99:7998–8002.28. Bredenbeck J, Helbing J, Sieg A, Schrader T, Zinth W, Renner C, Behrendt

R, Moroder L, Wachtveitl J, Hamm P (2003) Proc Natl Acad Sci USA100:6452–6457.

29. Chen E, Kumita JR, Woolley GA, Kliger DS (2003) J Am Chem Soc125:12443–12449.

30. Gruebele M (2002) Curr Opin Struc Biol 12:161–168.31. Doshi U, Munoz V (2004) J Phys Chem B 108:8497–8506.32. Doshi U, Munoz V (2004) Chem Phys 307:129–136.33. Hamm P, Helbing J, Bredenbeck J (2006) Chem Phys 323:54–65.34. Berezhkovskii A, Szabo A J Chem Phys 122:014503, 2005.35. Best RB, Hummer G (2005) Proc Natl Acad Sci USA 102:6732–6737.36. Henry ER, Eaton WA (2004) Chem Phys 307:163–185.37. Hummer G, Garcia AE, Garde S (2001) Proteins 42:77–84.38. Mu Y, Nguyen P, Stock G (2005) Proteins 58:343–357.39. Krivov SV, Karplus M (2004) Proc Natl Acad Sci USA 101:14766–14770.40. Rao F, Caflisch A (2004) J Mol Biol 342:299–306.41. Caflisch A (2006) Curr Opin Struc Biol 16:71–78.42. Mikhonin AV, Asher SA (2006) J Am Chem Soc 128:13789–13795.43. Barth A, Zscherp C (2002) Q Rev Biophys 35:369–430.44. Krimm S, Bandekar J (1986) Adv Protein Chem 38:181–364.45. Hamm P, Ohline SM, Zinth W (1997) J Chem Phys 106:519–529.46. Borisenko V, Woolley GA (2005) J Photochem Photobiol 173:21–28.47. van Stokkum IHM, Larsen DS, van Grondelle R (2004) Biochim Biophys Acta

1657:84–104.48. Zwanzig R (1988) Proc Natl Acad Sci USA 85:2029–2030.49. Krivov SV, Karplus M (2006) J Phys Chem B 110:12689–12698.50. Chekmarev SF, Krivov SV, Karplus M (2005) J Phys Chem B 109:

5312–5330.51. Bredenbeck J, Hamm P (2003) Rev Sci Instrum 74:3188–3189.52. Bredenbeck J, Helbing J, Hamm P (2004) Rev Sci Instrum 75:4462.53. Hamm P, Kaindl RA, Stenger J (2000) Opt Lett 25:1798–1800.

5388 � www.pnas.org�cgi�doi�10.1073�pnas.0607748104 Ihalainen et al.