J. Phys. Chem. B 2007, 111, 10481-10486
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Infrared Studies of Small Azobenzene Peptides: Unexpectedly Slow Reactions on the Time Range of Minutes Florian O. Koller,†,‡ Rossana Reho,† Tobias E. Schrader,†,‡ Luis Moroder,§ Josef Wachtveitl,| and Wolfgang Zinth*,†,‡ Lehrstuhl fu¨r BioMolekulare Optik Department fu¨r Physik, Ludwig-Maximilians-UniVersita¨t Mu¨nchen, Oettingenstrasse 67, D-80538 Mu¨nchen, Germany, Munich Center for Integrated Protein Science, Butenandtstrasse 5-13, D-81377 Mu¨nchen, Germany, Max-Planck-Institut fu¨r Biochemie, Am Klopferspitz 18A, 82152 Martinsried, Germany, and Institut fu¨r Physikalische und Theoretische Chemie and Institut fu¨r Biophysik, Johann Wolfgang Goethe-UniVersita¨t, Max Von Laue Strasse 7, 60438 Frankfurt, Germany ReceiVed: March 16, 2007; In Final Form: June 27, 2007
Infrared absorption experiments on light-triggered azobenzene peptides have been performed below and above the freezing point of the solvent dimethyl sulfoxide (DMSO). Even 20 K below the freezing point, illumination of the azobenzene chromophore resulted in IR absorption changes indicative of light-induced structural rearrangements of the peptide. In addition, new conformational states could be found at low temperature, which involve the formation of additional hydrogen bonds. In one sample the new low-temperature state survived melting and reheating and disappeared at 298 K only on the time scale of 10 min. The observations indicate that at low temperatures the peptide, together with traces of water present in the sample, forms a shell in the vicinity of the chromophore that facilitates internal motion even in the rigid cage of frozen DMSO.
1. Introduction Protein folding is one of the key processes in living organisms. However, even after many years of intense research activities, a molecular understanding of protein folding is still incomplete. In large proteins, folding from the amino acid chain synthesized by ribosomes into the correct three-dimensional structure, the native state, involves reactions that span many orders of magnitude in time, from the picosecond rotation of the molecular bonds and rearrangements to external forces over the formation of secondary structures in the nanosecond and microsecond range to the correct arrangement reached on the time scale of seconds.1-7 The many dimensions of the protein folding process and the wide range of relevant time scales do not allow for a simple solution of the folding problem. It is evident that on the way to a solution one has to separate complex proteins into prototypical substructures. Small model systems offer the chance to follow folding dynamics over the complete time range of the structural changes and to perform realistic simulations on a molecular level. In this respect, the combination of two approaches appears promising to gain more information on the molecular background and on the dynamics of folding reactions: (i) reducing the system size from large proteins to peptides containing only a small number of amino acids will shorten the overall folding time, and (ii) incorporating a photoswitchable dye molecule into the amino acid backbone will allow initiation of large-scale structural changes at a defined time. This combination should allow experimental studies over the whole (but limited) period of structural changes and to follow even the fastest parts of a folding reaction by means of IR * Corresponding author: phone +49 89 2180 9201; fax +49 89 2180 9202;
[email protected]. † Ludwig-Maximilians-Universita ¨ t Mu¨nchen. Mu¨nchen, Germany, ‡ Munich Center for Integrated Protein Science. § Max-Planck-Institut fu ¨ r Biochemie. | Johann Wolfgang Goethe-Universita ¨ t.
spectroscopy. Recently it has been shown by ultrafast experiments on azobenzene peptides that structural changes can be induced by the azobenzene chromophore and that these conformational changes occur on the time scale of a few tens of picoseconds.8-15 For the light-triggered cyclic octapeptide bicyclic 4-(aminomethyl)phenylazobenzoic acid (bcAMPB), it was demonstrated that the most prominent rearrangements of the amino acid chainsobserved by ultrafast spectroscopy in the infrared of the structure sensitive amide I bandsoccur within 20 ps:16,17 within that short period strong absorption changes take place, and finally the IR difference spectrum of the amide I range has adopted a very similar shape as compared to the steady state-difference spectrum. Only weak additional absorption changes have been found on longer time scales. For detailed understanding of the structural dynamics in these small cyclic chromopeptides, it would be interesting to know if the major structural dynamics always occur on the ultrafast time scale or if much slower processes may be found routinely or under certain environmental conditions. In this paper we present experimental results on different azobenzene peptides studied at lower temperatures as well as at ambient temperature. We will show that illumination may induce reversible and irreversible changes of the azobenzene peptides even below the freezing point of the solvent dimethyl sulfoxide (DMSO). Under certain sample conditions, changes of the absorption spectrum were generated at low temperatures that persist after warming to room temperature even for many minutes, pointing to very slow structural rearrangements. 2. Materials and Methods The investigated chromopeptides contain the azobenzene dye 4-(aminomethyl)phenylazobenzoic acid (AMPB) as a backboneintegrated photoswitch. The investigated peptide was the active site octapeptide fragment of thioredoxin reductase (H-Ala-Cys-
10.1021/jp072117g CCC: $37.00 © 2007 American Chemical Society Published on Web 08/10/2007
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Figure 1. Schematic structures of the bicyclic (bc), cyclic (c), and linear (l) AMPB peptides. In bcAMPB, the disulfide bridge between the two cysteines is closed. In cAMPB and lAMPB, reactive amino acid side chains are chemically protected. (Inset) Visible spectra of cis- and transbcAMPB. Gray areas indicate the spectral regions of illumination.
Ala-Thr-Cys-Asp-Gly-Phe-OH). The AMPB photoswitch allows induction of conformational changes in the peptide moiety: In the trans state AMPB has a longer end-to-end distance (∼12 Å) than in the cis state (∼9 Å). The light-induced isomerization is completely reversible. The trans isomer is stable. At room temperature the cis isomer thermally relaxes into the trans conformation on a time scale of several tens of hours. The syntheses of the chromophore 4-(aminomethyl)phenylazobenzoic acid (AMPB) as well as of the three peptides, linear (l), monocyclic (c), and bicyclic (bc) AMPB, have been reported elsewhere.18,19 A schematic view of the chromopeptides is given in Figure 1. In the linear peptide lAMPB with unprotected amino and carboxy termini, the thiol groups of both cysteines are chemically protected as tert-butylthio derivatives. In the monocyclic (cAMPB) peptide the two cysteine residues are still protected in order to avoid their oxidation to the disulfide and reduction of the azobenzene group by the thiol groups. The bicyclic (bcAMPB) peptide contains a disulfide bond between the two cysteines, thus forming a bicyclic structure. With the help of NMR methods, the structure of cAMPB and bcAMPB has been determined for the cis and trans isomers.20 In bcAMPB the two loops impose severe constraints on the structure. For the trans form of the AMPB dye one finds a well-defined straight arrangement of the peptide, while in the cis form the peptide acquires looplike structures. In cAMPB more flexible and less confined structures are found, and for lAMPB no well-defined structure is expected. Nevertheless there are weak but distinct differences in the peptide arrangement when the chromophore is switched between the cis and trans forms, as seen via differences in the amide I range of the IR absorption spectrum. For the experiments, the peptides were dissolved in nondeuterated DMSO (Aldrich 27,685-5, anhydrous, 99.9%, used as
delivered) at concentrations in the range 8-11 mM. At these concentrations, no indication of aggregation (characteristic absorption bands in the ranges 1610-1630 and 1680-1690 cm-1 21) were observed. The water (H2O and D2O) content of the samples was determined by the infrared absorbance at 3500 and 2550 cm-1, respectively, considering the known extinction coefficient of water and the solvent background. Samples with different water contents were investigated. The water content of the investigated “dry” samples was below 0.2% volume of water ( 400 nm) in the nπ* band, where the cis form of the AMPB chromophore has a larger absorption coefficient. The illumination in the two wavelength regions was performed by two different light sources coupled to the samples by means of light guides. A Hg(Xe) short arc lamp in combination with color glass filters (UG1, 1 mm, and WG320, 2 mm, from ITOS, Mainz, Germany) supplied light between 320 nm and about 385 nm (see shaded areas in Figure 1 inset) at a power of 120 mW,
Unexpectedly Slow Reactions of Azobenzene Peptides irradiating a cross-section of approximately 2 cm2 of the sample. For the production of the trans isomer, a cold light source (used color glass filters: KG2, 2 mm, and GG400, 2 mm, also from ITOS, transmission range between 400 and 800 nm) was applied (power 500 mW). With the two illumination procedures the peptides can be photoswitched efficiently, leading to the population of cis and trans forms with selectivities of > 80% in the photostationary state, which was reached after about 90 s of illumination. In the presented experiments shown below, an even longer illumination time of 180 s was chosen. The UV/vis spectra were measured with a Lambda 19 UV/ vis/near-IR spectrometer from Perkin-Elmer (Wellesley). For the infrared measurements, a Fourier transform infrared (FTIR) spectrometer from Bruker (Ettlingen, Germany, model IFS 66) was used. In order to record small absorbance changes, we averaged over 50 single scans. The time to measure one FTIR spectrum was about 60 s. This 60 s give also the highest achievable temporal resolution in our experiments. Many spectra (Figures 2-4) presented in this paper show differences between FTIR spectra recorded on one sample before and after illumination. They show the differences induced by the photoisomerization of the AMPB chromophore. Only bands that are influenced by the illumination procedure are observed. In the following, cis* denotes the difference spectrum one obtains when the photostationary trans spectrum is subtracted from the photostationary cis spectrum. Correspondingly, trans* denotes the photostationary trans spectrum minus the cis spectrum. The cis* difference spectrum displays cis bands with positive amplitudes and trans bands with negative amplitudes. During the measurement of the infrared spectra, the samples were kept in home-built, temperature-controlled cuvettes with 2 mm thick CaF2 windows and an effective aperture of 20 mm. A poly(tetrafluoroethylene) (PTFE) spacer defined the thickness of the sample solution to about 100 µm. The housing of the cuvettes was kept in good heat contact to the windows. The holder of the cuvettes could be tempered by flowing a thermostated water/ethylene glycol mixture through it or by a Peltier element. The temperature was controlled via a sensor (LM335, National Semiconductor, Santa Clara, CA) in the holder near the cuvette. This sensor was calibrated by a Pt100 thermometer placed within a test cuvette. The temperature accuracy and stability during the FTIR measurements was about 1 K in the applied temperature range from 260 to 310 K. Test experiments have shown that the sample volume reached the temperature of the holder within 2 min at an accuracy of (1 K. The temperature of the sample could be changed with a cooling rate of 1.5 K/min (Peltier element) and a heating rate of 10 K/min (water/ethylene glycol mixture). For the measurement of temperature dependencies, the samples were examined at 263 and 298 K. The value of 263 K was chosen in order to investigate the sample at a temperature well below its melting point. The melting point of pure DMSO is 292 K;19,22 DMSO containing the dissolved peptides freezes around 284 K, indicating that the peptide molecules slightly influence the structure of the frozen DMSO. From the IR absorbance spectra, the state of aggregation of the sample could be determined unambiguously: Spectral regions where the liquid sample shows negligible absorption have considerably lowered transmission in the frozen state, apparently due to increased scattering; between 2000 and 2400 cm-1, weak and very narrow bands appear in the frozen state. Freezing and heating were performed in the FTIR spectrometer without visible illumination. Before each measurement at the low-temperature value, 263 K, the samples were kept at this temperature for at least 30
J. Phys. Chem. B, Vol. 111, No. 35, 2007 10483
Figure 2. Typical FTIR difference spectra of dry bcAMPB at 298 K around the amide I (1620-1720 cm-1), amide II (1500-1550 cm-1), and AMPB chromophore (1550-1620 cm-1) regions. The spectral ranges of these distinct normal modes are shaded. The spectra were taken after 3 min of illumination.
min in the dark to allow equilibration. The heating from 263 to 298 K, including melting and thermal equilibration of the sample, took about 4.5 min. The difference spectra were taken only after equilibration at constant temperatures. For observation of temporal evolution after heating, a baseline spectrum was taken at the moment when the sample had equilibrated to room temperature (298 K). This was ca. 5 min after the heating began and about 3 min after the melting point was passed. That measured baseline spectrum also defines time 0 in Figure 5. Subsequently, every 5 min, spectra were recorded and the difference relative to the baseline spectrum was calculated. 3. Results In the following we present the results of three different experiments. The first set of data shows light-induced difference spectra recorded for the various dry and wet samples at two temperatures. In the second experiment, the dry bcAMPB was reheated after 30 min at low temperature in the dark, and the evolution of the IR spectrum is presented. Finally, we show the response of the reheated sample to illumination. 3.1. Photoswitching at Room Temperature and below the Freezing Point. The change of the IR absorption spectrum of bcAMPB after appropriate illumination is shown in Figure 2 for ambient temperature (298 K). We concentrate on the spectral range 1500-1750 cm-1, where most prominent bands of the AMPB peptides are seen: the amide I range between 1620 and 1720 cm-1, representing basically the CdO double bond stretching vibration of the amide group,23 the amide II region (1500-1550 cm-1, a combination of NH deformation and CN stretching vibrations), and the region from 1550 to 1620 cm-1, where difference bands from the AMPB chromophore appear.24,25 The trans* spectrum of bcAMPB reveals strong absorption changes in the amide I and II range, pointing to considerable structural variations upon photoisomerization of the chromophore. The absorption changes in the chromophore bands (e.g., around 1600 cm-1) are well visible but are significantly smaller than those in the peptide bands. The cis* and trans* spectra are symmetric to each other throughout the whole investigated spectral range. This points to a highly reversible switching of the bcAMPB peptide at room temperature. This was also controlled by repetitive switching of the bcAMPB sample: Even after 80 photoswitching cycles, the difference spectra show no indication for degradation.
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Figure 3. Light-induced trans* difference spectra of bc-, c-, and lAMPB (from left to right). Top: typical spectra of dry samples at 298 K. Middle: typical spectra of frozen wet samples at 263 K. Bottom: typical spectra of frozen dry samples at 263 K. The baseline is plotted as a dashed line.The cis* spectra (not shown) are symmetric to the trans* spectra, hence the light-induced processes are reversible. The spectral range with pronounced chromophore absorption is marked by the gray background.
The room-temperature trans* difference spectra of the dry samples can be seen in Figure 3, upper part. The cyclic cAMPB peptide (panel b) shows a trans* difference spectrum similar to that of bcAMPB. In the chromophore range, one finds absorption changes of similar size and shape as for bcAMPB. Alterations are evident in the peptide bands: the absorption changes in the amide I band are broader in bcAMPB, and the decrease in the amide II band of cAMPB has a much stronger modulation. The trans* spectrum of linear lAMPB peptide at room temperature (panel c, upper trace) deviates strongly from that of the other peptides at room temperature: The spectral features due to the AMPB chromophore still have the same frequency dependence as for cAMPB and bcAMPB. However, the signals in the peptide bands differ strongly. Taking the amplitude of the AMPB band as a reference, we find that the reaction of the peptide upon isomerization is much weaker in lAMPB. In addition, the shapes of the amide I and II bands are completely different from those of bc and cAMPB. These observations support the interpretation that the isomerization of the AMPB chromophore causes much smaller structural changes in lAMPB than in the cyclic and bicyclic AMPB peptides.8,17 When the samples are cooled down below the freezing point of the solvent DMSO, we also find pronounced changes in the difference spectra of the azobenzene peptides (see Figure 3, middle and lower traces). Surprisingly, the absorption spectra depend on small traces of water in the sample. For the lowest water concentration realized in the experiment (cwater< 0.2% vol, lowest traces in Figure 3; we call this condition “dry”), the cyclic peptide cAMPB shows negligible changes of the IR spectrum upon illumination. Only in the range of the chromophore bands are some very weak features visible. The dry bcAMPB shows also weak difference bands in the chromophore range. However, there is a significant new band in the amide I range at 1635 cm-1. This band has positive amplitude in the trans* spectrum, indicating that the peptide in the trans form shows increased absorption in this spectral range. Linear lAMPB peptide exhibits similar spectral features both at low (263 K) and at room temperature (298 K) in the amide II and chromophore ranges. Other relatively small absorption changes in the amide I range exist at low temperature, but they are different from those of the sample at room temperature. Surprisingly,
the lAMPB sample exhibits a very narrow and strong spike of increased absorption at 1635 cm-1 at low temperature. Samples with somewhat increased water content show interesting deviations in the spectra at low temperature: “wet” bcAMPB does not exhibit the pronounced 1635 cm-1 peak but displays absorption features resembling those of room-temperature difference spectra. Similar behavior is found for wet cAMPB. It should be noted that for both samples the relative amplitudes of the changes in the peptide bands as compared to those in the chromophore bands are different. It appears that the changes in the chromophore regionswhere the optical excitation is directly appliedsare larger than those in the peptide bands. The wet lAMPB at low temperature displays chromophore and amide II bands similar to those of the lowtemperature dry and the room-temperature sample. The amide I range differs, and there is a strong absorption increase again at 1635 cm-1. For the wet lAMPB sample, the 1635 cm-1 band is considerably broader than in the dry sample. Finally it should be noted that the AMPB chromophore without peptide undergoes isomerization at both room and low temperature independent of the water content, showing the typical bands between 1550 and 1620 cm-1 (data not shown). Upon going to even lower temperatures (∼250 K), also the remaining light-induced spectral differences decrease significantly, and hence switching behavior of the AMPB then vanishes at these temperatures. These observations may be summarized as follows: All three AMPB peptides show significant and reversible absorption changes at room temperature, not influenced by the water content (data not shown). Surprisingly, we also find absorption differences when the solvent is frozen. While all AMPB peptides feature low-temperature absorption differences (reflecting structural changes of the peptide and the chromophore part) when water is present, lAMPB and (to a smaller extent) bcAMPB show this behavior also for dry examples. Astonishingly, dry bcAMPB and lAMPB exhibit a novel band at about 1635 cm-1 at low temperature. 3.2. Slow Spectral Dynamics after a Freeze and Thaw Cycle. In a second set of experiments, we investigated the different samples after a thermal treatment: the samples in the photostationary trans state were frozen and cooled down to 263 K, kept at constant temperature for at least 30 min, and
Unexpectedly Slow Reactions of Azobenzene Peptides
Figure 4. Difference spectra of dry bcAMPB at 298 K after heating from 263 K. (b) Photoswitching shows an anomaly in the amide I region around 1635 cm-1. During the first illumination process (duration of 3 min), an absorption change cis*1 is observed, which deviates largely from the changes recorded at later switching processes. Here, the spectra are identical to those found for untreated samples. (a) Build-up of the absorption change after reheating in the dark. The sample was not illuminated at 263 or 298 K. Strong spectral changes are observed in the amide I region on the time scale of many minutes.
J. Phys. Chem. B, Vol. 111, No. 35, 2007 10485 of 1 h. The temporal and spectral behavior of this effect does not depend on deuteration; that is, we observe the same behavior for samples contaminated by H2O or D2O. 3.3. Photoinduced Spectral Dynamics after a Freeze and Thaw Cycle. In order to obtain more information on the nature of the long-lasting absorption changes observed in dry bcAMPB after the freeze and thaw cycle, we performed the following illumination experiment: After the reference spectrum was recorded, repetitive illumination cycles prepared the sample in the cis and trans photostationary states. For these cycles, cis* and trans* spectra were calculated from the spectra recorded before and after the respective illumination period. Four spectra are drawn in Figure 4b. The first light-induced difference spectrum obtained after the thermal treatment is the cis*1 spectrum (thick solid black line). One finds a strong decrease in absorption at 1635 cm-1 and an absorption increase around 1675 cm-1. This cis*1 spectrum differs completely from the cis* spectra recorded on untreated samples. On the other hand, this spectrum resembles the spectral differences observed after the slow relaxation of the frozen and heated-up sample (see Figure 4a). The first trans*1 spectrum is not at all mirrorsymmetric to the cis*1 spectrum. However, it is very similar to the trans* spectrum of the untreated sample and is mirrorsymmetric to the next spectrum (cis*2) obtained in the third illumination process. Later on, the same illumination-induced difference spectra are found in the untreated sample. The experiments have shown that the room-temperature sample bcAMPBth found after a thermal treatment not only decays spontaneously into the standard form of bcAMPB but also reaches it after one single illumination process. 4. Discussion
Figure 5. Absorbance behavior of dry bcAMPB at 298 K after heating from 263 K: temporal evolution at 1672 cm-1 (top) and 1635 cm-1 (bottom), corresponding to Figure 4. Time zero is set at the moment, when the sample had reached room temperature. The temporal resolution is approximately 1 min. Biexponential fits (lines) give time constants of about 7 and 60 min. The later time constant probably is due to drift of the spectrometer.
heated back to room temperature. After this freeze and thaw treatment, we investigated the absorption spectrum as a function of time after the melting point was passed. When the samples were equilibrated at room temperature (298 K), about 4.5 min after the melting point was passed, a reference IR spectrum was taken. Later on, absorption spectra were recorded and the difference relative to the reference spectrum was plotted. For the wet samples, as well as for dry cAMPB and lAMBP, we did not observe any changes in the IR spectrum in the investigated time range of t > 5 min. For dry bcAMPB, however, we observed absorption changes displayed in Figure 4a: 1 min after the time where the reference spectrum was taken, one finds a weak absorption decrease at 1635 cm-1 and an absorption increase around 1675 cm-1. These features grow on the time scale of 10 min. In Figure 5 we plotted the time dependence of the absorbance changes observed at 1635 and 1672 cm-1. At both wavenumbers one finds a fast and rapid absorbance change with an initial time constant of = 6-8 min. Further and weaker absorbance changes occur on the time scale
When an azobenzene peptide is triggered by illumination to acquire its new conformation, large parts of the chromophore and the peptide have to be moved through the viscous solvent. The influence of the solvent has been confirmed recently when initial reaction dynamics of azobenzene peptides have been investigated in different solvents.9 Here an acceleration of the reaction dynamics takes place if the molecule reacts in a less viscous solvent. One may argue that the incorporation of the peptide into a rigid, frozen solvent should modify or even prevent the structural rearrangements. In this respect it is highly interesting that the investigated chromopeptides show pronounced absorption transients even 20 K below the freezing point of the solvent DMSO and 10 K below the freezing point of water. The results presented above show that spectral changes of both the chromophore and the peptide occur in all investigated “wet” azobenzene peptides (containing minor traces of water) and in both samples of the linear peptide lAMPB as well as to a certain extent in the “dry” bcAMPB (Figure 3). Structural switching appears to be possible if the peptide can find enough volume within the rigid DMSO cage to accomplish conformational changes. Apparently water may act as a lubricant, facilitating the light-induced structural changes. In frozen DMSO the water content seems to be considerably concentrated around the dissolved peptides. From experiments on nanometer-size pores of water, it is known that water may remain liquidlike even well below the freezing point of bulk water.26 The action as a lubricant may originate from the liquid water itself or from the changes it induces on the surrounding DMSO molecules. In the case of dry bcAMPB, we find only very specific changes of the peptide absorption at 1635 cm-1 upon illumination. The rigid DMSO cage allows here only the motion of a small part
10486 J. Phys. Chem. B, Vol. 111, No. 35, 2007 of the peptide, leading to absorption changes strongly deviating from those in wet bcAMPB or in bcAMPB in liquid DMSO. The decreased signals in the chromophore region indicate that even the ability of the chromophore to isomerize seems to be reduced (see Figure 3a, lower part) due to the rigid surrounding. For the linear molecule, which does not exhibit major structural changes of the peptide part and where consequently only small spectral changes are visible even at room temperature in the liquid state, we still observe absorption changes of similar size in frozen DMSO. In the two linear peptide samples (wet and dry lAMPB) as well as in dry bcAMPB, we find a new band at 1635 cm-1 in the light-induced difference spectra, which represents an absorption increase for the trans form of the molecule at low temperatures. This band is in a spectral range where amide groups with intramolecular hydrogen bonds or amide groups with strong dipole interactions to other amide groups absorb. We call the new state characterized by the 1635 cm-1 absorption band transLT*. The small width of the 1635 cm-1 band in lAMPB indicates that the corresponding amide group is in a well-defined and rigid surrounding. The strong light-induced absorption changes in the frozen DMSO cage indicate that this (hydrogen-bonded) amide group should be located close to the azobenzene, since other parts of the peptide should not be influenced by the isomerization of the chromophore. Presumably this band belongs to one of the carbonyl groups forming the bridge between azobenzene and the peptide chain. The absorption band at 1635 cm-1 is broader in bcAMPB than in lAMPB. The geometrical restrictions from the cyclic peptide structure do not allow it to reach a single arrangement of the peptide in the solvent cage. The experiments presented in Figures 4 and 5 show that for dry bcAMPB the new conformation transLT* survives the thaw and heating procedure and persists for several minutes at room temperature. However, one single switching process to cis and back to trans immediately destroys the transLT* form. Upon disappearance of the 1635 cm-1 species, one finds increased absorption at 1675 cm-1, in a spectral range where amide groups absorb that are not hydrogen-bonded and that are in a weakly polar or unpolar surrounding.23 The 1635 cm-1 absorbing transLT* apparently involves a specific structure of the transbcAMPB peptide. The rigid DMSO cage found at low temperatures catalyzes the formation of the transLT* structure, which is energetically favored at low temperatures for the trans conformation of the azobenzene switch. Upon photoisomerization of the azobenzene, the cis conformation of the chromophore destroys the transLT* structure and the concomitant hydrogen bond. TransLT* is re-formed when the azobenzene is brought again to the trans form. Upon heating of the sample, the rigid DMSO cage is lost and the concentration of the transLT* structure of bcAMPB decreases. Thermal motions cause the transition to other configurations; the specific hydrogen bond is broken. However, the long lifetime of this state of >7 min indicates that transLT* is separated by a considerable barrier from other structures. In conclusion, we have presented IR absorption studies on different AMPB peptides in liquid and solid DMSO. We could show (i) that even 20 K below the freezing point of the solvent, structural changes of the azobenzene chromophore as well as the peptide part occur; (ii) that a new configuration with an absorption at 1635 cm-1 appears at low temperatures for some peptides; and (iii) that this conformation is stable on the time
Koller et al. scale of minutes, even at room temperature in liquid DMSO, for the dry bcAMPB sample. It is remarkable that such a longlived intermediate can exist in a small bicyclic peptide, when the dominant light-induced structural changes occur faster by 13 orders of magnitude within tens of picoseconds. Acknowledgment. This work was supported by Deutsche Forschungsgemeinschaft through the DFG-Cluster of Excellence Munich Centre for Advanced Photonics and through the Sonderforschungsbereich (SFB) 533.For assistance in the FTIR measurements, we thank Marion Kraus. References and Notes (1) Mayer, G.; Heckel, A. Angew. Chem., Int. Ed. 2006, 45, 4900. (2) Sadqi, M.; Fushman, D.; Munoz, V. Nature 2006, 442, 317. (3) Callender, R. H.; Dyer, R. B.; Gilmanshin, R.; Woodruff, W. H. Annu. ReV. Phys. Chem. 1998, 49, 173. (4) Gruebele, M. Annu. ReV. Phys. Chem. 1999, 50, 485. (5) Eaton, W. A.; Munoz, V.; Hagen, S. J.; Jas, G. S.; Lapidus, L. J.; Henry, E. R.; Hofrichter, J. Annu. ReV. Biophys. Biomol. Struct. 2000, 29, 327. (6) Kubelka, J.; Hofrichter, J.; Eaton, W. A. Curr. Opin. Struct. Biol. 2004, 14, 76. (7) Metzler, R.; Klafter, J.; Jortner, J.; Volk, M. Chem. Phys. Lett. 1998, 293, 477. (8) Wachtveitl, J.; Sporlein, S.; Satzger, H.; Fonrobert, B.; Renner, C.; Behrendt, R.; Oesterhelt, D.; Moroder, L.; Zinth, W. Biophys. J 2004, 86, 2350. (9) Satzger, H.; Root, C.; Renner, C.; Behrendt, R.; Moroder, L.; Wachtveitl, J.; Zinth, W. Chem. Phys. Lett. 2004, 396, 191. (10) Sporlein, S.; Carstens, H.; Satzger, H.; Renner, C.; Behrendt, R.; Moroder, L.; Tavan, P.; Zinth, W.; Wachtveitl, J. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 7998. (11) Nibbering, E. T. J.; Fidder, H.; Pines, E. Annu. ReV. Phys. Chem. 2005, 56, 337. (12) Loweneck, M.; Milbradt, A. G.; Root, C.; Satzger, H.; Zinth, W.; Moroder, L.; Renner, C. Biophys. J. 2006, 90, 2099. (13) Andruniow, T.; Fantacci, S.; De Angelis, F.; Ferre, N.; Olivucci, M. Angew. Chem., Int. Ed. 2005, 44, 6077. (14) Bredenbeck, J.; Helbing, J.; Kumita, J. R.; Woolley, G. A.; Hamm, P. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 2379. (15) Renner, C.; Moroder, L. ChemBioChem 2006, 7, 869. (16) Bredenbeck, J.; Helbing, J.; Behrendt, R.; Renner, C.; Moroder, L.; Wachtveitl, J.; Hamm, P. J. Phys. Chem. B 2003, 107, 8654. (17) Bredenbeck, J.; Helbing, J.; Sieg, A.; Schrader, T.; Zinth, W.; Renner, C.; Behrendt, R.; Moroder, L.; Wachtveitl, J.; Hamm, P. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 6452. (18) Renner, C.; Cramer, J.; Behrendt, R.; Moroder, L. Biopolymers 2000, 54, 501. (19) Cowie, J. M.; Toporowski, P. M. Can. J. Chem. 1961, 39, 2240. (20) Renner, C.; Behrendt, R.; Heim, N.; Moroder, L. Biopolymers 2002, 63, 382. (21) Singh, B. R. Infrared Analysis of Peptides and Proteins; American Chemical Society: Washington, DC, 2000. (22) D’Ursi, A.; Albrizio, S.; Fattorusso, C.; Lavecchia, A.; Zanotti, G.; Temussi, P. A. J. Med. Chem. 1999, 42, 1705. (23) Barth, A.; Zscherp, C. Q. ReV. Biophys. 2002, 35, 369. (24) Armstrong, D. R.; Clarkson, J.; Smith, W. E. J. Phys. Chem. 1995, 99, 17825. (25) Hamm, P.; Ohline, S. M.; Zinth, W. J. Chem. Phys. 1997, 106, 519. (26) Morishige, K.; Kawano, K. J. Chem. Phys. 1999, 110, 4867.
