J. Phys. Chem. B 2007, 111, 11297-11302
11297
Intramolecular Disulfide Bridges as a Phototrigger To Monitor the Dynamics of Small Cyclic Peptides Christoph Kolano,†,* Jan Helbing,† Go1 tz Bucher,§ Wolfram Sander,§ and Peter Hamm†,* Physikalisch-Chemisches Institut, UniVersita¨t Zu¨rich, Winterthurerstrasse 190, CH-8057 Zu¨rich, Switzerland, and Lehrstuhl fu¨r Organische Chemie II, Ruhr-UniVersita¨t Bochum, UniVersita¨tsstrasse 150, D-44801 Bochum, Germany ReceiVed: May 30, 2007; In Final Form: July 5, 2007
Two cyclic disulfide-bridged tetrapeptides [cyclo(Boc-Cys-Pro-Aib-Cys-OMe) (1) and cyclo(Boc-Cys-ProPhe-Cys-OMe) (2)] have been monitored by time-resolved mid-IR spectroscopy in the CdO vibrational range. A conformational change is induced by cleavage of the intramolecular disulfide bridge upon UV excitation (λexc ) 260 nm), giving rise to a pair of cysteinyl radicals (thiyl radicals), which diffuse apart allowing the peptide to change conformation before they undergo quenching. The amide I band reports on the dynamics of the peptide backbone, which evolves on a 100 ps time scale and then stays constant up to 10 µs at low enough concentrations (≈100 mM). To probe specifically the lifetime of the free cysteinyl radicals, timeresolved UV laser flash photolysis has been applied. The concentration of the cysteinyl radical decays nonexponentially, but about 50% are still present after 1 ms. The photocleavable disulfide bridge hence may serve as an intrinsic, naturally occurring phototrigger to study peptide dynamics that opens a wide timewindow from a few picoseconds to many hundreds of microseconds.
Introduction The process of peptide folding, which results in highly organized functional three-dimensional structures, remains one of the most challenging biophysical questions currently being investigated. Folding takes place on a widespread range of time scales, covering several orders of magnitude. The time domain larger than 50 ns is accessible by various time-resolved techniques like rapid-scan, step/scan, and laser flash photolysis (LFP) with IR and UV/vis detection.1-5 In addition, fast folding processes can also be monitored by resonance Raman6,7 and fluorescence techniques.8,9 These techniques provide fundamental insights on the dynamics taking place on the longer time scales. In order to distinguish between the controversially discussed models and to optimize algorithms of molecular dynamics program packages, it is of fundamental interest to also gain access to the earliest steps of peptide folding. In this perspective a variety of model systems have been investigated by fs-ps infrared spectroscopy.10-19 To study protein- or peptide folding, a triggering event that perturbs the protein/peptide from a “starting” conformation is required to initiate the process. The incorporation of a photoswitch in the peptide/protein sequence20 or the use of “caged compounds” (photolabile protecting groups)21-23 is one possibility, and a variety of these concepts have been introduced successfully into biomolecules and applied to time-resolved spectroscopies.19,24-29 A disulfide bridge can be understood as a predetermined breaking point, which can be used to initiate conformational dynamics. Disulfide bridges occur in natural peptides and proteins and play an important role in defining their threedimensional structure. They are weak covalent bonds (bond * Author to whom correspondence should be addressed. E-mail:
[email protected],
[email protected]. † Universita ¨ t Zu¨rich. § Ruhr-Universita ¨ t Bochum.
dissociation energy, 64.5 kcal mol-1), which can easily be cleaved by UV light, thereby generating two cysteinyl radicals. The generation and existence of radicals in proteins and peptides is not uncommon in biological processes. Although ultraviolet irradiation experiments on proteins have shown30,31 that structural defects upon irradiation are negligibly low, radical enzymes utilize free radicals of amino acid side chains for their catalytic function. Such radicals can be found on redox-active amino acids like tryptophane, tyrosine, gylcine, and cysteine. In particular the cysteinyl radical is believed to be important in biology: • Cysteinyl radicals are postulated to play a central role in the catalytic mechanisms of all three classes of ribonucleotide reductase (RNR).32 • There is evidence that radiation energy initially absorbed in other parts of the molecule is transferred to the sulfur groups. Presumably the sulfur groups act as sinks for the radiation energy.33 • The enzyme papain, a protease, which has a thiol at its active site, is inactivated by radiation.34
papain-SH + CyS• f papain - S• + CySH Disulfide bridges have further been used to trigger the folding of artificial model peptides. Pioneering work in this direction was carried out by Volk and Hochstrasser,18,35,36 who investigated the dynamics of an aryl disulfide-linked artificial R-helical polypeptidic backbone with 17 amino acids. The aryl disulfide linkage is a modified cysteine moiety, which can be cleaved with λexc ) 270 nm because of the influence of the adjacent aromatic ring. In contrast, here we investigate the photodynamics of a much smaller structural element, namely a β-turn structure, which contains a natural disulfide linkage consisting of two cysteine residues. Recently,37 we have presented the real-time observation of the ultrafast structural dynamics of a small cyclic peptide cyclo(Boc-Cys-Pro-Aib-Cys-OMe) (1) upon cleavage
10.1021/jp074184g CCC: $37.00 © 2007 American Chemical Society Published on Web 09/01/2007
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Figure 1. Top: Chemical structures of cyclo(Boc-Cys-Pro-Aib-Cys-OMe) (1) and cyclo(Boc-Cys-Pro-Phe-Cys-OMe) (2). The dashed line indicates the intramolecular hydrogen bond. Bottom: FTIR spectrum of the CdO region of 1 and 2 in CD3CN (spectral resolution 2 cm-1). Band assignments in the FTIR spectra correspond to the colored regions of the chemical structures of the peptides shown on top. Sample concentrations are given in the legends. The shoulders marked with an asterisk belong to ethyl acetate (eluate).
of the disulfide bridge using transient 2D-IR spectroscopy. The weakening of the intramolecular hydrogen bond and the concomitant collapse of the initial β-turn structure was inferred from a continuous change of the corresponding cross-peak between the two CdO vibrators of the hydrogen bond that takes place on a time scale of about 200 ps. In our first paper we did not observe any hint for recombination of the formed cysteinyl radicals within the first few nanoseconds, as has been reported in the paper of Volk and Hochstrasser.18
Consequently in the present work we focus on the long-time dynamics of the two cyclic disulfide-bridged peptides (cyclo(Boc-Cys-Pro-Aib-Cys-OMe) (1) and cyclo(Boc-Cys-Pro-PheCys-OMe) (2)).38 The accessible time window of such studies is governed by the fate of the chemically reactive cysteinyl radicals. Since the generated cysteinyl radicals exhibit just very weak and unspecific absorption bands in the accessible IR region, we extended our study into the UV, where cysteinyl radicals are known to exhibit a strong and characteristic band at 330 nm,39,40 using laser flash photolysis with nanosecond time resolution. Experimental Section Materials. The cyclic disulfide-bridged tetrapeptides (1 and 2) were synthesized according to the procedure published by Kolano et al.38 The samples were dissolved between ∼10 to ∼200 mM in CD3CN (99.8 atom% D, Armar Chemicals) and degassed by ultrasonication for at least 15 min and afterward stored in oxygen-free atmosphere. Time-Resolved IR Spectroscopy. Briefly, the system for UV-pump IR-probe spectroscopy consists of two synchronized41 commercially available Ti:sapphire-oscillator/regenerative am-
plifier femtosecond laser systems operating at 800 nm (Spectra Physics, duration ∼100 fs, repetition rate 1 kHz, energy ∼600 µJ/pulse), allowing us to cover the time range from 2 ps to 10 µs. Laser system 1 was frequency-doubled and -tripled with two BBO crystals. The obtained 266 nm pulses were stretched by a fused silica rod to approximately 1 ps and subsequently focused into the sample cell with a spotsize of 200 µm diameter. Measurements were carried out using parallel and perpendicular polarized pump pulses generated by a computer-controlled halfwave plate, from which the magic angle signal was calculated. Laser system 2 pumped a white light seeded two-stage BBO optical parametric amplifier (OPA),42 the signal and idler pulses of which were difference frequency mixed in an AgGaS2 crystal, producing broadband probe and reference pulses. The IR-probe pulses were focused into the sample cell in spatial overlap with the 266 nm pump pulse. Reference and probe pulse were dispersed in a monochromator (SPEX Triax Series) and imaged onto a 2 × 32 pixel MCT (mercury cadmium telluride) detector array (InfraRed Associates Inc.). By means of this technique it was possible to record transient absorption spectra with spectral resolution of 4 cm-1. The sample was circulated rapidly by a peristaltic pump (Ismatec MCP) through a closed cycle flow cell (path length 100 µm) to ensure efficient exchange of the excited volume. During the course of the measurement, unwanted photoproducts accumulated to less than 5%. Nanosecond Laser Flash Photolysis (LFP). A standard LFP setup was used, consisting of a Nd:YAG laser (Spectra Physics) operated at 1 Hz and 266 nm (50 mJ/pulse, 8 ns pulse duration), a pulsed Xe arc lamp, a monochromator coupled to a photoelectron multiplier tube, and a digital oscilloscope. In order to avoid depletion of the precursor and product build-up, a flow cell (1 cm × 1 cm) was used. The concentration of 1 and 2 was adjusted so that an optical density of 0.3 was achieved at the laser wavelength used for excitation (1: 0.4 mM; 2: 0.3 mM). Results The stationary FTIR absorption spectra of the CdO region of cyclo(Boc-Cys-Pro-Aib-Cys-OMe) (1) and cyclo(Boc-CysPro-Phe-Cys-OMe) (2) are shown in Figure 1 (bottom). In the
Dynamics of Small Cyclic Disulfide Peptides case of 1 we observe five well resolved bands, one for each CdO oscillator, whereas 2 shows only four resolved bands. Band assignment is based on previous work.37,38 According to NMR experiments38 cyclo(Boc-Cys-Pro-Aib-Cys-OMe) (1) exists in the energetically favored trans conformation of the CysPro peptide bond and is stabilized by an intramolecular hydrogen bond between NH Cys(4) and CO Cys(1) (β-turn). Consequently 1 adopts one rigid and strained conformation in solution exclusively. In contrast 2 lacks a strong intramolecular hydrogen bond and adopts several conformations in solution (all amide hydrogens of 2 are solvent exposed).38 The existence of several conformations is due to cis/trans isomerization of the Cys-Pro peptide bond and/or cis/trans isomerization at the urethane bond of Boc.43,44 Hence 2 is a much more floppy structure than 1. The strong interaction between solvent and the Phe- and Prooscillators as well as the existence of several interconverting conformations contribute to amide I band overlap giving rise to a broad absorption signal centered at 1682 cm-1. We have studied the FTIR spectra of the two peptides in deuterated acetonitrile over a wide range of concentrations in order to elucidate potential aggregation effects. While we do not observe any spectral changes in the CdO region with concentration in the case of cyclo(Boc-Cys-Pro-Phe-Cys-OMe) (2), we do find a significant change of the amplitude of the Pro-oscillator (∼1685 cm-1) in cyclo(Boc-Cys-Pro-Aib-CysOMe) (1) when the concentration exceeds ≈100 mM (Figure 1, bottom left, dashed trace). Our findings are in agreement with the IR spectroscopic observations that Piv-Pro-NHMe can adopt intramolecular hydrogen bonds at low concentrations and intermolecular hydrogen bonds at high concentrations.45 In order to rule out the existence of autocatalyzed proline isomerization at higher concentrations we measured 13C NMR spectra of 1 at a concentration of 193 mM. The obtained characteristic shifts of the β- and γ-Pro carbon atoms (γ: 23.24 ppm, β: 29.26 ppm) indicate that 1 exists in one conformation exclusively also at high concentration, namely trans.46 We conclude that 1 forms clusters/aggregates at high concentrations due to intermolecular hydrogen bonding. Figure 2c shows magic angle pump-probe spectra at different delay times after 266 nm excitation of cyclo(Boc-Cys-Pro-AibCys-OMe) (1) at three different concentrations. The underlying short-time dynamics has recently been investigated by transient 2D-IR spectroscopy (T2D)37 and can be described in the following way: Immediately after pulsed photolysis, a transient red shift of all CdO bands of the molecule is observed (Figure 2b left), which we assigned to transient heating of the molecule as a result of the excess energy released after ultrafast cleavage of the S-S bond by the UV photon, a common effect in UVIR experiments.15,19 The heating leads to an excitation of lowfrequency modes that are anharmonically coupled to the CdO vibrations,47 which causes the shift. The heat signal disappears on a 20 ps time scale upon dissipation of the excess energy into the solvent. After 20 ps, the initial red-shifted signal has transformed into a blue-shifted spectrum, which continues to grow on a 200 ps time scale. The blue shift is present for all CdO bands, nevertheless, a SVD analysis showed that the blue shift is associated with a second dynamic process occurring with a time constant of 160 ps that is most pronounced for the band at 1630 cm-1. We therefore assigned the second time constant to a conformational transition of the backbone, which is accompanied by the weakening of the intramolecular hydrogen bond. The initial cooling dynamics of cyclo(Boc-Cys-Pro-Phe-CysOMe) (2) (Figure 2g) resemble that of 1. However, a SVD analysis does not reveal any further spectral evolution afterward
J. Phys. Chem. B, Vol. 111, No. 38, 2007 11299 (>20 ps), which would be associated with a conformational change of the backbone. Presumably, this is since we do not start from a single, well-defined structure in the case of 2, but rather measure an unspecific average over several quickly interconverting conformers both before and after the photocleavage of the disulfide bridge. In addition, the absence of an intramolecular hydrogen bond in the disulfide-bridged form of 2, which acts as the most sensitive “reporter group” in the case of 1, prevents us from monitoring fast structural changes of the backbone in 2. The small blue shifts, which are visible throughout the spectrum of 2 at late times might be due to a charge redistribution due to the presence of the radicals (Stark effect).18 After this initial picosecond phase, both 1 and 2 do not reveal any decline of the signal within the first 10 µs, which could be associated with an intramolecular recombination of the two cysteinyl radicals (Figure 2d and 2h). This observation is valid for the low and the mid concentrated samples (Figure 2c+g, red and black spectra), whereas at higher concentrations (Figure 2c+g, blue dotted spectra) we observe a change of the amplitude and a continuing spectral evolution for time delays longer than 1 ns. In ref 37, we attributed this effect to a diffusion-controlled quenching of the cysteinyl radicals; however, we now refine this interpretation and conclude that this is an artifact caused by the formation of aggregates/clusters via intermolecular hydrogen bonds when the concentration exceeds approximately 100 mM. To probe specifically the lifetime of the free cysteinyl radicals, laser flash photolysis of 1 has been applied. Excitation at λexc ) 266 nm led to the formation of a long-lived transient species, which gives rise to an absorption maximum at λmax. ) 330 nm (Figure 3 A), a characteristic band for the cysteinyl radical.28,39,40,48,49 The transient appears within the ∼10 ns time resolution of the LFP instrument. The inset of Figure 3 A shows the corresponding time trace at 330 nm, which decreases nonexponentially and reaches about half of the initial value after 1 ms. Hence, the lifetime of the biradical is orders of magnitudes longer in our case as compared to that reported by Volk et al.18 Laser flash photolysis of cyclo(Boc-Cys-Pro-Phe-Cys-OMe) (2) under identical conditions also gives rise to a long-lived transient with an absorption maximum at λmax. ) 330 nm (Figure 3 B), which decays on a similar time scale. In addition we observed a broad absorption centered at λmax. ≈ 300 nm. Discussion In the IR measurement, the spectral evolution of 1 and 2 is finished after approximately 1 ns and remains constant up to 10 µs with respect to spectral shape. Within 10 µs, the maximum delay time of our setup, the amplitude of the IR signal decays by less than 10%, indicating that there is no significant recombination of the biradical structure on this time scale. These findings are in agreement with our time-resolved UV experiments, which cover the time range up to 1 ms, where we monitored the decay of the cysteinyl radicals at 330 nm. The LFP experiments show that the decay is not finished even after 1 ms. The cysteinyl signal decays nonexponentially, indicating diffusion-controlled intermolecular quenching, yet neither first nor second order kinetic fits are satisfactory (a plot of 1/∆OD vs time yields two different regimes). In the case of 2, we observed an additional broad absorption centered at λmax. ≈ 300 nm. As pointed out for example by Barro´n et al.,50 the photoproducts formed upon irradiation of a disulfide bridge can be complex. Possible explanations for the 300 nm band would be: • The formation of a charge transfer (CT) complex between the S• radical and the aromatic ring of the incorporated
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Figure 2. Each column consists of a chemical structure (top, a+e), FTIR spectra (middle, b+f), magic angle pump-probe spectra (c+g, spectral resolution 4 cm-1) at different time delays after excitation λexc ) 266 nm, and magic angle pump-probe spectra (d+h, spectral resolution 4 cm-1) of 1 (concn 53 mM, left) and 2 (concn 54 mM, right) in degassed CD3CN at delay times of 1 ns (solid) and 10 µs (dotted) after excitation λexc ) 266 nm. Carbonyl groups are colored to facilitate the band assignment The data of the left column belongs to cyclo(Boc-Cys-Pro-Aib-Cys-OMe) (1) (dashed line indicates the intramolecular hydrogen bond). The data of the right column belongs to cyclo(Boc-Cys-Pro-Phe-Cys-OMe) (2). Band assignments in the FTIR spectra (CD3CN, spectral resolution 2 cm-1) correspond to the colored regions of the chemical structures of the peptides shown on top. Bands appearing on irradiation are pointing upward; bands disappearing are pointing downward. (c) Red spectra: concn of 1 14 mM, degassed, black spectra: concn of 1 53 mM, degassed, blue dotted spectra: concn of 1 192 mM, degassed. The spectra of the low concentration measurement are magnified by a factor of ∼15, and the spectra of the mid concentration measurement are magnified by a factor of ∼2.8. (g) Red spectra: concn of 2 15 mM, degassed, black spectra: concn of 2 54 mM, degassed, blue dotted spectra: concn of 2 203 mM, degassed. The spectra of the low concentration measurement are magnified by a factor of ∼10, and the spectra of the mid concentration measurement are magnified by a factor of ∼2.5.
phenylalanine, explaining why the equivalent band is not observed in 1. The possibility of forming CT-complexes between heteroatoms and/or reactive intermediates with aromatic rings has been demonstrated in a variety of publications (e.g., O2benzene,51,52 SNS+-benzene,53 azidopentazole-benzene54).
• A disproportionation of the cysteinyl radicals yielding a thioaldehyde (≈280 nm).50,55 • Phenylalanine has a significant absorbance at 266 nm ( ∼ 200 M-1 cm-1). We cannot rule out that photolysis of 2 leads to some excitation of Phe. Bent and Hayon56 reported the
Dynamics of Small Cyclic Disulfide Peptides
J. Phys. Chem. B, Vol. 111, No. 38, 2007 11301 showed that chain cyclization is favorable for biradicals of very small rings, is most unfavorable for mid-sized rings, and becomes again more favorable for larger chains. In addition, the flexibility or, conversely, the rigidity of the amino acid sequence plays an important role. Calculated cyclization constants61 and the results derived from kinetic measurements of the collision frequency between the two ends of short randomcoil polypeptides62 allow arrangement of amino acids in a descending order of flexibility. Among these, Pro is the most rigid and Ala is one of the most flexible amino acids. This interpretation nicely fits to our observations and matches with our recent MD simulations,37 which indicate that the strain in the peptide backbone of 1 keeps the two S radicals apart and prevents recombination. In particular, the strain in our peptide sequence is much larger than in that of Volk et al.18 Moreover, we have to consider that aromatic thiyl radicals were generated in the experiments of Volk et al.,18 whereas aliphatic thiyl radicals are formed in our experiments. We cannot rule out that the different electronic structures of the radicals have an influence on the recombination dynamics. Our description of the peptide dynamics is valid for concentrations below ≈100 mM. For concentration exceeding this limit, we start to form clusters and/or aggregates via intermolecular hydrogen bonds, and intramolecular cysteinyl recombination might also become possible within these clusters. Conclusion
Figure 3. Top (A): Transient absorption spectra following 266 nm excitation [traces recorded after 375 ns (black dots), 8 µs (white dots), 32 µs (black triangles), 170 µs (white triangles), and 750 µs (black stars) in argon-purged acetonitrile] displaying the decay of the cysteinyl radical of 1. Inset: Corresponding time trace recorded at λ ) 330 nm upon laser flash photolysis. Bottom (B): Transient absorption spectra following 266 nm excitation [traces recorded after 409 ns (black dots), 7 µs (white dots), and 150 µs (black triangles), 170 µs (white triangles), and 750 µs (black stars) in argon-purged acetonitrile] displaying the decay of the cysteinyl radical of 2.
triplet-triplet absorption of Phe at λmax. ≈ 310 nm exhibiting a lifetime in the microsecond region. However, we can rule out the formation of several other reactive species that could arise from a secondary reaction. In our transient absorption spectra there is no evidence for the formation of perthiyl radicals (R-CH2-S-S•, λmax. ) 380 nm39,40). Moreover, because the samples have been degassed prior to irradiation and were kept under oxygen free atmosphere for the complete measurement, we can rule out the formation of sulfinyl (R-CH2-S•)O) and thiyl peroxyl (R-S-O-O•) radicals, too.49 With respect to the lifetime of the biradical state, our results are considerably different from those presented by Volk et al.,18 who reported 90% recombination within 2-5 ns. The molecular system of Volk et al.18 was designed to adopt an R-helical structure after photocleavage of the aryl disulfide linkage; however, they could not find any corresponding frequency shift of the amide I′ spectrum up to 2 ns. The observed small spectral changes were interpreted in terms of a vibrational Stark effect. Geminate recombination quenched the biradical too quickly before the molecule could fold. Our cyclic disulfide-bridged tetrapeptides (1 and 2) are entirely different in structure. The dynamics of flexible triplet biradicals arising from cyclic organic ring structures have been investigated in detail, taking into account the chain length and substituent effects.57-60 That work
Time-resolved infrared spectroscopy in combination with laser flash photolysis yields a consistent mechanistic picture of the backbone dynamics of two cyclic disulfide-bridged tetrapeptides upon photocleavage of an intramolecular disulfide bridge by short UV pulses. While the spectral evolution of the initially very rigid cyclo(Boc-Cys-Pro-Aib-Cys-OMe) (1) reports on the breaking of the intramolecular hydrogen bond, the backbone dynamics of the very floppy cyclo(Boc-Cys-Pro-Phe-Cys-OMe) (2) is hidden because of the conformational heterogeneity in both the cyclized and photocleaved form. The dynamics of 1 can be described by three individual time constants, reflecting cooling (20 ps), turn opening (160 ps), and a very slow quenching of the liberated cysteinyl radicals (> 1 ms). In the case of 2, the intermediate process is not observable, because of structural heterogeneity. The lifetime of the cysteinyl radicals is sufficiently long to monitor the nonequilibrium backbone dynamics of a peptide by time-resolved spectroscopic techniques. Thus, intramolecular disulfide bridges are suitable nonreversible phototriggers for small biological systems and potentially also for proteins. A modification of the parameter concentration, size of the molecule, and internal strain by variation of the amino acid sequence allows one to fine-tune the accessible time-window. In view of many biophysical problems, the combination of a native cysteine moiety as a photoactivateable “predetermined breaking point” and intramolecular hydrogen bonds offers new perspectives for the spectroscopic investigation of protein dynamics. Acknowledgment. C.K. thanks the Deutsche Forschungsgemeinschaft for a Postdoctoral Research Fellowship. This work was financially supported by Swiss Science Foundation (P.H. under grant 200020-107492/1), the Deutsche Forschungsgemeinschaft (W.S. & G.B.) and the Fonds der Chemischen Industrie (W.S.). References and Notes (1) Williams, S.; Causgrove, T. P.; Gilmanshin, R.; Fang, K. S.; Callender, R. H.; Woodruff, W. H.; Dyer, R. B. Biochemistry 1996, 35, 691.
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