Loop formation in unfolded polypeptide chains on the picoseconds to microseconds time scale
- Beat Fierz†,
- Helmut Satzger‡,§,
- Christopher Root‡,
- Peter Gilch‡,
- Wolfgang Zinth‡, and
- Thomas Kiefhaber†,¶
- †Division of Biophysical Chemistry, Biozentrum der Universität Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland; and
- ‡Lehrstuhl für BioMolekulare Optik, Department für Physik, Ludwig-Maximilians-Universität, Oettingerstrasse 67, D-80538 Munich, Germany
-
Communicated by Wolfgang Kaiser, Technical University of Munich, Garching, Germany, December 13, 2006 (received for review October 3, 2006)
Abstract
Intrachain loop formation allows unfolded polypeptide chains to search for favorable interactions during protein folding. We applied triplet–triplet energy transfer between a xanthone moiety and naphthylalanine to directly measure loop formation in various unfolded polypeptide chains with loop regions consisting of polyserine, poly(glycine–serine) or polyproline. By combination of femtosecond and nanosecond laserflash experiments loop formation could be studied over many orders of magnitude in time from picoseconds to microseconds. The results reveal processes on different time scales indicating motions on different hierarchical levels of the free energy surface. A minor (<15%) very fast reaction with a time constant of ≈3 ps indicates equilibrium conformations with donor and acceptor in contact at the time of the laserflash. Complex kinetics of loop formation were observed on the 50- to 500-ps time scale, which indicate motions within a local well on the energy landscape. Conformations within this well can form loops by undergoing local motions without having to cross major barriers. Exponential kinetics observed on the 10- to 100-ns time scale are caused by diffusional processes involving large-scale motions that allow the polypeptide chain to explore the complete conformational space. These results indicate that the free energy landscape for unfolded polypeptide chains and native proteins have similar properties. The presence of local energy minima reduces the conformational space and accelerates the conformational search for energetically favorable local intrachain contacts.
Understanding the protein folding process requires the characterization of the structure and dynamics of all states along the reaction coordinate and the transition states separating them. Properties of native proteins have been extensively characterized by x-ray crystallography and NMR spectroscopy. Detailed structural information on partially folded states has been obtained by hydrogen exchange and NMR techniques. The properties of unfolded proteins are much less understood, although a detailed characterization of the structure and dynamics of the unfolded state is essential for a better understanding of the early steps in protein folding and the free energy landscape of the folding reaction. A major problem in the characterization of unfolded proteins is the nonphysiological solvent conditions that are required to populate the unfolded state in equilibrium. NMR studies (1–9) and the analysis of the effect of mutations on the solvent accessibility of the unfolded state (10) have revealed the presence of both native and non-native interactions in unfolded states of several proteins. In addition, steric constraints and intramolecular hydrogen bonding were suggested to promote the folding reaction by restricting the conformational space of unfolded polypeptide chains (11, 12). Recently, the dynamics of the unfolded state have become accessible to experimental investigations using NMR spectroscopy and electron transfer reactions (13–15).
Intrachain loop formation between protein side chains is a particularly important process in unfolded proteins because it allows a folding polypeptide chain to search for energetically favorable interactions. Fast electron transfer reactions have recently been applied to investigate the kinetics of intrachain loop formation in polypeptide chains (13, 15–17). We have used triplet–triplet energy transfer (TTET) between a xanthone (Xan) donor moiety and a naphthylalanine (NAla) acceptor to measure loop formation in a variety of polypeptide chains (13, 15, 16, 18–20). TTET is a two-electron exchange reaction (Dexter mechanism) operating only between molecules in close contact and is thus fundamentally different from FRET, which is based on dipole–dipole interactions extending to the nanometer range. The photophysical reactions involved in TTET from Xan to Nala occur on the time scale of a few picoseconds (21, 22) and have a strong distance dependence with a reactive boundary of 4.4 Å (23). These properties allow the use of TTET to measure absolute rate constants for loop formation reactions slower than ≈5 ps (see Fig. 1) (15, 21). Applying TTET to study loop formation between the ends of unfolded polypeptide chains with repetitive sequences and in fragments derived from natural proteins revealed exponential kinetics with time constants on the 5-to 100-ns time scale depending on loop length and amino acid sequence (13, 15–17). The observed single exponential kinetics indicate fast interconversion processes between individual conformations of the polypeptide chains (24). All previous experiments on loop formation were restricted to the nanosecond time scale because of the experimental systems or setups applied (15). These experiments could, however, not rule out faster processes, which may occur in the experimental dead time. Because TTET from Xan to Nala allows the measurement of subnanosecond processes (Fig. 1) (21, 22) we combined femtosecond and nanosecond laserflash experiments to study loop formation reactions in several polypeptide chains with loop regions consisting of polyserine, poly(glycine–serine), or polyproline. This combination of experiments allowed us to monitor loop formation processes over six orders of magnitude in time from picoseconds to microseconds.
Results and Discussion
Nanosecond and Subnanosecond Intramolecular Contact Formation.
TTET between Xan and Nala can be monitored either by the decrease in the Xan triplet absorbance band centered at 590 nm or the increase in the Nala triplet absorbance at ≈420 nm (16). Typically the reaction is monitored at 590 nm because of the stronger Xan triplet absorbance band. Our previously performed nanoseconds laserflash TTET experiments had a dead time of ≈12 ns, which allowed us to measure nanosecond to microsecond loop formation dynamics in peptides of different length and sequence (16). To test for the existence of faster loop formation processes on the subnanosecond time scale, we recorded the donor triplet absorbance change at 590 nm in peptides containing donor and acceptor groups separated by polypeptide chains of different length and sequence. The observed kinetics of absorbance decay on the nanosecond to microsecond time scale were analyzed and the amplitudes of the decay were compared with the respective amplitudes measured in donor-only reference peptides (Fig. 2). In these peptides Nala was replaced by phenylalanine, which cannot undergo TTET with Xan (18). Fig. 2 shows that all chains containing Xan and naphthalene exhibit smaller amplitudes for the kinetics of the Xan triplet decay compared with the donor-only reference peptides. The effect is more pronounced for the Xan–Ser2–NAla and Xan–Ser6–NAla loops compared with the long and flexible Xan–(Gly-Ser)12–NAla loop (Fig. 2). When donor and acceptor are separated by five proline residues, which should result in a stiff and rod-like structure, only very little Xan triplet decay occurs within the dead time. These results reveal that subnanosecond dynamics of loop formation occur in all investigated polypeptide chains and that the extent of these fast reactions depends on loop size and amino acid sequence.
Kinetics of loop formation in different peptides measured by TTET from Xan to Nala induced by a 4-ns laserflash. The kinetics were monitored by the change in Xan triplet absorbance at 590 nm. Kinetics of a Xan-Ser2-NAla loop (red line), a Xan–Ser6–NAla loop (green line), a Xan–(Gly-Ser)12–NAla loop (magenta line), and a Xan–Pro5–NAla loop (blue line) are shown. For comparison, the triplet decay in a donor-only reference peptide (black line) with NAla replaced by Phe is displayed (for peptide sequences see Table 1). In the donor-only peptides the Xan triplet state decays by oxygen quenching and intersystem crossing pathways, which occurs on the 20- to 50-μs time scale. Data were corrected for small differences in peptide concentration and normalized to the absorbance of the reference peptide. The solid lines are exponential fits to the data with the time constants given in Table 1.
Femtosecond-Laserflash TTET Experiments.
To resolve the dynamics of the processes occurring within the dead time of the nanosecond experiments we induced TTET by a 100-fs laserflash. Formation of the Xan triplet state (ππ* → 3nπ* transition) and TTET from Xan to Nala were shown to have time constants ≈2 ps (21, 22). The photophysics of Xan involves an additional relaxation process from the initially formed 3nπ* state to the 3ππ* state, which has a time constant of ≈300 ps in water (22). However, the originally formed 3nπ* state can already undergo TTET with Nala (21). This fact allows the observation of loop formation reactions with time constants lower than ≈5 ps (see Fig. 1). To discriminate processes caused by TTET from photophysical reactions in the Xan donor group we compared changes in the Xan triplet absorbance band at ≈590 nm in peptides bearing donor and acceptor to the changes in a donor-only reference peptide. Fig. 3 shows time-resolved Xan triplet absorbance spectra for the Xan–Ser2–NAla peptide (Fig. 3 A) and the donor-only reference peptide (Fig. 3 B). The sum of four exponentials is required to fit the data. Analysis of the amplitude spectra (Fig. 3 C and D) reveals the spectral changes associated with the individual kinetic phases. In these plots the amplitude changes associated with a kinetic process are shown at different wavelengths. Negative values indicate an increase in absorbance, whereas positive values indicate a decrease in absorbance. In both peptides formation of the relaxed singlet state (ππ*) occurs with a time constant ≈0.3 ps. Subsequently, a high energy triplet state (3nπ*) is formed on the 2- to 3-ps time scale as seen from the blue shift of the absorbance spectrum associated with an increase in absorbance. The 3nπ* state is in equilibrium with the relaxed singlet state, which leads to delayed fluorescence processes (21, 22). In the donor-only reference peptide a significant increase in triplet absorbance in combination with a blue shift of the absorbance maximum is observed with a time constant of 280 ps (Fig. 3 B and D). As discussed in more detail in refs. 21 and 22, this process is caused by the 3nπ* → 3ππ* transition in Xan and shows an isosbestic point ≈630 nm. The Xan–Ser2–NAla peptide shows a similar blue shift in the Xan triplet absorbance band with a time constant of 320 ps. In contrast to the donor-only peptide, this process is associated with only very little change in absorbance at 590 nm and shows an isosbestic point ≈600 nm (Fig. 3 A and C). Subsequently, a slow decrease in Xan triplet absorbance on the nanosecond time scale is observed in the Xan–Ser2–NAla peptide in agreement with the nanosecond laserflash experiments (see Fig. 2). In the reference peptide no relaxation from the triplet state to the ground state is observed within the first 4 ns, because this process occurs on the 20- to 50-μs time scale (see Fig. 2). The time constant and the amplitude spectrum of this slow decay were determined in a nanosecond experiment and are indicated in Fig. 3 D.
TTET experiments induced by a 200-fs laser flash. (A) Kinetics of formation of the Xan–Ser2–NAla loop measured by TTET from Xan to Nala induced by an ultrashort laserflash (time resolution of the experiment, 200 fs). Spectral changes in the Xan triplet absorbance band are displayed on a split time base. The time range from −1 ps to 1 ps is shown on a linear time scale, and the time range from 1 ps to 4 ns is shown on a log time scale. The time of the laserflash is defined as t = 0. (B) Corresponding spectral changes in the Xan triplet absorbance band of the donor-only reference peptide. (C and D) Amplitude spectra obtained from a triple exponential fit of the data with the associated time constants as indicated. The slowest time constant for triplet decay in the Xan–Ser2–NAla loop corresponds to the kinetics observed in nanosecond experiments (Fig. 2). In the reference peptide the amplitude spectrum for τ = 20 μs was taken from the fit of the data shown in Fig. 2.
The time-dependent changes in the triplet absorbance bands reveal a major loss in the population of Xan triplet states on the time scale from 50 to 500 ps in the Xan–Ser2–NAla peptide as compared with the donor-only peptide. This loss in Xan triplet states is associated with an increase in naphthalene triplet absorbance at 420 nm (data not shown), confirming that this effect is caused by TTET between Xan and naphthalene in a subpopulation of conformations. However, the time dependence of the absorbance changes at 420 nm cannot be analyzed quantitatively, because a 1ππ* Xan fluorescence band (delayed fluorescence) and a second, weaker Xan triplet absorbance band are located in the same spectral region.
Loop Formation Dynamics from Picoseconds to Microseconds.
The kinetics of TTET between Xan and Nala differ significantly from the photophysics in the donor-only reference peptide (Fig. 3). There are two possibilities to separate TTET reactions from photophysics in Xan to gain information on the dynamics of
loop formation. One way is to monitor kinetics at the isosbestic point for the triplet relaxation at 630 nm. A second possibility
is to normalize the time-dependent spectral changes at the Xan triplet absorbance maximum (590 nm) in the Xan–Ser2–NAla peptide (Fig. 3
A) against the changes in the donor-only reference peptide (Fig. 3
B) according to
where A
590
D denotes the absorbance of the donor-only reference peptide at a given time and A
590
D/A the respective absorbance of the peptide-bearing donor and acceptor. Both procedures yield identical results. However, because
of the stronger absorbance changes at 590 nm the normalization of the data according to Eq. 1 gave better signal-to-noise ratios compared with measurements at the isosbestic point at 630 nm. Thus, in the following we
will present and discuss the time course of changes in A
590(rel). To gain information on loop formation in the time range from picoseconds to microseconds, we combined the data from the
femtosecond experiments with changes in A
590(rel) measured in nanosecond investigations. Fig. 4 shows changes in A
590(rel) from 1 ps to 30 μs. Several subnanosecond processes can be distinguished. For Xan–Ser2–NAla ≈12% of the Xan triplet states decay very fast with a time constant of ≈3 ps, indicating a process that is limited by the
photophysics of the system. This rapid decay may either be caused by loop formation reactions on the low picosecond or subpicosecond
time scale or, more likely, indicates that ≈12% of the peptide conformations have donor and acceptor in contact when Xan is
excited by the laserflash. Additional processes occur on the 50- to 500-ps time scale (Fig. 4), which can either be fitted by the sum of several exponentials or with a stretched exponential function (Kohlrausch–William–Watts
function) according to
where β indicates the stretching parameter and k
KWW denotes the apparent rate constant of this reaction. Accordingly after 1/k
KWW = τ app the signal has decreased to 37% (1/e) of the original signal (A0). Because the 3nπ* → 3ππ* relaxation occurs with a time constant of 300 ps (Fig. 3), kinetic coupling between triplet relaxation and TTET processes occurs, which complicates the quantitative evaluation of
the fast TTET kinetics. For a qualitative evaluation of the data and comparison of different loop sequences we used the τapp values of this reaction obtained by fitting the data according to Eq. 2. For the Xan–Ser2–NAla loop a τapp of 170 ps is observed and 32% of the total absorbance change occurs on this time scale. In the remaining molecules the triplet
states decay on the nanosecond time scale as observed in nanosecond laserflash experiments (see Table 1) (16).
Kinetics of loop formation on the time scale from 1 ps to 30 μs measured in TTET experiments and monitored by changes in Xan triplet absorbance at 590 nm. The kinetics of a Xan–Ser2–NAla loop (red dots), a Xan–Ser6–NAla loop (green dots), and a Xan–Pro5–NAla loop (blue dots) are shown. The kinetic traces were obtained from a combination of data measured in femtosecond and nanosecond laserflash experiments (Figs. 2 and 3). A 590(rel) was determined for each peptide according to Eq. 1 to correct for effects from photophysics in Xan (Fig. 3 B) and the different peptide concentrations required in nanosecond and femtosecond experiments. The lines represent the results of fits to the data to the sum of a stretched exponential function (see Eq. 2) and exponential functions with the parameters given in Table 1.
Time constants for loop formation obtained from the combination of femtosecond and nanosecond experiments
In an alternative analysis of the absorbance changes over the complete time range from picoseconds to microseconds (Fig. 4) we determined the distribution of time scales (25). The results indicate three separated time regimes on the low-picosecond, hundreds of picoseconds, and nanosecond time scale for TTET in the Xan–Ser2–NAla peptide (data not shown), which confirms the findings obtained by fitting the data to a combination of exponential and stretched exponential functions.
Effect of Chain Length and Chain Stiffness on Subnanosecond Dynamics of Loop Formation.
To further characterize the subnanosecond TTET reactions, we tested their sensitivity toward chain length and chain stiffness. Fig. 4 compares TTET kinetics in the Xan–Ser2–NAla peptide with the kinetics in the Xan–Ser6–NAla and the stiff Xan–Pro5–NAla peptides. All peptides show TTET reactions on the picosecond time scale, but the kinetics depend on the loop size and sequence (see Table 1). Similar to the Xan–Ser2–NAla peptide, the Xan–Ser6–NAla peptide shows ≈15% of very rapid Xan triplet decay with a time constant of ≈3 ps. Subsequently, a process with τapp = 260 ps is observed, which is on the same time scale as for the Xan–Ser2–NAla peptide. The amplitude of this reaction is 48%, which is larger than in the Xan–Ser2–NAla peptide, in agreement with the results from the dead-time absorbance changes observed in the nanosecond laserflash experiments (Fig. 2). The dynamics of nanosecond loop formation reactions are slightly slower for the Xan–Ser6–NAla loop compared with the Xan–Ser2–NAla loop. The Xan–Pro5–NAla peptide, which is significantly stiffer than the polyserine loops, does not show any evidence for very rapid TTET within the first 10 ps. It shows, however, subnanosecond loop formation kinetics with τapp = 210 ps and a largely reduced amplitude (13%) compared with the polyserine loops. The majority of Xan–Pro5–NAla conformations form loops on the 10-ns to μs time scale in a complex multiexponential process.
Origin of Loop Formation Processes on the Different Time Scales.
The combination of femtosecond and nanosecond TTET experiments shows that loop formation in a polypeptide chain occurs on different time scales. In both polyserine loops 10–15% of the molecules undergo very fast TTET limited by the ultrafast photophysics (intersystem crossing) of the system (Fig. 4). This finding suggests that a subpopulation of the ensemble of chain conformations have donor and acceptor in contact at the time point of the laserflash, which may be partly caused by the hydrophobic nature of the TTET labels. The absence of this reaction in the Xan–Pro5–NAla peptide indicates only a negligible fraction of closed loop conformations in equilibrium in this peptide, in agreement with the increased stiffness of polyproline chains (26).
In all peptides investigated in this study loop formation reactions that are not limited by photophysics occur on two well separated time scales. For all peptides reactions with an apparent time constant on the 200-ps time scale are observed (see Table 1). For the Xan–Pro5–NAla loop this process has a lower amplitude (13%) compared with the Xan–Ser2–NAla loop (32%) and the Xan–Ser6–NAla loop (48%). The time scale of these reactions is similar to the dynamics of local motions in the polypeptide backbone observed in a photoswitchable cyclic peptide (27, 28) and only slightly slower than expected for single-bond rotations in the polypeptide backbone in water. This observation suggests that a significant subpopulation of molecules can form loops by performing just a few bond rotations without having to explore the complete free energy landscape.
The remaining conformations in the ensemble of unfolded states show loop formation on the 10- to 100-ns time scale in all peptides. This reaction becomes slower with increasing loop length in agreement with our previous findings (16). The dynamics on the nanosecond time scale were shown to be caused by diffusional processes exploring the complete accessible free energy landscape for the polypeptide chains as shown by their length and sequence dependence, which are in agreement with predictions from polymer theory (24). These reactions are very slow and complex for the polyproline loop, which is caused by cis–trans equilibria at the five prolyl peptide bonds and leads to different populations of molecules that interconvert on the second to minute time scale. Thus, loop formation on the nanosecond time scale is observed for the individual populations giving rise to complex kinetics.
Hierarchy of Peptide Motions.
The observed separation of time scales in the dynamics of loop formation indicates motions on different hierarchical levels (tiers) of the free energy landscape for a flexible polypeptide chain (Fig. 5). The dynamics on the nanosecond time scale represent chain diffusion exploring different local minima on the free energy landscape (13, 15, 16). Loop formation on the 50- to 500-ps time scale, in contrast, most likely represents motions within local wells on the free energy landscape that contain loop structures (Fig. 5). Thus, all conformations within such a well can form a loop by undergoing local motions and do not have to cross major barriers. The amplitude of this reaction is larger for the Xan–Ser6–NAla loop compared with the Xan–Ser2–NAla loop, in agreement with the properties of polyserine chains, which have a persistence length of ≈5 aa (29). Thus, the Xan–Ser6–NAla peptide should be able to form a turn with less conformational strain as compared with the Xan–Ser2–NAla peptide. As a consequence, the Xan–Ser6–NAla peptide has a higher equilibrium population of conformations with close donor–acceptor distances. This model is confirmed by the results of the Xan–Pro5–NAla peptide, which shows largely increased chain stiffness and only a small fraction of conformations that can form a loop on the subnanosecond time scale. Another aspect is highlighted by the Xan–(Gly–Ser)12–NAla peptide. Here, the smaller amplitude of the subnanosecond reactions compared with the serine loops (Fig. 2 and Table 1) shows that a further increase in chain length leads to a smaller population of conformations that can form loops on the subnanosecond time scale, despite the larger flexibility of poly Gly-Ser.
Schematic representation of the hierarchical organization of the energy landscape for unfolded polypeptide chains. The highest tier of conformational substates contains different wells on the free energy landscape separated by energy barriers. Chain diffusion over the complete free energy surface is governed by transitions between these wells and includes major structural rearrangements of the polypeptide chain. Within each well a number of substates exist. Interconversion between these substates occurs by local structural changes. Motions on the highest tier lead to loop formation on the nanosecond time scale, whereas fluctuations within the local wells are much faster and result in loop formation on the 50- to 500-ps time scale. The dashed line indicates conformations with donor–acceptor in contact, which undergo TTET limited by the photophysical processes.
The observed organization of peptide motions in different hierarchical levels suggests that the free energy landscapes of unfolded polypeptide chains and folded proteins (30–36) have similar features. For native myoglobin the highest tier contains several taxonomic substates separated by significant energy barriers. Transitions between these substates require major structural rearrangements and are important for protein function (37, 38). Each taxonomic substate contains a large number of statistical substates. Transitions between statistical substates correspond to local rearrangements of the polypeptide chain and are much faster than transitions between taxonomic substates. Within the statistical substates further tiers with decreasing barrier height can be discriminated (36). Our results show that this concept does not only apply to folded proteins but also to the ensemble of unfolded peptide conformations (Fig. 5). Obviously, the functionally important and structurally well defined taxonomic substates are absent in flexible chains. However, the free energy landscape includes a number of local wells that are separated from each other by significant free energy barriers. At room temperature interconversion between substates from different wells is fast compared with the time scale of loop formation, which leads to the observed exponential dynamics on the nanosecond time scale (24). The observed rate constants for these reactions depend on chain stiffness and chain length, i.e., on the size of the available conformational space (19). The barriers separating the different wells may be caused by breakage of intramolecular hydrogen bonds, which were shown to exist in unfolded model polypeptide chains in water (23). Conformational space within a well can be explored without encountering major barriers, which leads to loop formation on the 50- to 500-ps time scale. These rate constants only weakly depend on chain length and amino acid sequence but the population of conformations within the different substates is strongly affected by chain length and chain stiffness.
The observation of complex free energy landscapes for unfolded polypeptide chains is supported by recent results from experiments using a combination of FRET and electron transfer reactions. Results on different donor/acceptor positions in α-synuclein, a natively unfolded protein, indicated complex pairwise distance distribution functions containing several maxima and minima (39).
Implications for Protein Folding.
The observed kinetics on different time scales show that loop formation in polypeptide chains is a hierarchical process and that the free energy landscape of an unfolded polypeptide chain is complex and contains local minima. This hierarchical organization may play a major role for the protein folding process by allowing a fraction of molecules to form loop structures very rapidly, on the 50- to 500-ps time scale, and producing a significant fraction of loop conformations within the ensemble of unfolded conformations. This large fraction of loop conformations might serve as initiation sites for the folding reaction. The frequently observed hydrophobic zipper motif in β-hairpins (40), where the interacting side chains have similar hydrophobicity as the TTET labels of our model loops, should lead to similar fractions of loop conformations in equilibrium. In addition to the effects on the equilibrium properties of the unfolded state, the presence of a number of local minima accelerates conformational search and barrier crossing during the folding process by reducing the entropic contributions to the free energy barriers (41).
It is unlikely that the fast loop formation reactions on the 50- to 500-ps time scale are strongly influenced by the nature of the side chains, because van der Waals interactions are short-range interactions and operate on similar distances as TTET. Fast loop formation rather reflects reactions that only require a few bond rotations or breakage of just a few hydrogen bonds to form a loop without the necessity for major rearrangements of the intramolecular hydrogen-bonding network. The dynamics of loop formation on different time scales should lead to kinetic heterogeneity in very fast folding processes like formation of α-helices and β-hairpins or acquisition of the 3D structure for fast folding proteins. Our results may explain the findings from temperature jump studies on λ-repressor, which was shown to fold in complex kinetics on the low microsecond time scale that could be described by either a double exponential or a stretched exponential function (42).
Materials and Methods
Peptide Synthesis.
Peptides and 9-oxoxanthen-2-carboxylic acid (Xan acid) were synthesized as described (16). Purity and mass were tested by analytical HPLC and mass spectrometry, respectively. For all measurements peptides were dissolved in 10 mM phosphate buffer, pH 7 or pure H2O at 22.5°C.
Laserflash Experiments.
The setup of the nanosecond laserflash experiments has been described (16). A Nd:YAG pulsed laser (354.6 nm, 4-ns pulse of 50 mJ; Quantel, Santa Clara, CA) was used to excite the samples, a Laser Flash Photolysis Reaction Analyzer (LKS.60; Applied Photophysics, Leatherhead, U.K.) was used to collect the absorption data. Peptide concentrations in the nanosecond experiments were ≈50 μM. Concentrations were determined by UV spectroscopy using Xan absorption at 343 nm (ε = 3,900 cm−1·M−1).
The setup of the femtosecond pump probe experiments has been described (43). A 1-kHz Ti:Sa laser/amplifier system was used as a light source. Pump pulses at 340 nm were generated by frequency doubling the output of a noncollinear optical parametic amplifier (44, 45). White light generation supplied the broadband probing pulses. The pertinent parameters for the pump probe experiment were the following: The pump light had an energy of 350 nJ per pulse and a diameter of ≈150 μm at the sample location. Its polarization plane was at magic angle with respect to the white light probe [experimental response time was ≈200 fs (FWHM)]. Multichannel probing recorded the absorption changes from 400 to 700 nm. The sample was exchanged between consecutive laser shots by a peristaltic pump. The fused silica sample cell had a path length of 0.5 mm. The concentrations in femtosecond experiments were ≈2 mM.
Test for Intermolecular Reactions.
To test for intermolecular TTET as the origin for the difference in kinetics between the Xan–Ser2–NAla peptide and the donor-only peptide we measured a 1:1 mixture of a donor-only and an acceptor-only peptide at the same concentrations as in the TTET experiments. In this control experiment the Xan triplet decay was identical to the kinetics in the donor-only peptide, ruling out intermolecular TTET in associated or aggregated peptides on the picosecond time scale.
Data Analysis.
Data were analyzed by using MATLAB (Mathworks, Natick, MA) and proFit (QuantumSoft, Uetikon am See, Switzerland).
Acknowledgments
We thank Hans Frauenfelder for discussion and comments on the manuscript. This work was supported by a grant from the Volkswagen Stiftung (to B.F.).
Footnotes
- ¶To whom correspondence should be addressed. E-mail: t.kiefhaber{at}unibas.ch
-
↵ §Present address: Steacie Institute for Molecular Sciences, National Council of Canada, 100 Sussex Drive, Ottawa, ON, Canada K1A OR6.
-
Author contributions: W.Z. and T.K. designed research; B.F., H.S., and C.R. performed research; B.F. and P.G. contributed new reagents/analytic tools; B.F., H.S., C.R., W.Z., and T.K. analyzed data; and B.F., W.Z., and T.K. wrote the paper.
-
The authors declare no conflict of interest.
- Abbreviations:
- TTET,
- triplet–triplet energy transfer;
- Xan,
- xanthone;
- Nala,
- naphthylalanine
-
Freely available online through the PNAS open access option.
- © 2007 by The National Academy of Sciences of the USA