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Fluorescence and phosphorescence of tryptophan in peptides of different length and sequence
Journal of Photochemistry & Photobiology, B: Biology 157 (2016) 120–128
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Journal of Photochemistry & Photobiology, B: Biology journal homepage: www.elsevier.com/locate/jphotobiol
Fluorescence and phosphorescence of tryptophan in peptides of different length and sequence Ksenija Radotić a,⁎, Thor Bernt Melø b, Roger M. Leblanc c, Yaser A. Yousef d, K. Razi Naqvi b a
Institute for Multidisciplinary Research, University of Belgrade, Kneza Višeslava 1, 11000, Belgrade, Serbia Department of Physics, Norwegian University of Science and Technology (NTNU), N-7491 Trondheim, Norway Department of Chemistry, University of Miami, FL, United States d Department of Chemistry, Yarmouk University, Irbid, Jordan b c
a r t i c l e
i n f o
Article history: Received 5 August 2015 Received in revised form 9 February 2016 Accepted 10 February 2016 Available online 15 February 2016 Keywords: Luminescence Peptides Phosphorescence Steady state fluorescence Time resolved fluorescence Tryptophan
a b s t r a c t To interpret accurately protein fluorescence and phosphorescence, it is essential to achieve a better understanding of the luminescence properties of tryptophan (Trp, or W) in peptides. In published literature data on luminescence of peptides of varied length are scarce. This article describes studies of fluorescence and phosphorescence properties of the eight Trp-containing synthetic peptides: WAK, AWK, SWA, KYLWE, AVSWK, WVSWAK, WAKLAWE, and AVSWAKLARE. The aim was to investigate which factors influence the fluorescence yield and phosphorescence-spectra and lifetimes. Absorption spectra, room temperature fluorescence emission and corresponding excitation spectra and time-resolved phosphorescence spectra (77 K) have been recorded; the dependence of the fluorescence quantum yield on the specific peptide and its variation with the wavelength of excitation has been studied. The changes in fluorescence yield and shape of phosphorescence spectra are explained in terms of internal electron and proton transfer. The structured phosphorescence spectrum originates from proton transfer occurring upon excitation of Trp, while electron transfer gives rise to a non-structured luminescence spectrum. There is also electron transfer from higher vibronic S1 states. In the peptides there is higher probability of electron transfer than in Trp alone. The obtained data are interpreted in light of the peptides' sequence, length and conformation. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Two factors have contributed to the immense popularity of luminescence measurements among workers interested in studying protein folding. First, the overwhelming contribution to the luminescence of the polypeptides and proteins which contain at least one tryptophan (Trp) residue comes from Trp itself; secondly, some of the key spectroscopic properties (lifetimes and quantum yields of emission, spectra) of the Trp molecule undergo, more often than not, large changes in response to subtle variation in its environment. When the protein under investigation contains a Trp residue whose properties in the unfolded form differ markedly from those in the folded conformation, fluorometric detection allows the experimenter to investigate folding with comparatively small quantities of the sample; accordingly, it has been suggested that, when one is dealing with a protein that does not contain an intrinsic Trp residue, it would be profitable to add one [1]. Unfortunately, an unambiguous interpretation of the photophysical data of Trp-bearing proteins has turned out to be far from simple, because even proteins with a single ⁎ Corresponding author at: Institute for Multidisciplinary Research, University of Belgrade, Kneza Višeslava 1, 11000, Belgrade, Serbia. E-mail addresses: [email protected] (K. Radotić), [email protected], [email protected] (T.B. Melø), [email protected] (R.M. Leblanc), [email protected] (Y.A. Yousef).
http://dx.doi.org/10.1016/j.jphotobiol.2016.02.011 1011-1344/© 2016 Elsevier B.V. All rights reserved.
Trp residue fail to show a monoexponential decay of the intensity of emission (whether fluorescence or phosphorescence). In fact, even fluorescence from solutions of Trp has been found to deviate, in general, from a monoexponential decay [2–5]. Several suggestions, including one about electron transfer from, and another about proton transfer to the N-atom of, the indole nucleus of Trp have been put forward to account for the heterogeneity of the lifetimes of Trp in solution, in proteins, and in synthetic polypeptides, but a comprehensive picture is yet to emerge. On the other side, there are some studies of Trp phosphorescence, as a delicately sensitive monitor of protein conformation [6–8]. There appears to be a general consensus that, in order to interpret data on protein luminescence, it is essential to acquire a better understanding of similar data on Trp-containing peptides. Some excellent investigations of the fluorescence spectroscopy of peptides have appeared in the literature [1,9], but there seems to be a dearth of studies in which both fluorescence and phosphorescence of peptides of different lengths are examined, and it is clear that such data are sorely needed. The present study was undertaken with a view to taking a first step towards this goal, an aim that could not be accomplished without developing a new approach to recording timeresolved emission spectra of long-lived triplets. Fluorescence and luminescence of the eight synthesized peptides of different length,
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Fig. 1. Upper: Absorption, corrected fluorescence excitation and relative fluorescence yield of sample WVSWAK. Middle: Fluorescence yields of all samples, divided by that of tryptophan (circles), as a function of excitation wavelength. Lower: The graphs in the middle inset are subtracted from their respective terminal levels; and inset: The heights at 240 nm in relation to the yield at 280 nm. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)
containing three to ten amino acids with Trp at different positions in the peptide, have been investigated. The fluorescence and phosphorescence luminescence properties of the peptides are related to their sequence, length and conformation. 2. Materials and Methods 2.1. Materials Peptides were synthesized by China Peptides Co., Ltd. (Shanghai, China) with N 99% purity. All other reagents were of analytical grade. Three peptides are composed of three amino acids: WAK, AWK and SWA. One of them, Trp (W) is located at the end of peptide, while at the other end is a charged amino acid (K) and in the middle is a non-polar amino acid (A). For the other two peptides Trp is located in the middle of peptide, one at the end being non-polar amino acid and one at the
other end being a polar or charged, basic, amino acid.One of the two five-amino acid peptides, KYLWE, contains Tyr (Y) separated from Trp by one amino acid. Y is less strictly polar, and the other amino acids are non-polar or charged, one basic and one acidic located at the two peptide ends. The second five-amino acid peptide, AVSWK, contains Trp at the same position as the other five amino-acid peptide, and also the non-polar amino acids, one neutral/polar and one charged (basic) amino acid at the end of peptide. In the six amino-acid peptide, WVSWAK, and in the seven amino-acid peptide, WAKLAWE, there are two Trps, one of them being at the peptide end; the other amino acids are non-polar or charged, basic or acidic at the peptide end. The ten amino acid peptide, AVSWAKLARE, contains one Trp inside the peptide, the other amino acids being non-polar, polar and charged, two basic and one acidic. The sequence of this peptide is the same as the WAKLAWE, except for the amino acid next to the end amino acid, R, which is polar and basic, instead of W in the former amino acid. Thus in these peptides
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of varied length Trp is located either at the end or within the peptide, and neighbored by amino acids of different polarity: non-polar, polar or charged, basic or acidic. 2.2. Absorption, Fluorescence Excitation and Emission Spectra Absorption spectra were measured on a Shimadzu UV-1601PC, UV– visible spectrophotometer. Fluorescence spectra were recorded by an OLIS RSM 1000 Spectrophotometer. When emission spectra were measured, the excitation wavelength was set to 280 nm and the absorbance (A) of the samples at 280 nm was in the 0.1–0.15 range. The slit widths at the excitation and emission were 1.24 mm and 0.6 mm, respectively. The fluorescence spectra of the samples were nearly identical, and the fluorescence yields from the various samples were calculated as fluorescence intensity at 350 nm, F(350), divided by the absorbance at 280 nm, A(280), multiplied by a factor to take care of the small inner filter effects: η f ¼
Fð350Þ . Að280Þ10ðAð280Þ=2Þ
When corrected fluorescence excitation spec-
tra were measured, the wavelength of the emission monochromator was set to 350 nm and the excitation wavelength (λx) was scanned and the ratio of fluorescence intensity to the excitation intensity, I(λx), is obtained from the instrument; Fc(λx) = F(350)/I(λx). Later on the corrected fluorescence excitation is divided by the absorption spectrum and the inner filter effect, which compensates for the loss of excitation intensity from the entrance to the middle part of the cuvette: η f ðλx Þ ¼ Fcðλx Þ . Aðλx Þ10ðAðλx Þ=2Þ
This was done in a worksheet, and the fluorescence excita-
of the laser pulse; this was achieved by choosing the following values for the three parameters: f = 2 Hz, td = 100 μs, tv = 100 ms. To record a set of time-resolved spectra, the ungated spectrometer was run in the so called timeline mode, which permits one to acquire a number of spectra (up to 100) in a single scan. The laser was run at 10 Hz for a few minutes (in order to build up a standing concentration of long-lived species), a trigger was manually sent to the spectrometer (to start the scan immediately) the laser was stopped (also manually) as soon afterwards as possible. By following this strategy we were able to acquire a set of spectra (with an excellent signal-to-noise ratio) at regularly spaced intervals. It need hardly be added that, since the procedure involves manual acts, a precise value for td cannot be stated; moreover, the first few members of the acquired set (typically two or three) were rejected because the intensity of the incident light at some wavelengths exceeded, perforce and by design, the permissible limit. The indeterminate time at which the first usable spectrum is acquired, is estimated to be 0–0.4 s, and will be denoted by the symbol t0. A cold finger quartz Dewar (filled with liquid nitrogen) was used for refrigerating the sample. A capillary quartz tube was partially immersed into the sample solution, and its upper end was closed with a plastic stopper. The tube (containing the sample at its lower end) was then withdrawn and gently immersed into a reservoir of liquid nitrogen and allowed one to reach the temperature of the surrounding coolant. Finally, the sample was quickly removed from the reservoir and immersed into the cold finger Dewar.
tion as a function of wavelength was obtained. 2.4. Time Resolved Fluorescence 2.3. Measurement of Phosphorescence The excitation source for phosphorescence measurements was a tunable pulsed laser (EKSPLA NT342A-SH-10-WW). The wavelength was 280 nm and the duration and energy of each pulse were approximately 7 ns and 2 mJ, respectively. The resulting phosphorescence emission was viewed (along a direction perpendicular to that of the laser beam) by two multichannel spectrometers, which received part of the emitted light through two optical fibers whose receiving ends were placed on opposite sides of the sample. One spectrometer (Hamamatsu PMA 12 Series; Model C110029-01) uses a gated and intensified array detector; the other was an ungated multichannel detector (B&W Tek Spectrometer Model BRC642E). The gated detector is capable of providing a high time resolution (N10 ns) but it can record only one spectrum after it is triggered (by the laser pulse); the other instrument (to be called the ungated spectrometer) has a much slower time response (see below) but it can record a set of spectra at regular intervals after the arrival of the trigger signal. In a particular run, only one spectrometer was allowed to view the emitted light. Let td and tv denote the delay time (the interval between the firing of the laser and the opening of the gate) and the viewing time (the width of the gate pulse), respectively, and let f be the repetition rate of the excitation pulse. The gated detector was used for viewing the emission during the first 100 ms after the end
Fluorescence decay times were measured using a Jobin Yvon IBM FluoroCube photon counting spectrometer in the time-correlated single photon counting mode (TC-SPC). A nano-LED excitation source (pulse
Table 1 The short (sh) and long (lo) lifetimes of fluorescence (F) and luminescence (P), and ratio of the long- and short-lifetime amplitudes from Hamamatsu spectra (Rlo/sh). Sample
τ sh-F,ns
τ lo-F,ns
τ sh-P,s
τ lo-P,s
Rlo/sh-H
W WAK AWK SWA KYLWE AVSWK WVSWAK WAKLAWE AVSWAKLARE
0.1 1.17 0.69 0.6 1.34 0.91 1.24 1.4 0.82
3.15 5.28 2.02 1.7 2.96 2.78 3.92 4.53 2.54
0.48 0.44 0.47
2.4 3.6 5.0
0.44 0.41 0.56 0.47
4.6 4.0 2.9 5.2
2.60 0.32 0.54 0.04 0.04 0.66 0.60 0.22 0.04
Fig. 2. Upper: The yields, calculated from lifetimes, as a function of yields calculated from steady state emission spectra. Lower: Relation between short and long fluorescence lifetimes. The samples are dissolved in buffer.
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duration 150 ps) was used emitting at 285 nm with a pulse repetition frequency of 1 MHz. 3. Results and Discussion 3.1. Absorption, Fluorescence Excitation and Fluorescence Yields In the upper part of Fig. 1, the absorption-and the corrected fluorescence excitation spectrum of one of the peptides, WVSWAK, is shown. The fluorescence excitation spectrum has been scaled to coincide with the maximum of the absorption spectrum. The graph also contains the relative fluorescence yield, which is the ratio of corrected fluorescence excitation and absorption spectrum. As seen from the graph, it is fairly constant up to 250 nm, where it starts to decrease. That is also the case for tryptophan (W) itself [10]. In the middle part of Fig. 1, the fluorescence yields of the peptides under investigation in relation to that of W are shown. For WAKLAWE the original result is shown (red circles) and for all of them the original result has been replaced by smooth curves. They are parallel lines up to 250 nm, and further up on the wavelength scale there is a U shaped dip of variable depth. In the lower part of Fig. 1, the difference (Δ (λ)) between the terminal level of the yield, called Y (300 nm), and the yield as a function of wavelength, Y (λ); Δ (λ) = Y (300 nm)-Y (λ) is shown. The differences are bell shaped with variable heights and in the inset of the lower part of Fig. 1 the maximum values of Δ are shown, Δ (240 nm), plotted versus the terminal values, Y (300 nm). For the tripeptides, SWA, AWK and WAK, Δ (240 nm) change in proportion to Y (300 nm), for WAK being the highest value. Maximum value of Δ is blue shifted for the two peptides containing polar amino acid K at their end, in comparison with SWA. However, for penta-, hepta- and deca-peptides Δ (240 nm) and Y (300 nm) are anti-correlated.
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which are relatively small for W and hence easily influenced by the local environment. Samples WAK, SWA, KYLWE, WVSWAK and WAKLAWE are on a straight line, which is to be expected when the radiative lifetime is the same. The other samples, W, AVSWK, AVSWAKLARE and AWK, can be assembled on a line which is not passing through the origin. One obvious reason to this is that kr is sample dependent, which means that the radiation transition probability of W also depends upon its environment. In Fig. 2 (lower part) the size of the long lifetime as a function of the short lifetime is shown. Except for W, WAK and KYLWE, they are on a straight line, which means that the two lifetimes are influenced in proportion by the microenvironments. For all peptides the short lifetimes are longer than that of Trp, and (with the above exceptions) increasing with peptide length. The bi-exponential fluorescence decay of Trp originates in the three different stereoconformers, also called rotamers, of W. Because the Cα\\Cβ bond in W is a single bond, see the figure below, the amino acid part of W can rotate with respect to the indole part. It is a restricted rotation and the three different minimal energy rotamers, shown in the figure below, can interconvert by passage over a rotation barrier.
3.2. Relation Between Fluorescence Yield and Fluorescence Lifetimes The nature and probabilities (in parenthesis) of the various deactivation channels open for a molecule in the first excited electronic state (the S1 state) are: fluorescence emission (kr), radiationless decay to the ground state (kn) and intersystem crossing (kisc). Often there are additional deactivation channels, in general called quenching (kq), which could be proton or electron transfer. The fluorescence yield, which is the ratio of emitted to absorbed photons, is related to these probabilities as follows:
Chemical structure of tryptophan.
kr , that is; the ratio of probability of the radiative chanη f ¼ kr þkn þk isc þkq
nel in relation to the sum of all other probabilities from the S1 level. The fluorescence lifetime is as follows: 1 , which means that: ηf = kr ⋅ τf. As long as the radiaτ f ¼ kr þkn þk isc þkq tive lifetime (kr) is unchanged, yields calculated from steady state (CW) fluorescence spectra and lifetimes (τ) should be in proportion to one another. The shapes of the measured fluorescence emission spectra were very similar, and accordingly, fluorescence yields from steady state measurements were taken as the peak heights of the fluorescence spectra divided by the absorption at the exciting wavelength, which is 280 nm, and compensated for the small inner filter effects. The measured fluorescence decays (f(t)) could always be fitted to a sum of two exponential decays with different lifetimes. f(t) = sh αf ⋅ exp(−t/τlo f ) + (1 − αf) ⋅ exp(−t/τf ). lo and sh symbolize long (lo) and short (sh) lifetime and αf is the relative weight of the first contribution. The lifetimes are given in Table 1 and the amplitudes in Fig. 5. The yields from lifetime measurements were calculated as the sum of amplitudes multiplied by their respective lifetimes, or the average lifetime. The fluorescence yields from lifetimes versus steady state measurements are shown in the Fig. 2 (upper) using W as a standard (ηf(CW) = 1, ηf(τ) = 1). The yields from either method agree quite well, as seen by the linearity of the graph. The differences have to be explained according to the differences in the radiative probabilities (kr),
The three stereoconformers, or rotamers, of tryptophan. R is the planar indole group and the projection is along the Cα-Cβ bond (Newman projection). According to Robbins et al. (1980) [11], the fluorescence of W in buffer (called naked W) is quenched by internal proton transfer (ipt) from the –NH+ 3 groups to the indole part of W. In the rotamer having the shortest distance between COO− and indole, according to GudginTempleton and Ware (1984) [5], the fluorescence decay is due to quenching by electron transfer (et) from –COO− to indole. Because of symmetry, two W rotamers have similar –NH+ 3 indole distances, and accordingly, only two lifetimes are expected. When the rotamer interconversion times are longer than fluorescence lifetimes, the two rotamer populations will have different fluorescence lifetimes. According to Robbins et al. (1980) [11], quenching is mainly by ipt, and conformation I and III have the largest populations. In naked W photo ionization can
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also occur, by which a photo ionized electron settles in surrounding water as solvated electrons. The yield of photoionization is constant (about 0.04) and wavelength independent up to 250 nm [11,12], and increases below this value [10]. In peptides and proteins there is additional internal electron transfer (iet). An excited electron on the indole group is transferred to the peptide bond [13], and it is this internal electron transfer that causes the great variability in fluorescence quantum yields of W in a peptide or protein. Furthermore, as seen from Fig. 1, in all peptides the fluorescence yield below 245 nm decreases more than in W. In W the drop means more photoionization, in the peptides it means more electron transfer inside the peptide from higher vibronic states around 245 nm. It is the distance between the W residue and the peptide group and the energy difference between the initial (S1) and final state (excited peptide bond) that are the most crucial factors for
the size of rate constant of the electron transfer. These factors depend on sequence and thus conformation of a particular peptide, which creates unique microenvironment for Trp residue. The fact that the peptides, containing the same motif (for example SWA or WAK) but differing in the rest of sequence, have different yields/lifetimes (Fig. 2) may indicate the influence of conformation/length of the peptides on the distance between indole and peptide bond. Increase in fluorescence yield with peptide length may indicate that distance between indole and peptide group increases and thus there is an increase in electron transfer in comparison with photoionization/electron ejection to the solvent. The result obtained for the tri-peptides, SWA, AWK and WAK, that Δ (240 nm) change in proportion to Y (300 nm), shows a possible influence of the conformation of these tripeptides by decreasing electron transfer to the surrounding water. These peptides have similar
Fig. 3. Luminescence spectra of tryptophan (W) and AVSWK detected at different times (see legend) after the laser pulses had been turned off, using the BWTek array detector. Upper part: The spectra from W and inset: The integrated intensity of each spectrum plotted versus delay time with a double exponential fit. Lower part: Corresponding spectra from sample AVSWK and inset: The component spectra resulting from singular value decomposition and global analysis of the measured spectra with an exponential decay for each of them.
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order of polar and non-polar amino acids regarding Trp, i.e. Trp is neighbored with a non-polar amino acid (A) on one side and with a polar amino acid on the other (K) or with the solvent in the case of WAK. The fact that for penta-, hepta- and deca-peptide Δ (240 nm) and Y (300 nm) are anti-correlated may be explained through effect of peptide conformations, increasing the COO−/indole distance, which consequently increases electron transfer to the solvent, approaching to the value for Trp alone. The amino acid polarity may also have influence on peptide lifetime. For example WAK has the highest fluorescence yield, which may be explained by feasibility of hydrogen bonds formation between terminal lysine and solvent, in this peptide. This may affect distance between indole and peptide bond and result in considerably higher value than those for similar peptides SWA and AWK, but where Trp is located in the middle of peptide. A similar interpretation may be given for the shift of the maximum of Δ to the high energy part of the spectrum, for AWK and WAK comparing to SWA. Also, addition of lysine on SWA increases the yield (SWA b AVSWAKLARE b WVSWAK). This may be due
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to the influence of this charged amino acid on the distance between indole and peptide bond, and on corresponding radiation probability. The presence of Tyr decreases the yield comparing with the peptide of the same length but lacking Tyr (KYLWE b AVSWK — Fig. 2) and causes blue shift of the emission spectra and increasing of the short lifetime. This may indicate extra deactivation channel in the peptide containing Tyr which reduces fluorescence yield. 3.3. 77 K Phosphorescence Emission; Spectra Recorded in the Time Line Mode (BWTek) In Fig. 3 (upper part) successively recorded phosphorescence spectra every 0.6 s from W with a detection window of 0.2 s and dwell time of 0.4 s, after the pulsed laser had been turned off are shown. The shape of the spectrum remains unchanged, but its intensity decays with time. In the inset of the figure, it is shown how the integrated spectrum, done in the worksheet, decays with time. The decay can be well fitted as a double exponential decay with time, and the two amplitudes
Fig. 4. Spectra obtained by global analysis. Lower: Spectra associated with the short lifetime. Upper: Spectra associated with the long lifetime, and Inset: Expanded in the 420–450 nm region.
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with corresponding lifetimes are shown in Fig. 5 and the ratio of amplitudes for the long and short lifetime in Table 1. In the lower part of Fig. 3 the corresponding spectra for AVSWK is shown. In this case, as well as for the other peptides, the overall luminescence spectrum consists of a structured, like that of W, and a nonstructured spectrum, decaying with different lifetimes, after stopping the laser pulses. The spectra recorded in the timeline mode were subjected to singular value decomposition (SVD), which showed that only two principle components were present. The SVD analysis was succeeded by a global analysis, in which both component spectra were assumed to have a single exponential decay with different lifetimes. The result of the global analysis is shown in the inset, the unstructured spectrum having the shortest lifetime (sh) and the structured spectrum the longest lifetime (lo). The lifetimes are given in Table 1. Except for samples SWA and KYLWE, the remaining samples were subjected to a similar treatment as AVSWK. The decay is well described as a sum of two spectra, a structured and a non-structured spectrum with different lifetimes. The result of four samples is collected in Fig. 4. In the lower part, the non-structured spectra, which are normalized to unity at the peak is shown. All non-structured spectra have the same shape. In the upper part of Fig. 4, the structured spectra of the investigated samples as well as that of W are shown. They are also normalized to unity at the maximum. Although quite similar, it is seen that they are slightly different. AVSWK has the narrowest lines and is somewhat displaced to longer wavelength compared to the other spectra. In the inset, enlarged spectra in the 400–450 nm region is shown. The lifetimes obtained from the global analysis are shown in Table 1.
3.4. Phosphorescence Spectra with a Fixed Delay Between Laser and Gate (Hamamatsu) In the above BWTek measurements, the initial delay time between the last laser pulse and detection of the first phosphorescence spectrum might have been anywhere in the interval between 0 and 0.4 s, which was the dwell time of BWTek in the timeline mode. The Hamamatsu detector offers the possibility to detect the phosphorescence spectrum at a certain delay with respect to the laser. For the spectra shown in Fig. 6 the delay is set to 100 μs. As seen from these spectra, by comparing to the BWTek spectra, the Hamamatsu instrument does not have the same spectral resolution as BWTek. Furthermore, by comparing the spectra of W recorded by the two instruments, one sees a drop in wavelength sensitivity in Hamamatsu compared to BWTek below 420 nm. The phosphorescence spectra recorded using the Hamamatsu detector consist of a structured and a non-structured part, as was the case for the BWTek detector, however, a fixed delay makes the determination of the initial intensity ratio of the structured to the non-structured spectrum more precise. The spectra from the Hamamatsu detector are shown in the lower part of Fig. 6, and it is seen that the contribution of structured spectrum to the overall spectrum varies from sample to sample. The phosphorescence spectrum of W (PW) is purely structured, and accordingly, was taken to represent the structured part of the overall spectrum of any sample. As also observed from Fig. 6, the spectrum of SWA (PSWA) is nearly non-structured, and therefore was chosen to represent the non-structured part. In the upper part of Fig. 6 the spectrum of AWK has been resolved into a sum of the W and SWA spectrum: Ps = Alolo·Pw + Ash, using Solver, which is a toolbox in Excel. When Solver returns the Alo and Ash coefficients, their ratio can be obtained: Rlo/sh = Alo/Ash and is given in Table 1 for the various samples. The relative amplitudes can be found, usingAso + Alo = 1, to be: Alo = Rlo/sh/(1 + Rlo/sh) and are given in Fig. 5. The spectra of KYLWE and AVSWAKLARE are somewhat displaced to longer wavelengths than the rest of the non-structured spectra and have very small contributions of the structured spectrum.
Upper: The spectrum of AWK has been resolved into a sum of a structured (W) and a non-structured (SWA) spectrum, using Solver in Excel. 3.5. Origin of Structured and Non-Structured Phosphorescence Emission When a peptide is formed, by linking amino acids through peptide bonds, the free rotation around the Cα\\Cβ bond is restricted, due to the increased sizes of the two parts on either side of the Cα\\Cβ bond. However, there may still be two non-rotating conformers (stereoisomers) of the peptide and the distance between the indole group and the peptide bond might be different in the two conformers. For each conformer (C1 and C2) the rate constant for quenching (kq) is composed of two parts, internal proton transfer (kipt) and electron transfer C1 C1 (kiet). kC1 q = kipt + kiet , which might be different in the two conformers. In all peptides and W double exponential decay of fluorescence and phosphorescence lifetimes were observed. For naked W the two phosphorescence spectra are only distinguishable by lifetime, while the phosphorescence spectra of the peptides are a sum of two distinctly different spectra with different shapes and lifetimes. Except for W, the amplitudes of the long fluorescence lifetime and the short phosphorescence lifetime were roughly similar, indicating that they arise from the same conformer. According to Robbins et al. (1980) [11] and Tsentalovich et al. (2004) [5], a protonated form of the triplet is formed when naked W is photo excited, because prior to intersystem crossing, there is excited singlet state protonation of the indole group. The majority spectrum of W with long lifetime is thought to be due to protonated W, because its relative abundance is nearly the same as that of protonated triplets of W, as seen in transient absorption (Robbins et al. [ref. 11] −0.65, Tsentalovich et al. [ref. 12] −0.58). The short-lived spectrum of W is due to electron transfer from COO− to the indole group, in opposite direction as for internal electron transfer in a peptide, where it is from the indole group to the amide bond. Both proton and electron transfer can happen in one conformer, but their contributions might be different compared to the conformer in the other peptide. Accordingly, the weight of the structured/nonstructured phosphorescence spectrum is C2 a sum of contributions from each conformer: APlo = αC1 ipt + αipt, and: C2 + α . The differences in fluorescence and phosphorescence APsh = αC1 iet iet amplitudes as seen in Fig. 5 might be explained from this; the initial fluorescence amplitudes are in proportion to the weights of two conformers, while initial phosphorescence amplitudes are a sum of amplitudes of ipt and iet, respectively. The fact that the spectra SWA, KYLWE and AVSWAKLARE have very small contributions of the structured spectrum (Fig. 6) may indicate
Fig. 5. Both fluorescence and luminescence decays are a sum of two exponential decays with different lifetimes. The figure shows relative amplitudes of the short (sh) and long (lo) fluorescence (F) /luminescence (P) lifetimes. The luminescence amplitudes were calculated from Hamamatsu spectra (called H) followed by a decomposition into a structured and non-structured spectrum (see text).
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that in these peptides, upon excitation predominates electron transfer, i.e. conformer with unprotonated indole. In these peptides there is no ipt, since W is not a terminal amino acid. Although the amplitudes of the two phosphorescence spectra change from sample to sample, their lifetimes are remarkably invariant; see Table 1. The longest lifetime is 3–5 s, and the shortest lifetime about 0.3–0.4 s. For all peptides ratio between amplitudes of long- and short-phosphorescence lifetimes is similar and significantly lower than for Trp. It means that the same conformer predominates in all peptides, with higher probability of electron transfer than in Trp alone. The photophysics of smaller peptides is also under theoretical investigations. For instance, Shemesh and Domcke (2010) [14] have done density functional calculation (DFT) on the Trp-Gly dipeptide in which the position of the H atom belonging to COOH group has been changed to approach the O atom belonging to the peptide group. After a vertical excitation to the 1Lb state, it relaxes to the 1La state from which it makes a radiationless transition over a low barrier to a locally excited state (1LE). The lowest adiabatic excitation is not the 1La state, but a 1LE
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state localized on the peptide group. This transition can be considered as energy transfer. The 1LE state develops into a charge transfer (CT) state, which can be considered as an electron transfer to the peptide bond, and finally, the CT state has conical intersections with the ground state. It means that the fluorescence quenching is a multistep process and is delicately dependent upon the geometry of the peptide. When the lifetime of the CT state is short, due to conical intersection, it is non-luminescent. It might be suggested that the non-structured phosphorescence spectrum in fact is a luminescent CT state. In donor-acceptor compounds, tuned so that the triplet CT level is lower than triplet level, there is triplet CT emission [15]. The characteristics of triplet CT emission are a non-structured spectrum with a short lifetime. All peptides have similar values of fluorescence long lifetimes, but differ in short lifetimes (Table 1), so that longer peptides have longer short lifetimes. This gives relation between short- and long-lifetimes as shown in Fig. 2, which increases with increasing peptide length. This indicates that population of Trp rotamer with the smaller indole – peptide bond distance is increasing in the peptides with increasing
Fig. 6. Lower: Luminescence spectra of the peptides using the Hamamatsu detector with a fixed delay of 100 μs between laser and detector gate. The gate width was 0.1 ms.
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peptide length, while population of the rotamer with longer indole – peptide distance stays relatively unchanged. The fact that probability of electron ejection from Trp (photoionization) and probability of electron transfer in peptides are dependent on peptide sequence and length and thus on their conformation, may enable fine regulation of certain processes in very narrow Trp environments in a protein. It means that light-induced electron ejection and/ or electron transfer may be one of the ways of fine regulation of protein conformation changes, being connected with Trp rotameric switch. Such fine regulation, involving Trp located in protein conserved domains, is known also for some other kinds of stimuli [16,17]. 3.6. The Conformer Model and Beyond The conformer model was established to explain the multi exponential fluorescence decay of W in solution. In their work on substituents on the 3-ethyl chain of W (the amino and acid group of W), Petrich et al. [4] introduced the socalled modified conformer model (MCM) in which they postulated electron transfer from the indole group of W to the electrophilic substituents. Differences in lifetimes of the three conformers were not only due to populations of the conformers but also to differences in quenching abilities of the electrophilic substituents. GudginTempleton and Ware [12] modified the MCM even further to state that the charge (electron) donor is the solvated indole chromophore, which could be a charge to solvent complex (CTTS). This is in line with the fact that the emitting state is always 1La and fluorescence quenching could be a dissociation of the complex, or in other words, an electron transfer. These ideas, originally used to explain the multiexponential fluorescence decay of W and substituents on the 3– ethyl chain of W, are also in line with the results for the peptides studied here, and are most clearly seen for the peptides where W is exposed to the solvent. It is believed that charge transfer (CT) takes place at 77 K and that part of the luminescence spectrum seen at that temperature is due to emission from a stabilized CT complex. Most probably this CT complex originates from electron transfer to the peptide bond and not from the CTTS complex. This has bearings on the use of nomenclature; phosphorescence should be reserved for proton transfer (ipt), in which the number of electrons on the indole group is unchanged, and luminescence should be used for electron transfer (iet), in which case the number of electrons is changed. The spectra seen at 77 K are partly a phosphorescence and luminescence spectrum. 4. Conclusions In conclusion, our results show that there is an influence of conformation/length of the peptides on the distance between indole and peptide bond, which has crucial influence on luminescence emission. Both proton and electron transfer can happen in one Trp conformer, but their contributions might be different in different peptides. In the peptides there is higher probability of electron transfer than in Trp alone. Population of Trp rotamer with the smaller indole–peptide
bond distance is increasing in the peptides with increasing peptide length. The obtained results show that fluorescence and phosphorescence properties of Trp even in small peptides are complex, and that studies on peptides are unavoidable for deep understanding of protein luminescence. Acknowledgments The authors acknowledge receiving financial support from the Research Council of Norway (Project No. 191102), as well as from the Ministry of Education, Science and Technological Development of the Republic of Serbia (Project No. 173017). The authors are grateful to Prof. Mikael Lindgren for providing them access to the instrument for time resolved fluorescence measurements. References [1] R.W. Alston, M. Lasagna, G.R. Grimsley, J.M. Scholtz, G.D. Reinhart, C.N. Pace, Peptide sequence and conformation strongly influence tryptophan fluorescence, Biophys. J. 94 (2008) 2280–2287. [2] R.J. Robbins, J.W. Petrich, D.B. McDonald, G.R. Fleming, G.S. Beddard, G.W. Robinson, P.J. Thistlewhaite, G.J. Woolfe, Photophysics of aqueous tryptophan: pH and temperature effects, J. Am. Chem. Soc. 102 (1980) 6271–6279. [3] M.C. Chang, J.W. Petrich, D.B. McDonald, G.R. Fleming, Nonexponential fluorescence decay of tryptophan, tryptophylglycine, and glycyltryptophan, J. Am. Chem. Soc. 88 (1983) 3819–3824. [4] J.W. Petrich, M.C. Chang, D.B. McDonald, G.R. Fleming, On the origin of nonexponential fluorescence decay in tryptophan and its derivatives, J. Am. Chem. Soc. 105 (1983) 3824–3832. [5] E.F. Gudgin-Templeton, W.R. Ware, The time dependence of the low-temperature fluorescence of tryptophan, J. Phys. Chem. 88 (1984) 4626–4631. [6] G. Strambini, E. Gabellieri, Temperature dependence of tryptophan phosphorescence in proteins, Photochem. Photobiol. 51 (1990) 643–648. [7] M. Gonnelli, G.B. Strambini, Phosphorescence lifetime of tryptophan in proteins, Biochemistry 34 (1995) 13847–13857. [8] J. Broos, E. Gabellieri, G. I. van Boxel, J. B. Jackson, G. B. Strambini, Tryptophan phosphorescence spectroscopy reveals that a domain in the NAD(H)-binding component (dI) of transhydrogenase from Rhodospirillum rubrum has an extremely rigid and conformationally homogeneous protein core, J. Biol. Chem. 278 (278) 47578–47584. [9] C.-P. Pan, M.D. Barkley, Conformational effects on tryptophan fluorescence in cyclic hexapeptides, Biophys. J. 94 (2004) 3828–3835. [10] H.B. Steen, Wavelength dependence of the quantum yield of fluorescence and photoionization of indoles, J. Chem. Phys. 61 (1974) 3997–4002. [11] R.J. Robbins, G.R. Fleming, G.S. Beddard, G.W. Robinson, P.J. Thistlewaite, G.J. Woolfe, Photophysics of aqueous tryptophan: pH and temperature effects, J. Am. Chem. Soc. 102 (1980) 6271–6279. [12] Y.P. Tsentalovich, O.A. Snytnikova, R.Z. Sagdeev, Properties of excited states of aqueous tryptophan, J. Photochem. Photobiol. A Chem. 162 (2004) 371–379. [13] P.R. Callis, A. Petrenko, P.L. Muino, J.R. Tusell, Ab Initio prediction of tryptophan quenching by protein electric field enabled electron transfer, J. Pys. Chem. B 111 (2007) 10335–10339. [14] D. Shemesh, W. Domcke, in: Wagner, et al., (Eds.), High Performance Computing in Science and Engineering, Springer-Verlag Berlin Heidelberg 2010, pp. 641–649. [15] J. Herbich, A. Kapturkiewics, J. Nowacki, Phosphorescent intramolecular charge transfer triplet states, Chem. Phys. Lett. 262 (1996) 633–642. [16] R. Pankov, E. Cukierman, K. Clark, K. Matsumoto, C. Hahn, B. Poulin, K.M. Yamada, Specific β1 integrin site selectively regulates Akt/protein kinase B signaling via local activation of protein phosphatase 2 A, J. Biol. Chem. 278 (2003) 18671–18681. [17] M. Sotomayor, K. Schulten, The allosteric role of the Ca2+ switch in adhesion and elasticity of C-cadherin, Biophys. J. 94 (2008) 4621–4633.
Contents lists available at ScienceDirect
Journal of Photochemistry & Photobiology, B: Biology journal homepage: www.elsevier.com/locate/jphotobiol
Fluorescence and phosphorescence of tryptophan in peptides of different length and sequence Ksenija Radotić a,⁎, Thor Bernt Melø b, Roger M. Leblanc c, Yaser A. Yousef d, K. Razi Naqvi b a
Institute for Multidisciplinary Research, University of Belgrade, Kneza Višeslava 1, 11000, Belgrade, Serbia Department of Physics, Norwegian University of Science and Technology (NTNU), N-7491 Trondheim, Norway Department of Chemistry, University of Miami, FL, United States d Department of Chemistry, Yarmouk University, Irbid, Jordan b c
a r t i c l e
i n f o
Article history: Received 5 August 2015 Received in revised form 9 February 2016 Accepted 10 February 2016 Available online 15 February 2016 Keywords: Luminescence Peptides Phosphorescence Steady state fluorescence Time resolved fluorescence Tryptophan
a b s t r a c t To interpret accurately protein fluorescence and phosphorescence, it is essential to achieve a better understanding of the luminescence properties of tryptophan (Trp, or W) in peptides. In published literature data on luminescence of peptides of varied length are scarce. This article describes studies of fluorescence and phosphorescence properties of the eight Trp-containing synthetic peptides: WAK, AWK, SWA, KYLWE, AVSWK, WVSWAK, WAKLAWE, and AVSWAKLARE. The aim was to investigate which factors influence the fluorescence yield and phosphorescence-spectra and lifetimes. Absorption spectra, room temperature fluorescence emission and corresponding excitation spectra and time-resolved phosphorescence spectra (77 K) have been recorded; the dependence of the fluorescence quantum yield on the specific peptide and its variation with the wavelength of excitation has been studied. The changes in fluorescence yield and shape of phosphorescence spectra are explained in terms of internal electron and proton transfer. The structured phosphorescence spectrum originates from proton transfer occurring upon excitation of Trp, while electron transfer gives rise to a non-structured luminescence spectrum. There is also electron transfer from higher vibronic S1 states. In the peptides there is higher probability of electron transfer than in Trp alone. The obtained data are interpreted in light of the peptides' sequence, length and conformation. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Two factors have contributed to the immense popularity of luminescence measurements among workers interested in studying protein folding. First, the overwhelming contribution to the luminescence of the polypeptides and proteins which contain at least one tryptophan (Trp) residue comes from Trp itself; secondly, some of the key spectroscopic properties (lifetimes and quantum yields of emission, spectra) of the Trp molecule undergo, more often than not, large changes in response to subtle variation in its environment. When the protein under investigation contains a Trp residue whose properties in the unfolded form differ markedly from those in the folded conformation, fluorometric detection allows the experimenter to investigate folding with comparatively small quantities of the sample; accordingly, it has been suggested that, when one is dealing with a protein that does not contain an intrinsic Trp residue, it would be profitable to add one [1]. Unfortunately, an unambiguous interpretation of the photophysical data of Trp-bearing proteins has turned out to be far from simple, because even proteins with a single ⁎ Corresponding author at: Institute for Multidisciplinary Research, University of Belgrade, Kneza Višeslava 1, 11000, Belgrade, Serbia. E-mail addresses: [email protected] (K. Radotić), [email protected], [email protected] (T.B. Melø), [email protected] (R.M. Leblanc), [email protected] (Y.A. Yousef).
http://dx.doi.org/10.1016/j.jphotobiol.2016.02.011 1011-1344/© 2016 Elsevier B.V. All rights reserved.
Trp residue fail to show a monoexponential decay of the intensity of emission (whether fluorescence or phosphorescence). In fact, even fluorescence from solutions of Trp has been found to deviate, in general, from a monoexponential decay [2–5]. Several suggestions, including one about electron transfer from, and another about proton transfer to the N-atom of, the indole nucleus of Trp have been put forward to account for the heterogeneity of the lifetimes of Trp in solution, in proteins, and in synthetic polypeptides, but a comprehensive picture is yet to emerge. On the other side, there are some studies of Trp phosphorescence, as a delicately sensitive monitor of protein conformation [6–8]. There appears to be a general consensus that, in order to interpret data on protein luminescence, it is essential to acquire a better understanding of similar data on Trp-containing peptides. Some excellent investigations of the fluorescence spectroscopy of peptides have appeared in the literature [1,9], but there seems to be a dearth of studies in which both fluorescence and phosphorescence of peptides of different lengths are examined, and it is clear that such data are sorely needed. The present study was undertaken with a view to taking a first step towards this goal, an aim that could not be accomplished without developing a new approach to recording timeresolved emission spectra of long-lived triplets. Fluorescence and luminescence of the eight synthesized peptides of different length,
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Fig. 1. Upper: Absorption, corrected fluorescence excitation and relative fluorescence yield of sample WVSWAK. Middle: Fluorescence yields of all samples, divided by that of tryptophan (circles), as a function of excitation wavelength. Lower: The graphs in the middle inset are subtracted from their respective terminal levels; and inset: The heights at 240 nm in relation to the yield at 280 nm. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)
containing three to ten amino acids with Trp at different positions in the peptide, have been investigated. The fluorescence and phosphorescence luminescence properties of the peptides are related to their sequence, length and conformation. 2. Materials and Methods 2.1. Materials Peptides were synthesized by China Peptides Co., Ltd. (Shanghai, China) with N 99% purity. All other reagents were of analytical grade. Three peptides are composed of three amino acids: WAK, AWK and SWA. One of them, Trp (W) is located at the end of peptide, while at the other end is a charged amino acid (K) and in the middle is a non-polar amino acid (A). For the other two peptides Trp is located in the middle of peptide, one at the end being non-polar amino acid and one at the
other end being a polar or charged, basic, amino acid.One of the two five-amino acid peptides, KYLWE, contains Tyr (Y) separated from Trp by one amino acid. Y is less strictly polar, and the other amino acids are non-polar or charged, one basic and one acidic located at the two peptide ends. The second five-amino acid peptide, AVSWK, contains Trp at the same position as the other five amino-acid peptide, and also the non-polar amino acids, one neutral/polar and one charged (basic) amino acid at the end of peptide. In the six amino-acid peptide, WVSWAK, and in the seven amino-acid peptide, WAKLAWE, there are two Trps, one of them being at the peptide end; the other amino acids are non-polar or charged, basic or acidic at the peptide end. The ten amino acid peptide, AVSWAKLARE, contains one Trp inside the peptide, the other amino acids being non-polar, polar and charged, two basic and one acidic. The sequence of this peptide is the same as the WAKLAWE, except for the amino acid next to the end amino acid, R, which is polar and basic, instead of W in the former amino acid. Thus in these peptides
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of varied length Trp is located either at the end or within the peptide, and neighbored by amino acids of different polarity: non-polar, polar or charged, basic or acidic. 2.2. Absorption, Fluorescence Excitation and Emission Spectra Absorption spectra were measured on a Shimadzu UV-1601PC, UV– visible spectrophotometer. Fluorescence spectra were recorded by an OLIS RSM 1000 Spectrophotometer. When emission spectra were measured, the excitation wavelength was set to 280 nm and the absorbance (A) of the samples at 280 nm was in the 0.1–0.15 range. The slit widths at the excitation and emission were 1.24 mm and 0.6 mm, respectively. The fluorescence spectra of the samples were nearly identical, and the fluorescence yields from the various samples were calculated as fluorescence intensity at 350 nm, F(350), divided by the absorbance at 280 nm, A(280), multiplied by a factor to take care of the small inner filter effects: η f ¼
Fð350Þ . Að280Þ10ðAð280Þ=2Þ
When corrected fluorescence excitation spec-
tra were measured, the wavelength of the emission monochromator was set to 350 nm and the excitation wavelength (λx) was scanned and the ratio of fluorescence intensity to the excitation intensity, I(λx), is obtained from the instrument; Fc(λx) = F(350)/I(λx). Later on the corrected fluorescence excitation is divided by the absorption spectrum and the inner filter effect, which compensates for the loss of excitation intensity from the entrance to the middle part of the cuvette: η f ðλx Þ ¼ Fcðλx Þ . Aðλx Þ10ðAðλx Þ=2Þ
This was done in a worksheet, and the fluorescence excita-
of the laser pulse; this was achieved by choosing the following values for the three parameters: f = 2 Hz, td = 100 μs, tv = 100 ms. To record a set of time-resolved spectra, the ungated spectrometer was run in the so called timeline mode, which permits one to acquire a number of spectra (up to 100) in a single scan. The laser was run at 10 Hz for a few minutes (in order to build up a standing concentration of long-lived species), a trigger was manually sent to the spectrometer (to start the scan immediately) the laser was stopped (also manually) as soon afterwards as possible. By following this strategy we were able to acquire a set of spectra (with an excellent signal-to-noise ratio) at regularly spaced intervals. It need hardly be added that, since the procedure involves manual acts, a precise value for td cannot be stated; moreover, the first few members of the acquired set (typically two or three) were rejected because the intensity of the incident light at some wavelengths exceeded, perforce and by design, the permissible limit. The indeterminate time at which the first usable spectrum is acquired, is estimated to be 0–0.4 s, and will be denoted by the symbol t0. A cold finger quartz Dewar (filled with liquid nitrogen) was used for refrigerating the sample. A capillary quartz tube was partially immersed into the sample solution, and its upper end was closed with a plastic stopper. The tube (containing the sample at its lower end) was then withdrawn and gently immersed into a reservoir of liquid nitrogen and allowed one to reach the temperature of the surrounding coolant. Finally, the sample was quickly removed from the reservoir and immersed into the cold finger Dewar.
tion as a function of wavelength was obtained. 2.4. Time Resolved Fluorescence 2.3. Measurement of Phosphorescence The excitation source for phosphorescence measurements was a tunable pulsed laser (EKSPLA NT342A-SH-10-WW). The wavelength was 280 nm and the duration and energy of each pulse were approximately 7 ns and 2 mJ, respectively. The resulting phosphorescence emission was viewed (along a direction perpendicular to that of the laser beam) by two multichannel spectrometers, which received part of the emitted light through two optical fibers whose receiving ends were placed on opposite sides of the sample. One spectrometer (Hamamatsu PMA 12 Series; Model C110029-01) uses a gated and intensified array detector; the other was an ungated multichannel detector (B&W Tek Spectrometer Model BRC642E). The gated detector is capable of providing a high time resolution (N10 ns) but it can record only one spectrum after it is triggered (by the laser pulse); the other instrument (to be called the ungated spectrometer) has a much slower time response (see below) but it can record a set of spectra at regular intervals after the arrival of the trigger signal. In a particular run, only one spectrometer was allowed to view the emitted light. Let td and tv denote the delay time (the interval between the firing of the laser and the opening of the gate) and the viewing time (the width of the gate pulse), respectively, and let f be the repetition rate of the excitation pulse. The gated detector was used for viewing the emission during the first 100 ms after the end
Fluorescence decay times were measured using a Jobin Yvon IBM FluoroCube photon counting spectrometer in the time-correlated single photon counting mode (TC-SPC). A nano-LED excitation source (pulse
Table 1 The short (sh) and long (lo) lifetimes of fluorescence (F) and luminescence (P), and ratio of the long- and short-lifetime amplitudes from Hamamatsu spectra (Rlo/sh). Sample
τ sh-F,ns
τ lo-F,ns
τ sh-P,s
τ lo-P,s
Rlo/sh-H
W WAK AWK SWA KYLWE AVSWK WVSWAK WAKLAWE AVSWAKLARE
0.1 1.17 0.69 0.6 1.34 0.91 1.24 1.4 0.82
3.15 5.28 2.02 1.7 2.96 2.78 3.92 4.53 2.54
0.48 0.44 0.47
2.4 3.6 5.0
0.44 0.41 0.56 0.47
4.6 4.0 2.9 5.2
2.60 0.32 0.54 0.04 0.04 0.66 0.60 0.22 0.04
Fig. 2. Upper: The yields, calculated from lifetimes, as a function of yields calculated from steady state emission spectra. Lower: Relation between short and long fluorescence lifetimes. The samples are dissolved in buffer.
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duration 150 ps) was used emitting at 285 nm with a pulse repetition frequency of 1 MHz. 3. Results and Discussion 3.1. Absorption, Fluorescence Excitation and Fluorescence Yields In the upper part of Fig. 1, the absorption-and the corrected fluorescence excitation spectrum of one of the peptides, WVSWAK, is shown. The fluorescence excitation spectrum has been scaled to coincide with the maximum of the absorption spectrum. The graph also contains the relative fluorescence yield, which is the ratio of corrected fluorescence excitation and absorption spectrum. As seen from the graph, it is fairly constant up to 250 nm, where it starts to decrease. That is also the case for tryptophan (W) itself [10]. In the middle part of Fig. 1, the fluorescence yields of the peptides under investigation in relation to that of W are shown. For WAKLAWE the original result is shown (red circles) and for all of them the original result has been replaced by smooth curves. They are parallel lines up to 250 nm, and further up on the wavelength scale there is a U shaped dip of variable depth. In the lower part of Fig. 1, the difference (Δ (λ)) between the terminal level of the yield, called Y (300 nm), and the yield as a function of wavelength, Y (λ); Δ (λ) = Y (300 nm)-Y (λ) is shown. The differences are bell shaped with variable heights and in the inset of the lower part of Fig. 1 the maximum values of Δ are shown, Δ (240 nm), plotted versus the terminal values, Y (300 nm). For the tripeptides, SWA, AWK and WAK, Δ (240 nm) change in proportion to Y (300 nm), for WAK being the highest value. Maximum value of Δ is blue shifted for the two peptides containing polar amino acid K at their end, in comparison with SWA. However, for penta-, hepta- and deca-peptides Δ (240 nm) and Y (300 nm) are anti-correlated.
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which are relatively small for W and hence easily influenced by the local environment. Samples WAK, SWA, KYLWE, WVSWAK and WAKLAWE are on a straight line, which is to be expected when the radiative lifetime is the same. The other samples, W, AVSWK, AVSWAKLARE and AWK, can be assembled on a line which is not passing through the origin. One obvious reason to this is that kr is sample dependent, which means that the radiation transition probability of W also depends upon its environment. In Fig. 2 (lower part) the size of the long lifetime as a function of the short lifetime is shown. Except for W, WAK and KYLWE, they are on a straight line, which means that the two lifetimes are influenced in proportion by the microenvironments. For all peptides the short lifetimes are longer than that of Trp, and (with the above exceptions) increasing with peptide length. The bi-exponential fluorescence decay of Trp originates in the three different stereoconformers, also called rotamers, of W. Because the Cα\\Cβ bond in W is a single bond, see the figure below, the amino acid part of W can rotate with respect to the indole part. It is a restricted rotation and the three different minimal energy rotamers, shown in the figure below, can interconvert by passage over a rotation barrier.
3.2. Relation Between Fluorescence Yield and Fluorescence Lifetimes The nature and probabilities (in parenthesis) of the various deactivation channels open for a molecule in the first excited electronic state (the S1 state) are: fluorescence emission (kr), radiationless decay to the ground state (kn) and intersystem crossing (kisc). Often there are additional deactivation channels, in general called quenching (kq), which could be proton or electron transfer. The fluorescence yield, which is the ratio of emitted to absorbed photons, is related to these probabilities as follows:
Chemical structure of tryptophan.
kr , that is; the ratio of probability of the radiative chanη f ¼ kr þkn þk isc þkq
nel in relation to the sum of all other probabilities from the S1 level. The fluorescence lifetime is as follows: 1 , which means that: ηf = kr ⋅ τf. As long as the radiaτ f ¼ kr þkn þk isc þkq tive lifetime (kr) is unchanged, yields calculated from steady state (CW) fluorescence spectra and lifetimes (τ) should be in proportion to one another. The shapes of the measured fluorescence emission spectra were very similar, and accordingly, fluorescence yields from steady state measurements were taken as the peak heights of the fluorescence spectra divided by the absorption at the exciting wavelength, which is 280 nm, and compensated for the small inner filter effects. The measured fluorescence decays (f(t)) could always be fitted to a sum of two exponential decays with different lifetimes. f(t) = sh αf ⋅ exp(−t/τlo f ) + (1 − αf) ⋅ exp(−t/τf ). lo and sh symbolize long (lo) and short (sh) lifetime and αf is the relative weight of the first contribution. The lifetimes are given in Table 1 and the amplitudes in Fig. 5. The yields from lifetime measurements were calculated as the sum of amplitudes multiplied by their respective lifetimes, or the average lifetime. The fluorescence yields from lifetimes versus steady state measurements are shown in the Fig. 2 (upper) using W as a standard (ηf(CW) = 1, ηf(τ) = 1). The yields from either method agree quite well, as seen by the linearity of the graph. The differences have to be explained according to the differences in the radiative probabilities (kr),
The three stereoconformers, or rotamers, of tryptophan. R is the planar indole group and the projection is along the Cα-Cβ bond (Newman projection). According to Robbins et al. (1980) [11], the fluorescence of W in buffer (called naked W) is quenched by internal proton transfer (ipt) from the –NH+ 3 groups to the indole part of W. In the rotamer having the shortest distance between COO− and indole, according to GudginTempleton and Ware (1984) [5], the fluorescence decay is due to quenching by electron transfer (et) from –COO− to indole. Because of symmetry, two W rotamers have similar –NH+ 3 indole distances, and accordingly, only two lifetimes are expected. When the rotamer interconversion times are longer than fluorescence lifetimes, the two rotamer populations will have different fluorescence lifetimes. According to Robbins et al. (1980) [11], quenching is mainly by ipt, and conformation I and III have the largest populations. In naked W photo ionization can
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also occur, by which a photo ionized electron settles in surrounding water as solvated electrons. The yield of photoionization is constant (about 0.04) and wavelength independent up to 250 nm [11,12], and increases below this value [10]. In peptides and proteins there is additional internal electron transfer (iet). An excited electron on the indole group is transferred to the peptide bond [13], and it is this internal electron transfer that causes the great variability in fluorescence quantum yields of W in a peptide or protein. Furthermore, as seen from Fig. 1, in all peptides the fluorescence yield below 245 nm decreases more than in W. In W the drop means more photoionization, in the peptides it means more electron transfer inside the peptide from higher vibronic states around 245 nm. It is the distance between the W residue and the peptide group and the energy difference between the initial (S1) and final state (excited peptide bond) that are the most crucial factors for
the size of rate constant of the electron transfer. These factors depend on sequence and thus conformation of a particular peptide, which creates unique microenvironment for Trp residue. The fact that the peptides, containing the same motif (for example SWA or WAK) but differing in the rest of sequence, have different yields/lifetimes (Fig. 2) may indicate the influence of conformation/length of the peptides on the distance between indole and peptide bond. Increase in fluorescence yield with peptide length may indicate that distance between indole and peptide group increases and thus there is an increase in electron transfer in comparison with photoionization/electron ejection to the solvent. The result obtained for the tri-peptides, SWA, AWK and WAK, that Δ (240 nm) change in proportion to Y (300 nm), shows a possible influence of the conformation of these tripeptides by decreasing electron transfer to the surrounding water. These peptides have similar
Fig. 3. Luminescence spectra of tryptophan (W) and AVSWK detected at different times (see legend) after the laser pulses had been turned off, using the BWTek array detector. Upper part: The spectra from W and inset: The integrated intensity of each spectrum plotted versus delay time with a double exponential fit. Lower part: Corresponding spectra from sample AVSWK and inset: The component spectra resulting from singular value decomposition and global analysis of the measured spectra with an exponential decay for each of them.
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order of polar and non-polar amino acids regarding Trp, i.e. Trp is neighbored with a non-polar amino acid (A) on one side and with a polar amino acid on the other (K) or with the solvent in the case of WAK. The fact that for penta-, hepta- and deca-peptide Δ (240 nm) and Y (300 nm) are anti-correlated may be explained through effect of peptide conformations, increasing the COO−/indole distance, which consequently increases electron transfer to the solvent, approaching to the value for Trp alone. The amino acid polarity may also have influence on peptide lifetime. For example WAK has the highest fluorescence yield, which may be explained by feasibility of hydrogen bonds formation between terminal lysine and solvent, in this peptide. This may affect distance between indole and peptide bond and result in considerably higher value than those for similar peptides SWA and AWK, but where Trp is located in the middle of peptide. A similar interpretation may be given for the shift of the maximum of Δ to the high energy part of the spectrum, for AWK and WAK comparing to SWA. Also, addition of lysine on SWA increases the yield (SWA b AVSWAKLARE b WVSWAK). This may be due
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to the influence of this charged amino acid on the distance between indole and peptide bond, and on corresponding radiation probability. The presence of Tyr decreases the yield comparing with the peptide of the same length but lacking Tyr (KYLWE b AVSWK — Fig. 2) and causes blue shift of the emission spectra and increasing of the short lifetime. This may indicate extra deactivation channel in the peptide containing Tyr which reduces fluorescence yield. 3.3. 77 K Phosphorescence Emission; Spectra Recorded in the Time Line Mode (BWTek) In Fig. 3 (upper part) successively recorded phosphorescence spectra every 0.6 s from W with a detection window of 0.2 s and dwell time of 0.4 s, after the pulsed laser had been turned off are shown. The shape of the spectrum remains unchanged, but its intensity decays with time. In the inset of the figure, it is shown how the integrated spectrum, done in the worksheet, decays with time. The decay can be well fitted as a double exponential decay with time, and the two amplitudes
Fig. 4. Spectra obtained by global analysis. Lower: Spectra associated with the short lifetime. Upper: Spectra associated with the long lifetime, and Inset: Expanded in the 420–450 nm region.
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with corresponding lifetimes are shown in Fig. 5 and the ratio of amplitudes for the long and short lifetime in Table 1. In the lower part of Fig. 3 the corresponding spectra for AVSWK is shown. In this case, as well as for the other peptides, the overall luminescence spectrum consists of a structured, like that of W, and a nonstructured spectrum, decaying with different lifetimes, after stopping the laser pulses. The spectra recorded in the timeline mode were subjected to singular value decomposition (SVD), which showed that only two principle components were present. The SVD analysis was succeeded by a global analysis, in which both component spectra were assumed to have a single exponential decay with different lifetimes. The result of the global analysis is shown in the inset, the unstructured spectrum having the shortest lifetime (sh) and the structured spectrum the longest lifetime (lo). The lifetimes are given in Table 1. Except for samples SWA and KYLWE, the remaining samples were subjected to a similar treatment as AVSWK. The decay is well described as a sum of two spectra, a structured and a non-structured spectrum with different lifetimes. The result of four samples is collected in Fig. 4. In the lower part, the non-structured spectra, which are normalized to unity at the peak is shown. All non-structured spectra have the same shape. In the upper part of Fig. 4, the structured spectra of the investigated samples as well as that of W are shown. They are also normalized to unity at the maximum. Although quite similar, it is seen that they are slightly different. AVSWK has the narrowest lines and is somewhat displaced to longer wavelength compared to the other spectra. In the inset, enlarged spectra in the 400–450 nm region is shown. The lifetimes obtained from the global analysis are shown in Table 1.
3.4. Phosphorescence Spectra with a Fixed Delay Between Laser and Gate (Hamamatsu) In the above BWTek measurements, the initial delay time between the last laser pulse and detection of the first phosphorescence spectrum might have been anywhere in the interval between 0 and 0.4 s, which was the dwell time of BWTek in the timeline mode. The Hamamatsu detector offers the possibility to detect the phosphorescence spectrum at a certain delay with respect to the laser. For the spectra shown in Fig. 6 the delay is set to 100 μs. As seen from these spectra, by comparing to the BWTek spectra, the Hamamatsu instrument does not have the same spectral resolution as BWTek. Furthermore, by comparing the spectra of W recorded by the two instruments, one sees a drop in wavelength sensitivity in Hamamatsu compared to BWTek below 420 nm. The phosphorescence spectra recorded using the Hamamatsu detector consist of a structured and a non-structured part, as was the case for the BWTek detector, however, a fixed delay makes the determination of the initial intensity ratio of the structured to the non-structured spectrum more precise. The spectra from the Hamamatsu detector are shown in the lower part of Fig. 6, and it is seen that the contribution of structured spectrum to the overall spectrum varies from sample to sample. The phosphorescence spectrum of W (PW) is purely structured, and accordingly, was taken to represent the structured part of the overall spectrum of any sample. As also observed from Fig. 6, the spectrum of SWA (PSWA) is nearly non-structured, and therefore was chosen to represent the non-structured part. In the upper part of Fig. 6 the spectrum of AWK has been resolved into a sum of the W and SWA spectrum: Ps = Alolo·Pw + Ash, using Solver, which is a toolbox in Excel. When Solver returns the Alo and Ash coefficients, their ratio can be obtained: Rlo/sh = Alo/Ash and is given in Table 1 for the various samples. The relative amplitudes can be found, usingAso + Alo = 1, to be: Alo = Rlo/sh/(1 + Rlo/sh) and are given in Fig. 5. The spectra of KYLWE and AVSWAKLARE are somewhat displaced to longer wavelengths than the rest of the non-structured spectra and have very small contributions of the structured spectrum.
Upper: The spectrum of AWK has been resolved into a sum of a structured (W) and a non-structured (SWA) spectrum, using Solver in Excel. 3.5. Origin of Structured and Non-Structured Phosphorescence Emission When a peptide is formed, by linking amino acids through peptide bonds, the free rotation around the Cα\\Cβ bond is restricted, due to the increased sizes of the two parts on either side of the Cα\\Cβ bond. However, there may still be two non-rotating conformers (stereoisomers) of the peptide and the distance between the indole group and the peptide bond might be different in the two conformers. For each conformer (C1 and C2) the rate constant for quenching (kq) is composed of two parts, internal proton transfer (kipt) and electron transfer C1 C1 (kiet). kC1 q = kipt + kiet , which might be different in the two conformers. In all peptides and W double exponential decay of fluorescence and phosphorescence lifetimes were observed. For naked W the two phosphorescence spectra are only distinguishable by lifetime, while the phosphorescence spectra of the peptides are a sum of two distinctly different spectra with different shapes and lifetimes. Except for W, the amplitudes of the long fluorescence lifetime and the short phosphorescence lifetime were roughly similar, indicating that they arise from the same conformer. According to Robbins et al. (1980) [11] and Tsentalovich et al. (2004) [5], a protonated form of the triplet is formed when naked W is photo excited, because prior to intersystem crossing, there is excited singlet state protonation of the indole group. The majority spectrum of W with long lifetime is thought to be due to protonated W, because its relative abundance is nearly the same as that of protonated triplets of W, as seen in transient absorption (Robbins et al. [ref. 11] −0.65, Tsentalovich et al. [ref. 12] −0.58). The short-lived spectrum of W is due to electron transfer from COO− to the indole group, in opposite direction as for internal electron transfer in a peptide, where it is from the indole group to the amide bond. Both proton and electron transfer can happen in one conformer, but their contributions might be different compared to the conformer in the other peptide. Accordingly, the weight of the structured/nonstructured phosphorescence spectrum is C2 a sum of contributions from each conformer: APlo = αC1 ipt + αipt, and: C2 + α . The differences in fluorescence and phosphorescence APsh = αC1 iet iet amplitudes as seen in Fig. 5 might be explained from this; the initial fluorescence amplitudes are in proportion to the weights of two conformers, while initial phosphorescence amplitudes are a sum of amplitudes of ipt and iet, respectively. The fact that the spectra SWA, KYLWE and AVSWAKLARE have very small contributions of the structured spectrum (Fig. 6) may indicate
Fig. 5. Both fluorescence and luminescence decays are a sum of two exponential decays with different lifetimes. The figure shows relative amplitudes of the short (sh) and long (lo) fluorescence (F) /luminescence (P) lifetimes. The luminescence amplitudes were calculated from Hamamatsu spectra (called H) followed by a decomposition into a structured and non-structured spectrum (see text).
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that in these peptides, upon excitation predominates electron transfer, i.e. conformer with unprotonated indole. In these peptides there is no ipt, since W is not a terminal amino acid. Although the amplitudes of the two phosphorescence spectra change from sample to sample, their lifetimes are remarkably invariant; see Table 1. The longest lifetime is 3–5 s, and the shortest lifetime about 0.3–0.4 s. For all peptides ratio between amplitudes of long- and short-phosphorescence lifetimes is similar and significantly lower than for Trp. It means that the same conformer predominates in all peptides, with higher probability of electron transfer than in Trp alone. The photophysics of smaller peptides is also under theoretical investigations. For instance, Shemesh and Domcke (2010) [14] have done density functional calculation (DFT) on the Trp-Gly dipeptide in which the position of the H atom belonging to COOH group has been changed to approach the O atom belonging to the peptide group. After a vertical excitation to the 1Lb state, it relaxes to the 1La state from which it makes a radiationless transition over a low barrier to a locally excited state (1LE). The lowest adiabatic excitation is not the 1La state, but a 1LE
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state localized on the peptide group. This transition can be considered as energy transfer. The 1LE state develops into a charge transfer (CT) state, which can be considered as an electron transfer to the peptide bond, and finally, the CT state has conical intersections with the ground state. It means that the fluorescence quenching is a multistep process and is delicately dependent upon the geometry of the peptide. When the lifetime of the CT state is short, due to conical intersection, it is non-luminescent. It might be suggested that the non-structured phosphorescence spectrum in fact is a luminescent CT state. In donor-acceptor compounds, tuned so that the triplet CT level is lower than triplet level, there is triplet CT emission [15]. The characteristics of triplet CT emission are a non-structured spectrum with a short lifetime. All peptides have similar values of fluorescence long lifetimes, but differ in short lifetimes (Table 1), so that longer peptides have longer short lifetimes. This gives relation between short- and long-lifetimes as shown in Fig. 2, which increases with increasing peptide length. This indicates that population of Trp rotamer with the smaller indole – peptide bond distance is increasing in the peptides with increasing
Fig. 6. Lower: Luminescence spectra of the peptides using the Hamamatsu detector with a fixed delay of 100 μs between laser and detector gate. The gate width was 0.1 ms.
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peptide length, while population of the rotamer with longer indole – peptide distance stays relatively unchanged. The fact that probability of electron ejection from Trp (photoionization) and probability of electron transfer in peptides are dependent on peptide sequence and length and thus on their conformation, may enable fine regulation of certain processes in very narrow Trp environments in a protein. It means that light-induced electron ejection and/ or electron transfer may be one of the ways of fine regulation of protein conformation changes, being connected with Trp rotameric switch. Such fine regulation, involving Trp located in protein conserved domains, is known also for some other kinds of stimuli [16,17]. 3.6. The Conformer Model and Beyond The conformer model was established to explain the multi exponential fluorescence decay of W in solution. In their work on substituents on the 3-ethyl chain of W (the amino and acid group of W), Petrich et al. [4] introduced the socalled modified conformer model (MCM) in which they postulated electron transfer from the indole group of W to the electrophilic substituents. Differences in lifetimes of the three conformers were not only due to populations of the conformers but also to differences in quenching abilities of the electrophilic substituents. GudginTempleton and Ware [12] modified the MCM even further to state that the charge (electron) donor is the solvated indole chromophore, which could be a charge to solvent complex (CTTS). This is in line with the fact that the emitting state is always 1La and fluorescence quenching could be a dissociation of the complex, or in other words, an electron transfer. These ideas, originally used to explain the multiexponential fluorescence decay of W and substituents on the 3– ethyl chain of W, are also in line with the results for the peptides studied here, and are most clearly seen for the peptides where W is exposed to the solvent. It is believed that charge transfer (CT) takes place at 77 K and that part of the luminescence spectrum seen at that temperature is due to emission from a stabilized CT complex. Most probably this CT complex originates from electron transfer to the peptide bond and not from the CTTS complex. This has bearings on the use of nomenclature; phosphorescence should be reserved for proton transfer (ipt), in which the number of electrons on the indole group is unchanged, and luminescence should be used for electron transfer (iet), in which case the number of electrons is changed. The spectra seen at 77 K are partly a phosphorescence and luminescence spectrum. 4. Conclusions In conclusion, our results show that there is an influence of conformation/length of the peptides on the distance between indole and peptide bond, which has crucial influence on luminescence emission. Both proton and electron transfer can happen in one Trp conformer, but their contributions might be different in different peptides. In the peptides there is higher probability of electron transfer than in Trp alone. Population of Trp rotamer with the smaller indole–peptide
bond distance is increasing in the peptides with increasing peptide length. The obtained results show that fluorescence and phosphorescence properties of Trp even in small peptides are complex, and that studies on peptides are unavoidable for deep understanding of protein luminescence. Acknowledgments The authors acknowledge receiving financial support from the Research Council of Norway (Project No. 191102), as well as from the Ministry of Education, Science and Technological Development of the Republic of Serbia (Project No. 173017). The authors are grateful to Prof. Mikael Lindgren for providing them access to the instrument for time resolved fluorescence measurements. References [1] R.W. Alston, M. Lasagna, G.R. Grimsley, J.M. Scholtz, G.D. Reinhart, C.N. Pace, Peptide sequence and conformation strongly influence tryptophan fluorescence, Biophys. J. 94 (2008) 2280–2287. [2] R.J. Robbins, J.W. Petrich, D.B. McDonald, G.R. Fleming, G.S. Beddard, G.W. Robinson, P.J. Thistlewhaite, G.J. Woolfe, Photophysics of aqueous tryptophan: pH and temperature effects, J. Am. Chem. Soc. 102 (1980) 6271–6279. [3] M.C. Chang, J.W. Petrich, D.B. McDonald, G.R. Fleming, Nonexponential fluorescence decay of tryptophan, tryptophylglycine, and glycyltryptophan, J. Am. Chem. Soc. 88 (1983) 3819–3824. [4] J.W. Petrich, M.C. Chang, D.B. McDonald, G.R. Fleming, On the origin of nonexponential fluorescence decay in tryptophan and its derivatives, J. Am. Chem. Soc. 105 (1983) 3824–3832. [5] E.F. Gudgin-Templeton, W.R. Ware, The time dependence of the low-temperature fluorescence of tryptophan, J. Phys. Chem. 88 (1984) 4626–4631. [6] G. Strambini, E. Gabellieri, Temperature dependence of tryptophan phosphorescence in proteins, Photochem. Photobiol. 51 (1990) 643–648. [7] M. Gonnelli, G.B. Strambini, Phosphorescence lifetime of tryptophan in proteins, Biochemistry 34 (1995) 13847–13857. [8] J. Broos, E. Gabellieri, G. I. van Boxel, J. B. Jackson, G. B. Strambini, Tryptophan phosphorescence spectroscopy reveals that a domain in the NAD(H)-binding component (dI) of transhydrogenase from Rhodospirillum rubrum has an extremely rigid and conformationally homogeneous protein core, J. Biol. Chem. 278 (278) 47578–47584. [9] C.-P. Pan, M.D. Barkley, Conformational effects on tryptophan fluorescence in cyclic hexapeptides, Biophys. J. 94 (2004) 3828–3835. [10] H.B. Steen, Wavelength dependence of the quantum yield of fluorescence and photoionization of indoles, J. Chem. Phys. 61 (1974) 3997–4002. [11] R.J. Robbins, G.R. Fleming, G.S. Beddard, G.W. Robinson, P.J. Thistlewaite, G.J. Woolfe, Photophysics of aqueous tryptophan: pH and temperature effects, J. Am. Chem. Soc. 102 (1980) 6271–6279. [12] Y.P. Tsentalovich, O.A. Snytnikova, R.Z. Sagdeev, Properties of excited states of aqueous tryptophan, J. Photochem. Photobiol. A Chem. 162 (2004) 371–379. [13] P.R. Callis, A. Petrenko, P.L. Muino, J.R. Tusell, Ab Initio prediction of tryptophan quenching by protein electric field enabled electron transfer, J. Pys. Chem. B 111 (2007) 10335–10339. [14] D. Shemesh, W. Domcke, in: Wagner, et al., (Eds.), High Performance Computing in Science and Engineering, Springer-Verlag Berlin Heidelberg 2010, pp. 641–649. [15] J. Herbich, A. Kapturkiewics, J. Nowacki, Phosphorescent intramolecular charge transfer triplet states, Chem. Phys. Lett. 262 (1996) 633–642. [16] R. Pankov, E. Cukierman, K. Clark, K. Matsumoto, C. Hahn, B. Poulin, K.M. Yamada, Specific β1 integrin site selectively regulates Akt/protein kinase B signaling via local activation of protein phosphatase 2 A, J. Biol. Chem. 278 (2003) 18671–18681. [17] M. Sotomayor, K. Schulten, The allosteric role of the Ca2+ switch in adhesion and elasticity of C-cadherin, Biophys. J. 94 (2008) 4621–4633.