Journal of Photochemistry and Photobiology B: Biology 149 (2015) 243–248

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Fluorescence kinetics of Trp–Trp dipeptide and its derivatives in water via ultrafast fluorescence spectroscopy Menghui Jia a, Hua Yi a, Mengfang Chang a, Xiaodan Cao a, Lei Li a, Zhongneng Zhou a, Haifeng Pan a, Yan Chen b, Sanjun Zhang a,⇑, Jianhua Xu a a b

State Key Laboratory of Precision Spectroscopy, East China Normal University, Shanghai 200062, China Tongji Hospital Affiliated to Tongji University, 389 Xincun Road, Shanghai 200065, China

a r t i c l e

i n f o

Article history: Received 13 November 2014 Received in revised form 23 May 2015 Accepted 16 June 2015 Available online 17 June 2015 Keywords: Tryptophan dipeptides Time resolved fluorescence Decay associated spectra (DAS) Quasi static self-quenching (QSSQ) Electron/proton transfer (ET/PT)

a b s t r a c t Ultrafast fluorescence dynamics of Tryptophan–Tryptophan (Trp–Trp/Trp2) dipeptide and its derivatives in water have been investigated using a picosecond resolved time correlated single photon counting (TCSPC) apparatus together with a femtosecond resolved upconversion spectrophotofluorometer. The fluorescence decay profiles at multiple wavelengths were fitted by a global analysis technique. Nanosecond fluorescence kinetics of Trp2, N-tert-butyl carbonyl oxygen-N0 -aldehyde group-L-tryptophan-Ltryptophan (NBTrp2), L-tryptophan-L-tryptophan methyl ester (Trp2Me), and N-acetyl-L-tryptophan-Ltryptophan methyl ester (NATrp2Me) exhibit multi-exponential decays with the average lifetimes of 1.99, 3.04, 0.72 and 1.22 ns, respectively. Due to the intramolecular interaction between two Trp residues, the ‘‘water relaxation’’ lifetime was observed around 4 ps, and it is noticed that Trp2 and its derivatives also exhibit a new decay with a lifetime of 100 ps, while single-Trp fluorescence decay in dipeptides/proteins shows 20–30 ps. The intramolecular interaction lifetime constants of Trp2, NBTrp2, Trp2Me and NATrp2Me were then calculated to be 3.64, 0.93, 11.52 and 2.40 ns, respectively. Candidate mechanisms (including heterogeneity, solvent relaxation, quasi static self-quenching or ET/PT quenching) have been discussed. Ó 2015 Published by Elsevier B.V.

1. Introduction As an intrinsic fluorescence probe, tryptophan (Trp) has been widely used to study the conformational changes of peptides/proteins since it possesses the highest UV extinction coefficient and quantum yield, high sensitivity to the polarity and dynamics of the immediate environment [1,2]. Over the past decades, a large number of biomolecules including Trp peptides [3], proteins [4– 8] or other molecules containing Trp [9–11], and complex solutions with Trp [12,13], have been investigated by using Trp fluorescence spectroscopy. In order to obtain a deep insight into the protein dynamic and functional properties, many mutants of Trp residue were reported [14,15]. However, peptides/proteins usually contain more than one Trp residue and different Trp residues are close to each other, thus leading to the interaction among the nearby Trp residues and it may complicate the Trp fluorescence characterizations [16–18]. Moreover, the studies on Trp–Trp interaction and dynamics are becoming essential because of the difficulty and

⇑ Corresponding author. E-mail address: [email protected] (S. Zhang). http://dx.doi.org/10.1016/j.jphotobiol.2015.06.014 1011-1344/Ó 2015 Published by Elsevier B.V.

limitation of site-directed mutagenesis in proteins. It is known that even single Trp can always yield multiexponential fluorescence decays mainly because of the ground state heterogeneity, relaxation of the peptide/protein matrix (or solvent water) [19,20], and electron/proton transfer (ET/PT) within the excited-state of Trp [21–24]. Compared to Trp or single-Trp peptides, Trp–Trp (Trp2) dipeptide has two indoles connected by a peptide bond, resulting in more flexible conformations and more complex lifetime components. The charge transfer between the carbonyl group, amino group and peptide bond as well as intramolecular interaction of two indoles will all contribute to the decay kinetics of Trp2 dipeptide. In previous studies [25,26], the time constant of ‘‘solvent relaxation’’ for Trp, NATA, single-Trp dipeptides in water was 1–2 ps, with a pre-exponential amplitude that was positive at the blue side and negative at the red side in the decay associated spectra (DAS). An ultrafast decay around 30 ps with a positive amplitude has been found for the single-Trp dipeptides, which verifies the predictions of dipeptide QSSQ (quasi static self quenching) – the loss of quantum yield to sub-100 ps decay process. However, there is no report in studying the ultrafast solvent and intramolecular interaction of Trp2 dipeptide and its derivatives.

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Understanding the interaction of Trp–Trp in water or peptides/proteins is critical, we have designed and synthesized three Trp2 derivatives (Scheme 1) as an intramolecular electron transfer system. The samples differ in the substituted parts of amino and carbonyl groups. Trp plays an important role in the electron transfer during lots of biological reactions [27–29] and acts as an excellent electron donor after excited by a UV beam [30]. The excited indoles are usually the electron donors, while the peptide bond and carbonyl groups act as the electron acceptors. In this paper, time resolved fluorescence spectroscopy of Trp– Trp dipeptides including fluorescence decay kinetics and DAS, are collected from both an upconversion spectrophotofluorometer and a time correlated single photon counting (TCSPC) apparatus coupled to fs and ps laser sources. The full dynamics are dissected to explore contributions from the QSSQ of heterogeneous environs and water relaxation. We also studied the intramolecular interaction of Trp2 dipeptide caused by photon induced electron transfer. 2. Experimental section 2.1. Material L-Tryptophan-L-tryptophan dipeptide (Trp2), N-(tert-Butoxycarbonyl)-L-tryptophan-L-tryptophan (NBTrp2), L-tryptophan-Ltryptophan methyl ester (Trp2Me), N-acetyl-L- tryptophan-Ltryptophan methyl ester (NATrp2Me) were prepared by condensing single L-tryptophan or its derivatives, and further purifying (see Scheme 1). L-tryptophan and its derivatives N-(tert-Butoxy carbonyl)-L-tryptophan, L-tryptophan methyl ester and N-acetyl -L-tryptophan were purchased from Sigma and used without further purification. The solutions were prepared in deionized water at pH 6.7 with a resistivity of 18.2 MX cm1. A typical concentration of Trp2 in water for the femtosecond upconversion fluorescence measurement was 1 mM. The concentrations of NBTrp2, Trp2Me and NATrp2Me for upconversion were 0.2, 1 and 0.1 mM, respectively, due to the low solubility of NBtrp2 and NATrp2Me. The concentration of all samples for other measurements was 0.1 mM. All sample solutions were fresh in each time-resolved measurement, and all the measurements were made at room temperature.

2.2. Steady-state absorption and fluorescence spectra Steady-state absorption spectra were obtained with a UV–Vis spectrophotometer (TU1901, Beijing Purkinje General Instrument Co. Ltd.) from 230 to 320 nm with a step of 0.2 nm. Steady-state

R1

O

O

C HN

O

fluorescence measurements were acquired by a FluoroMax-4 spectrofluorometer (Horiba, Jobin Yvon). The excitation wavelength was set at 280 nm, and both bandwidth of the excitation and emission were 2.5 nm. The steady-state emission spectra were recorded from 305 nm to 480 nm with a step of 0.5 nm. Under the same circumstance, the baseline was measured by water in the sample cells without any Trp2 dipeptides. All spectra were calibrated by the baseline and the wavelength-dependent instrumental profiles. 2.3. Upconversion spectrophotofluorometer In this experimental setup, a mode locked Ti:sapphire laser (Tsunami, Spectra-Physics) generated a 400 mW seed pulse train with a typical pulse duration of 50 fs at a repetition rate of 80 MHz, pumped by a CW diode-pumped solid state lasers (Millennia Pro@532 nm, Spectra-Physics). The femtosecond laser pulses were used to seed a Ti:sapphire regenerative amplifier (Spitfire Pro, Spectra-Physics), generating a 2 W pulse train centered at 800 nm with a pulse width of 50 fs and a repetition rate of 1 kHz. 60 percent of the fundamental pulse energy was then used to pump a parametric amplifier (Topas-C, Spectra-Physics) to generate the ultraviolet excitation pulses at 295 nm with an average power of 4 mW. The UV pump pulse was purified by a pair of UV prisms and attenuated to less than 0.5 mW to avoid undesirable effects such as photo degradation and sample damage. Leftover 40 percent of the pulse energy was attenuated to be less than the average power of 500 mW, and used as a gate pulse to probe the fluorescence decay. The sample was injected into a continuously spinning UV quartz disk, 80 mm in diameter, rotating with a tangential velocity greater than 1 m/s at the beam entrance. In the fluorescence upconversion experiments, the fluorescence was collected by a pair of parabolic focus mirrors and focused into a 0.2 mm BBO mixing crystal. The frequency upconverted signal from 234 nm to 258 nm was obtained via type I sum frequency generation with the gate pulse. The gate pulse and the fluorescence of samples were arranged at noncollinear configuration to reduce background signals from the gate beam, fluorescence and residual pump UV beam. The polarization of gated fluorescence (vertical) was fixed by the BBO crystal and the excitation was set at the magic angle (54.7°) by a motor-controlled zero-order half-wave plate. The upconversion signal was directed into a monochromator (Omni-k500, Zolix), and then detected by a solar blind photomultiplier tube (R7154, Hamamatsu). The upconversion signal was integrated/amplified by a fast gated integrator (BOXCAR SR250, Stanford Instrument) and was recorded by a 16-bit analog-to-digital converter (BNC2120, National Instruments). The instrument response function (IRF) under the same circumstances was about 300 fs, determined by the cross correlation between Raman scattering of water at 328 nm and the gate pulses. 2.4. TCSPC apparatus

R2

C N H

HN

HN

Scheme 1. Chemical structures of Trp2 dipeptide and its derivatives. The groups R1 and R2 are as follows. R1 = H, R2 = H in Trp2; R1 = H, R2 = CH3 in Trp2Me; R1 = COOC(CH3)3, R2 = H in NBTrp2; R1 = COCH3, R2 = CH3 in NATrp2Me.

In this system, the excitation pulses centered at 295 nm had a repetition rate of 10 MHz, and were provided by a picosecond pulsed diode laser (PDL 800-B, PicoQuant). The fluorescence measurement was recorded by a stand-alone time correlated single photon counting (TCSPC) module (PicoHarp 300, PicoQuant) and a single photon counting photomultiplier (PMA165A-N-M, PicoQuant). All the TCSPC measurements were made at the magic angle (54.7°). A monochromator (7ISW151, Sofn Instruments) with a slit width of 1 mm was used to select the emission wavelength. The Rayleigh scattering from silica nanoparticle aqueous solution was used as the instrument response function (IRF). Long pass fluorescence filters with OD 4 or greater were set at the entrance slit of the monochromator, to block the Rayleigh scattering from the excitation pulses. All the fluorescence decays from both the sample

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Iðk; tÞ ¼

X

ai ðkÞet=si ;

ð1Þ

Goodness of fit was evaluated via the v value, which was 1.01–1.3 in this work. The baseline (or dark counts) was removed automatically by the fitting algorithm. All the experimental decay profiles at different wavelengths from 320 to 410 nm were analyzed by global fit [31,32]. It was performed by constraining the 3 lifetime components globally, and fitting all the experimental curves with the same model described by Eq. (1). The decay associated spectra (DAS) were then obtained from the results of the global fit. 2 R

3. Results and discussion 3.1. Steady-state absorption and fluorescence spectra Fig. 1 shows the normalized steady-state absorption and emission spectra of Trp2 dipeptide and its derivatives (Trp2, NBTrp2, Trp2Me and NATrp2Me). All the samples measured here display a typical indole absorption band around the peak of 280 nm. The emission peak of Trp2 is at 362 nm, which is red shifted relative to that of Trp in water (358 nm). NBTrp2 shows a 3-nm red shift but Trp2Me shows a 3-nm blue shift with respect to Trp2. It should be noted that the emission spectra do not show an obvious difference between Trp2 and NATrp2Me, as well as the emission intensities. The quantum yields of Trp2 and its derivatives were determined through equation, Q m ¼ Q R IIRs

AR n2s As n2 R

where Q is the quan-

tum yield, I is the integrated fluorescence intensity, A is the absorbance, and n is the refractive index. The subscripts s and R represent the sample and reference standard, respectively. The reference was L-Trp, the quantum yield of which was reported to be 0.13 [33]. Thus, the quantum yields of Trp2, NBTrp2, Trp2Me and NATrp2Me were obtained to be 0.049, 0.12, 0.033 and 0.041, respectively. Compared to the quantum yield of L-Trp, the relative low quantum yields indicate a strong fluorescence quenching in Trp2 dipeptides except for NBTrp2. In the previous work [26,34], the emission spectra of Trp-X zwitterion show a blue shift and those of X-Trp zwitterion show a red shift relative to NATA, where X is another amino acid without a fluorophore. This blue or red shift can be explained as the result of the charge difference in the tryptophanyl moiety, that is, the charge transfer between the

Normalized Intensity (a.u.)

Absorption

Trp2 Trp2 Me NBTrp2 NATrp2 Me

Emission

λ ex =280nm

indole and the amino group. In this work, it is found that, with a free amino in the Trp residue, Trp2Me was blue shifted relative to NATrp2Me as the same as Trp2 did to NBTrp2; with a free carboxyl on the Trp residue, Trp2 was red shifted to Trp2Me and so did NBTrp2 to NATrp2Me. Therefore, this is in accordance with the previous observation [26,34]. 3.2. Picosecond-resolved fluorescence decay kinetics The time resolved emission spectra of Trp2 and its derivatives were measured by the TCSPC apparatus on the time scale from 200 ps to 20 ns. In this work, the decay curves of Trp2, NBTrp2, Trp2Me and NATrp2Me exhibit tri-exponential profiles at a typical wavelength of 360 nm (see Fig. 2), with the average lifetimes of 1.99, 3.04, 0.72 and 1.22 ns, respectively. For comparison, Trp in water yields a biexponential decay with the components of 0.6 and 3.2 ns (data not shown), resulting in an average lifetime of 3 ns. Reported in the previous work [35], the excited indole of Trp is close to the amino and carbonyl quenching group, leading to the proton transfer and electron transfer respectively. Trp dipeptide in water presents as a zwitterion, namely the substituted group attached to carbonyl group is –O. According to the quenching order (–CH3 > –OH  –OCH3 > –NH2  –O) [36], the Trp2 should have a longer lifetime relative to Trp2Me as well as NBTrp2 compared with NATrp2Me. With substituted groups attached to the amino group, NBTrp2 should a longer lifetime compared with Trp2 as well as NATrp2Me compared with Trp2Me. Those predictions have been demonstrated compatible with our experimental results. Decay associated spectra (DAS) are usually very useful to analyze the heterogeneity versus relaxation through the full emission spectra of Trp. The decay profile of Trp2, NBTrp2, Trp2Me and NATrp2Me could be globally fitted by three lifetimes as stated above, showing different decay associated spectra from 320 nm to 420 nm (see Fig. 3). In this work, all dipeptides have three exponential decay terms, but they differ drastically in the component amplitudes except for NBTrp2 with almost similar amplitudes on all the three components. In effect, the

Fluorescence kinetics of Trp-Trp dipeptide and its derivatives in water via ultrafast fluorescence spectroscopy.

Ultrafast fluorescence dynamics of Tryptophan-Tryptophan (Trp-Trp/Trp2) dipeptide and its derivatives in water have been investigated using a picoseco...
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