Spectral Properties of Thioflavin T in Solvents with Different

Spectral Properties of Thioflavin T in Solvents with Different Dielectric Properties and in a Fibril-Incorporated Form Alexander A. Maskevich,*,† Vitali I. Stsiapura,† Valeriy A. Kuzmitsky,‡ Irina M. Kuznetsova,§ Olga I. Povarova,§ Vladimir N. Uversky,*,|,⊥ and Konstantin K. Turoverov*,§ Yanka Kupala Grodno State University, Grodno 230023, Belarus, Institute of Molecular and Atomic Physics, National Academy of Sciences of Belarus, Minsk 220072, Belarus, Institute of Cytology, Russian Academy of Sciences, St. Petersburg 194064, Russia, Institute of Biological Instrumentation, Russian Academy of Sciences, Pushchino 142290, Russia, and Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202 Received October 21, 2006

The increase in the solvent polarity induces a significant shift of the long-wavelength absorption band of the thioflavin T (ThT) to the shorter wavelengths. This is due to the fact that the positive charge of the ThT molecule (Z ) +1e) is unequally and very differently distributed between the benzthiazole and aminobenzene rings in the ground and excited states. Therefore, ThT ground state is stabilized by the orientational interactions of the polar solvent dipoles with the positively charged ThT fragments, whereas the configuration of the solvation shell of the ThT molecule in the excited Franck-Condon state is likely far from being equilibrium. ThT absorption spectrum has the shortest (412 nm) and the longest (450 nm) wavelengths in water and in water being incorporated to the amyloid fibrils, respectively. Intriguingly, the position of the ThT fluorescence spectrum depends on the polarity of solvent to a significantly lesser degree than its absorption spectrum: being excited at 440 nm, ThT has emission with maxima at 493 and 478 nm in water and fibrils, respectively. This can be due to the fact that, in the excited state, the rotational oscillations of the ThT fragments relative to each other prevent establishing equilibrium with the solvent and fluorescence occurs from the partially equilibrium excited stated to the partially equilibrium ground state. For the fibril-incorporated ThT, the maximum of the fluorescence excitation spectrum coincides with the maximum of the long wavelength absorption band (450 nm), whereas for ThT in aqueous and alcohol solutions, additional short-wavelength bands of fluorescence and fluorescence excitation spectra were described (Naiki et al. Anal. Biochem. 1989, 177, 244-249; Le Vine Methods Enzymol. 1999, 309, 274-284). These bands could result either from some fluorescent admixtures (including free benzthiazole and aminobenzene) or from the specific ThT conformers in which benzthiazole and aminobenzene rings, being oriented at φ angle close to 90 or 270°, serve as independent chromophores. On the basis of the results of the quantum-chemical calculations, it is proposed that at φ ) 90° (270°), the relatively low barrier (only 700 cm-1) of the internal rotation of the benzthiazole and aminobenzene rings relative to each other gives rise to a subpopulation of ThT molecules possessing a violated system of the π-conjugated bonds of the benzthiazole and aminobenzene rings. Keywords: thioflavin T • amyloid fibril • absorption • fluorescence • fluorescence excitation • solvent polarity • solvent orientational polarizability • quantum chemical calculations

Introduction The sequences of globular proteins have evolved in such a way that their unique native states can be found very efficiently even in the complex and crowded environment inside a living cell. However, under some conditions, proteins misfold; i.e., * To whom correspondence should be addressed. Vladimir N. Uversky, Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, 635 Barnhill Drive, MS#4021, Indianapolis, IN 46202; Phone, 317-278-9194; Fax, 317-274-4686; E-mail, [email protected]. Konstantin K. Turoverov, Institute of Cytology, Russian Academy of Sciences, Tikhoretsky av., 4, St. Petersburg 194064, Russia; E-mail, [email protected]. Alexander A. Maskevich, Yanka Kupala Grodno State University, ul. Ozheshko, 22, Grodno 230023, Belarus; E-mail, [email protected]. † Yanka Kupala Grodno State University. ‡ National Academy of Sciences of Belarus. § Institute of Cytology, Russian Academy of Sciences. | Institute of Biological Instrumentation, Russian Academy of Sciences. ⊥ Indiana University School of Medicine.

1392

Journal of Proteome Research 2007, 6, 1392-1401

Published on Web 02/17/2007

they fail to fold properly, or to remain correctly folded. This misfolding can lead to the development of different pathological conditions. A number of human diseases, including the amyloidoses and several neurodegenerative disorders, such as Alzheimer’s disease, Parkinson’s disease, transmissible spongiform encephalopathies, etc., originate from the deposition of stable, ordered, and filamentous protein aggregates, commonly known as amyloid fibrils. In each of these pathological states, a specific protein or protein fragment changes from its natural soluble form into insoluble fibrils, which accumulate in a variety of organs and tissues.1-16 Approximately 20 proteins, which are unrelated structurally or at the level of primary structure, are known to be involved in the amyloidoses (extracellular deposits). In addition, a number of diseases also originate from the intracellular deposits, i.e., accumulation of fibrillar proteins within cells. Prior to fibrillation, amyloidogenic 10.1021/pr0605567 CCC: $37.00

 2007 American Chemical Society

research articles

Spectral Properties of Thioflavin T

polypeptides may be rich in β-sheet, R-helix, β-helix, or contain both R-helices and β-sheets. They may be well folded or belong to a class of natively unfolded (or intrinsically unstructured) proteins.8,10 Despite these differences, the fibrils from different pathologies display many common properties including a core cross-β-sheet structure in which continuous β-sheets are formed with β-strands running perpendicular to the long axis of the fibrils.17 Amyloid fibrils have been shown to form in vitro from disease-associated,18-25 as well as from disease-unrelated, proteins and peptides.26-44 Furthermore, there is an increasing belief that the ability to form fibrils is a generic property of the polypeptide chain, i.e., many proteins, perhaps all, are able to form amyloid fibrils under appropriate conditions.3,34,39 For a long time, since Virchow’s original description in 1854 of the waxy deposits in tissue that stained in a characteristic way with certain dyes until very recently, atomic level resolution structure of amyloid fibrils was an elusive goal.45 Recently, significant progress has been achieved in understanding the structure of amyloid fibril at the atomic level both from experimental studies and from computer modeling.46 It has been shown that a seven-residue peptide segment, GNNQQNY, derived from the yeast protein Sup35 (a protein whose fibril formation represents the basis of protein-based inheritance and prion-like infectivity in yeast47-51) is not only able to assemble into the amyloid-like fibrils but can also form closely related microcrystals, from which the atomic structure of the cross-β spine was determined.52 It has been shown that GNNQQNY molecules were extended and were hydrogen bonded to each other forming standard parallel β-sheets, where each GNNQQNY molecule represented a single β-strand. The overall structure of a fibril was shown to represent a double β-sheet, which is stabilized by side chains protruding from the two sheets via a dry, tightly self-complementing steric zipper.52 Currently, studies on protein misfolding in vitro and specific staining of amyloid fibrils in vivo rely heavily on the characteristic changes in spectral parameters of the benzothiazole dyes, thioflavin S (ThS), and thioflavin T (ThT), which are known to form highly fluorescent complexes with amyloid-like fibrils.53-60 Due to these unique spectral properties, ThS and ThT have been used for the histological demonstration of amyloid fibrils in tissues and organs since the 1960s.61 ThS is a complex mixture of methylated and sulfonated primuline polymers. Its emission intensity is enhanced in the presence of amyloid fibrils.53 Despite being a heterogeneous mixture, the anionic ThS is commonly used for histology. Its cationic counterpart, ThT, on the other hand, is a single molecular species of low molecular weight. ThT (also known as Basic Yellow 1 or CI 49005) is a benzothiazole salt binding of which to amyloid fibrils is accompanied by the characteristic changes in its spectroscopic properties giving rise to the wide use of ThT for biochemical studies of the formation of amyloid fibrils in vitro.39,53,55,57,60,62-64 Importantly, ThT does not interact with folded or partially folded monomeric proteins, soluble oligomers, or amorphous aggregates, or if such interaction does occur, it is not accompanied by noticeable changes in ThT fluorescence. Despite the fact that the number of papers describing the ThT application for the analysis of different aggregating systems is rapidly rising, the molecular mechanism of specific ThT binding to amyloid fibrils and the reasons for the dramatic changes in its fluorescence properties in such complexes is poorly understood. Furthermore, there are a number of inconsistencies in the description of the spectral properties of this dye. For

example, in some papers, it is mentioned that the ThT fluorescence spectrum in aqueous solutions is significantly short-wavelength shifted, that its excitation and absorption spectra do not coincide, and that the ThT fluorescence excitation spectrum in glycerol possesses two maxima.60 At the same time, the solvent polarity influence on the positions of ThT absorption, excitation, and fluorescence spectra were not systematically analyzed. The main goal of the current study was an attempt to fill this gap and study the effect of solvent polarity on ThT spectral properties. These experimental studies are supported by the quantum chemistry calculations aiming for elucidation of the structural peculiarities of ThT molecule in its ground and excited states.

Materials and Methods Materials. We have analyzed ThT samples from SigmaAldrich (USA) and Fluka (Switzerland) dissolved in distilled water or organic solvents, such as methanol, acetonitrile, ethanol, acetone, chloroform, 2-propanol, ethylene glycol, N,Ndimethylformamide, dimethylsulfoxide (DMSO), pyridine, glycerol (all from Merck, Germany), and 1-butanol (from Baker). Glycerol concentration in water-glycerol mixtures was measured using the Abbe refractometer (LOMO, Russia). Amyloidlike fibrils were produced by the incubation of insulin (Sigma) in 20% acetic acid solution in the presence of 100 mM NaCl (pH 2.0) at 37 °C with constant stirring. ThT was dissolved in 20 mM Tris-HCl buffer (pH 7.7) containing 150 mM NaCl. Spectral Measurements. Fluorescence emission and excitation spectra were measured using the CM2203 spectrofluorometer (Solar, Belarus) and the fluorescence spectrophotometer described in ref 65. Absorption spectra were analyzed using EPS-3T (Hitachi, Japan), Specord 200PC (Analytik Jena, Germany) and PV 1251A (Solar, Belarus) spectrophotometers. Computational Approaches. Semiempirical (AM166 and PM367 and ab initio methods [basis sets 3-21G68 and 6-31G69] were used to calculate the geometry of ThT cation (Z ) +1) in the ground S0 state. More accurate description of the ground state conformers corresponding to the minima and the saddle points at the potential energy surface was achieved via the additional optimization using the extended basis 6-31G**, which includes d-orbitals of all the heavy atoms and p-orbitals of the hydrogen atoms.70 The energy of the excited singlet Franck-Condon (S1*) state, the oscillator strength for the S0 f S1* transition, and the efficiency of the charge transfer in the excited-state were determined by the semiempirical method INDO/S,71 which was specially elaborated to describe the properties of the excited states. All calculations (with the exception for INDO/S) were performed using PC GAMESS 6.372 and Wingamess (version R2, 12 December 2003) versions of the set of quantum-chemistry programs Gamess-US.73 The energies of the excited states, together with the charge-transfer probabilities and the oscillator strengths of the electronic transitions, were calculated using a set of programs developed by one of us (Kuzmitsky, V. A., personal communication). All calculations were performed for the molecules in the gas phase. Conformers’ geometries and the molecular orbitals were visualized using the Molekel program.74

Results and Discussion Quantum-Chemistry Analysis of the ThT Properties in the Ground and the Excited States. Figure 1 represents the ThT Journal of Proteome Research • Vol. 6, No. 4, 2007 1393

research articles

Figure 1. ThT structure. (A) Chemical structure of the ThT cation. (B) Three-dimensional model of the ThT cations. S, C, and N atoms are shown in yellow, light blue, and dark blue, respectively, whereas hydrogen atoms are gray. Three ThT fragments are shown: benzthiazole ring (I), benzene ring (II), and dimethylamino group (III). Atoms forming torsion angles φ (N5-C6-C12C13) and ψ (C14-C15-N18-C19) are numbered in B. These angles are used to characterize the potential ThT configurations in the ground and the excited states.

structure and shows that the molecule can be divided on three fragments, a benzthiazole ring (fragment I), a benzene ring (fragment II), and a dimethylamino group (fragment III). As these three groups are rather rigid, both structure and photophysical behavior of ThT in gas phase or in the solution should mostly be determined by their spatial orientations. On the basis of these assumptions, we chose a couple of torsion angles, φ (N5-C6-C12-C13) and ψ (C14-C15-N18-C19), to detect and analyze the plausible conformations of the ThT molecule in the ground state. Fixed values from 0 to 360° (with a step of 30°) were assigned to these angles, and the semiempirical methods AM1 and PM3 were used to search for the stable states characterized by the geometries with the minimal energies. Besides the fixed angles φ and ψ, all other parameters were allowed to vary. This approach was used to determine the geometry of the stable conformers of the ThT molecule in the ground state (data not shown) and to build the energy surface of the ground state S0, i.e., the dependence of the ThT energy on φ and ψ (Figure 2A). This analysis revealed that the dependence of the ThT energy on the torsion angle φ between the benzthiazole and aminobenzene rings possesses four minima separated by relatively low-energy barriers (700 cm-1 or 1200-1500 cm-1 according to the semiempirical (AM1) and ab initio methods (basis sets 3-21G, 6-31G), respectively), whereas the barrier restricting the mobility of the dimethylamino group is considerably higher (2800 cm-1). The presence of the methyl group bound to the nitrogen N5 of the benzthiazole ring prevents the formation of the strictly planar conformation of ThT molecule and determines the existence of the energy barrier at φ ) 0 (180)°. The barriers at φ ) 0 (180)° and φ ) 90 (270)° have different nature. The overcoming of the barrier at φ ) 0 (180)° does not affect the 1394

Journal of Proteome Research • Vol. 6, No. 4, 2007

Maskevich et al.

electron structure of ThT (i.e., forms of molecular orbitals), whereas the barrier at φ ) 90 (270)° is associated with the breakdown of the conjugation between π-electron systems of the benzthiazole and dimethylaminobenzene rings. As a result of this distortion, the π-conjugated systems of the benzthiazole and dimethylaminobenzene rings behave as independent chromophores. The existence of the mentioned above methyl group bound to the benzthiazole ring not only prevents the formation of the planar configuration of the ThT molecule, but also makes tense conformations at φ ) 37, 145, 217, and 325° energetically favorable and decreases the energy barrier at φ ) 90 (270)° between the ThT conformers corresponding to the energy minima. Quantum-chemical calculations of the ThT analogue in which the methyl group at the benzthiazole ring N5 atom is substituted to hydrogen revealed that this molecule possess energy minima at φ ) 0 and 180°, and the energy barriers at φ ) 90 and 270° are as high as 4000 cm-1 (see Figure 2B). Ab initio calculations 3-21G/RHF revealed that for ThT in the ground state, the positive charge is mostly located at the benzthiazole ring (fragment I), being equal to +0.6e at φ ) 0°, +0.7e at φ ) 37° and +0.8e at φ ) 90°. At the same time, fragments II and III possess the summed charges of +0.4e, +0.3e, and +0.2e, respectively. The transition to the excited Franck-Condon state S1* is accompanied by a dramatic redistribution of the electron density resulting from the transfer of the negative charge to fragment I (see Figure 2C, D). In fact, in the excited state, the charge located at the benzthiazole ring is +0.5e, +0.3e, and -0.1e at φ )0, 37, and 90°, respectively, whereas at the similar angles, the charge at the aminobenzene ring and dimethylamino group (i.e., at the fragments II and III) equals to +0.5e, +0.7e, and +1.1e. Therefore, ThT molecule is a cation (Z ) +1e) with the nonuniform distribution of charges between the fragments I and (II + III). The excitation of the ThT molecule or the change of the angle φ between the fragments I and II is accompanied by the significant redistribution of the charge between the fragments. ThT Absorption Spectra. Both, the molar extinction coefficient and the position of the long-wavelength band in the ThT absorption spectra depend on the solvent polarity (see Table 1). Figure 3A represents ThT absorption spectra measured in distilled water, glycerol, and different water-glycerol mixtures. It can be seen that the ThT molar extinction coefficient in aqueous solutions is almost 2-fold smaller than that in glycerol. The increase in the glycerol concentration is accompanied by the shift of the long-wavelength absorption band from 412 nm in water to 421 nm in 99% glycerol. Importantly, the ThT absorption spectrum in water was characterized by the most short-wavelength shifted position. The results of the analysis of the dependence of the ThT spectral properties (νabs, cm-1) on the solvent polarity (characterized by the solvent orientational polarizability, ∆f ) ( 1)/(2 + 1) - (n2 - 1)/(2n2 + 1)75) are presented in Figure 3B, which shows that the increase in the solvent polarity is accompanied by hypsochromic shift of the absorption spectrum. The dependence of the maximum of absorption spectrum on ∆f is almost linear. Analysis of the ThT interaction with amyloid fibrils was complicated by the significant light scattering. However, even under such unfavorable conditions, a small long-wavelength shift of absorption spectrum was clearly detected. The difference absorption spectrum (which is a result of the subtraction of the spectrum of ThT measured in aqueous solution from the dye spectrum measured in the presence of amyloid fibrils) revealed that the fibril-bound ThT

research articles

Spectral Properties of Thioflavin T

Figure 2. Peculiarities of the ThT structure elucidated via the quantum-chemical calculations. (A) Dependence of the ThT potential energy on the φ and ψ angles. Calculations were done using AM1 method. (B) Effect of the methyl group substitution at the N5 atom of the benzthiazole on the dependence of the ThT potential energy on the torsion angle φ between the planes of the benzthiazole and aminobenzene rings. Analysis was performed using the AM1 method for ThT (curve 1) and ThT analogue in which the methyl group at N5 was substituted to hydrogen (curve 2). (C) Highest occupied molecular orbital of the ThT conformers with the torsion angle φ ) 37°. (D) Lowest unoccupied molecular orbital of the ThT conformers with the torsion angle φ ) 37°. Table 1. Effect of Different Solvents on Spectral Properties of ThTa no.

solvent

∆fb

νjabsc, cm-1 (λabs, nm)

νjfld, cm-1 (λfl, nm)

νj0-0e, cm-1

∆νjf, cm-1

qg

1 2 3 4 5 6 7 8 9 10 11 12 13 14

water methanol acetonitrile ethanol acetone 2-propanol ethyleneglycol N,N-dimethyl-formamide dimethyl-sulfoxide 1-butanol glycerol 1-octanol piridine chlorophorm

0.320 0.309 0.306 0.290 0.284 0.277 0.276 0.275 0.265 0.263 0.263 0.226 0.214 0.149

24270 (412) 24040 (416) 24040 (416) 23920 (418) 24100 (415) 23920 (418) 23810 (420) 23980 (417) 23920 (418) 23920 (418) 23750 (421) 23870 (419) 23470 (426) 23580 (424)

20280 (493) 20490 (488) 20280 (493) 20410 (490) 20450 (489) 20490 (488) 20160 (496) 20160 (496) 19880 (503) 20450 (489) 20280 (493) 20490 (488) 20160 (496) 20410 (490)

22275 22265 22160 22165 22275 22205 21985 22070 21900 22185 22015 22180 21815 21995

3990 3550 3760 3510 3650 3430 3650 3820 4040 3470 3470 3380 3310 3170

0.0003 0.0004 0.0002 0.0012 0.0003 0.0025 0.0006 0.0009 0.0043 0.0992 0.0139 0.0113

a Corresponding wavelength values are given in brackets. All measurements were performed at 298 K. b ∆f ) (-1)/(2+1)-(n2-1)/(2n2+1) is the solvent orientational polarazability. c νjabs is a wavenumber of the maximum of the long-wavelength absorption band. d νjfl is a wavenumber of the fluorescence maximum. e νj0-0 ) (νjabs + νjfl)/2 is a wavenumber of the 0-0 transition. f ∆νj is a shift of the fluorescence maximum relative to the maximum of the long-wavelength absorption band. g q is a quantum yield.

molecule are characterized by the maximal absorption at 450 nm (Figure 3C,D). This strong long-wavelength shift of the absorption spectrum accompanying the ThT incorporation into the amyloid fibrils fits to the established νabs vs ∆f dependence. The absorption is associated with the transition of the ThT molecule from the equilibrium ground state to the nonequilibrium Franck-Condon excited state. Described above, quantum-chemistry calculations revealed that the ground state of ThT at φ ) 37° is characterized by benzthiazole ring charge of +0.7e. Furthermore, the degree of the ground state stabilization by orientation of the solvent molecules around ThT increases with the solvent polarity. The transition to the excited-state is accompanied by the decrease of the benzthiazole ring’s charge from +0.7e to +0.3e, whereas the charge associated with the

benzene ring and the dimethylamino group increases from +0.3e to +0.7e. Therefore, the Franck-Condon excited state in the polar solvent should have essentially higher energy in comparison with the equilibrium excited state (Figure 4). The unequal distribution of the charges between the ThT fragments and the noticeable charge re-distribution associated with the transition to the excited-state might explain why the increase in solvent polarity was accompanied by the short-wavelength shift of the ThT absorption spectrum. ThT Fluorescence Spectra. Contrarily to the absorption spectra, the position of the fluorescence of ThT excited at the long-wavelength absorption band (λex ) 440 nm) possesses negligible dependence on the solvent polarity. In fact, ThT fluorescence measured in water and in ethanol or 99% glycerol Journal of Proteome Research • Vol. 6, No. 4, 2007 1395

research articles

Maskevich et al.

Figure 4. Model describing the localization of the ground and excited energy levels for the ThT molecule in the polar solvent. E and N are energy levels corresponding to the equilibrium and nonequilibrium states. Vertical arrows correspond to the transitions that are accompanied by the light absorption. The density of the force lines are determined by the charge size of the ThT molecule fragment. The size of the symbol “plus” reflects the charge value. S0 and S1 are the levels of the ground and excited states of the isolated ThT molecule. W0 and W1 are the stabilization energies of the ThT molecule in the ground and excited equilibrium states due to the orientational interaction of the positively charged ThT fragments with the dipoles of the polar solvent.

Figure 3. Effect of environment on ThT absorption spectra. (A) ThT absorption spectra in water-glycerol mixtures. Curves 1, 2, 3, 4, and 5 correspond to the glycerol content of 0, 50, 60, 88, and 97%, respectively. (B) Dependence of the absorption maximum position on the solvent orientational polarizability ∆f ) ( - 1)/(2 + 1) - (n2 - 1)/(2n2 + 1), where  is the dielectric constant and n is the refraction index. Numbers in circles correspond to the different solvents (see Table 1). (C) Absorption spectra of ThT solution in water (curve 1) and in water in the presence of amyloid fibrils (curve 2). Curve 3 represents the apparent optical density determined by the light scattering (Dscat ) aλ-n), whereas curve 4 corresponds to the ThT absorption spectrum in the presence of the amyloid fibrils corrected for the light scattering. (Inset) Curves used to determine Dscat. Curve 1 represents the lg(D) vs lg(λ) dependence, whereas curve 2 is a straight line obtained by the extrapolation of the linear region of the lg(D) ) f(lg(λ)) curve, which corresponds to the spectral region where the active absorption is absent; lg(Dscat) ) a - n lg(λ), a ) 4.57, n ) 2. (D) Differential spectrum between the ThT absorption in water (curve 1 in C) and in water in the presence of fibrils (curve 4 in C). The measurements were done for 20 µM ThT in the absence or presence of 0.01 mg/mL amyloid fibrils.

is characterized by λfl,max of 493 and 490 nm, respectively (see Figure 5A-C). Figure 5D shows that the addition of the amyloid fibrils produces a dramatic increase (several orders of magnitude) in the ThT fluorescence intensity in the concentrationdependent manner. The fluorescence intensity measured for 1396

Journal of Proteome Research • Vol. 6, No. 4, 2007

the 0.01 µM ThT solution in the presence of insulin fibrils (0.02 mg/mL) exceeds that measured in the aqueous solution in ∼1000 times. Furthermore, the incorporation of ThT into the amyloid fibrils produces small short-wavelength shift of the fluorescence maximum (λfl,max ) 478 nm). Figure 5D shows that among all conditions studied, the comparable blue-shifted position of the ThT fluorescence is observed only in the presence of 1-butanol. This is due to the fact that the fibrilincorporated ThT molecules are located in the rigid nonpolar environment whereas the emission occurs from the FranckCondon state that is energetically close to the equilibrium one. Overall, the position of the ThT fluorescence maximum is less sensitive to the solvent polarity than the position of the corresponding absorption spectrum. Relaxation processes taking place in the excited-state should be considered to explain this phenomenon. ThT fluorescent properties are determined by the relation among the radiation lifetime of the excited state (τr), the rotational isomerization time, i.e., the characteristic time required to establish the equilibrium distribution of the ThT molecules by the torsion angle φ in the excited S1 state (τor), and the time required to establish the equilibrium between the solvent molecules and excited ThT molecule (τsolv). The situation when ThT is located in viscous or rigid media will be considered separately. In solvents with low viscosity τor, τr, the excited ThT molecule have enough time to adopt configurations with φ close to 90 or 270°, which were shown to be characterized by the specific distortion of conjugated π-electron systems of the benzthiazole and dimethylaminobenzene rings

research articles

Spectral Properties of Thioflavin T

and possess high probability of the radiationless transition of the ThT molecule to the ground state.76 The rate constant for the transition between the two conformational states is determined by the value of the energy barrier separating these conformations: k ) (kBT/h) exp(-∆E/RT)

(1)

where T is the Kelvin’s temperature, whereas kB ) 1.3807 × 10-23(J/deg K), h ) 6.6261 × 10-34 (J‚sec), and R ) 8.32 (J‚mol-1‚deg‚K-1) are the Boltsman, Plank, and the universal gas constants, respectively. Factor (kBT/h) ) 6.25 × 1012 sec-1 determines the per-second number of attempts undertaken by a molecule to overcome the energy barrier. In the absence of such a barrier (∆E ≈ 0), this parameter determines the rate of achieving the lower energy state. The magnitude of the energy difference between the two states does not affect the transition rate but determines the relative populations of these states at the equilibrium. It is likely that in the ThT excited state, the equilibrium distribution by the torsion angle φ will not be reached, as transition to the states with φ close to 90 or 270° will be accompanied by the radiationless transition to the ground state. Obviously, the equilibrium distribution of the ThT molecules by the torsion angle φ can be reached only in cases when τor , τr. It is necessary to emphasize that the estimated above rate of the reaching of a state with φ close to 90 and 270° was obtained for the isolated ThT molecule in vacuo. Even for the low viscosity solvents (such as water and alcohols), the time required to overcome the energy barrier between the two states might be significantly longer. This is because of the fact that in this case the rate constant of this process is determined by the solvent viscosity and hydrodynamic properties of the ThT fragments rather than by the energy barrier of the internal rotation. The rotational relaxation of the ThT fragments relative to each other requires the simultaneous movement of the solvent molecules preventing the fragment rotation. The rotational relaxation time is given by the well-known DebyeStokes-Einstein equation: F)

Figure 5. Peculiarities of ThT fluorescence at various environmental conditions. Fluorescence excitation spectra (curve 1, λfl ) 440 nm; curve 2, λfl ) 480 nm) and fluorescence emission spectra (curve 3, λex ) 340 nm; curve 4, λex ) 440 nm) of ThT in water (A), ethanol (B), and 99% glycerol (C). The measurements were done for 5 µM ThT. (D) Fluorescence excitation (λfl ) 480 nm) and emission spectra (λex ) 440 nm) of ThT incorporated in amyloid fibrils. Spectra were measured in the presence of different concentrations of insulin fibrils ranging from 0.02 to 0.14 mg/mL with the step of 0.02 mg/mL (Curves 1-7). (Inset) Dependence of the fluorescence intensity (λex ) 440 nm, λfl ) 480 nm) on amyloid fibril concentration.

4πa3 η 3kB T

(2)

where a is the hydrodynamic radius of the molecule (in our case, a corresponds to the effective hydrodynamic radius of the ThT fragment). Although the rotational relaxation time of the water diffusion at 293 K estimated by this equation is F ) 0.013 nsec (a ) 2.35 Å, η ) 1.0 ‚10-3 Pa‚sec), is significantly higher than the experimentally determined value of F ) 2-2.5 ps,77,78 eq 2 can still be used for estimation of the rotational correlation time. The molecular mass of the ThT molecule (319 Da) is ∼18-times greater than that of water. Therefore, the time of the rotational relaxation of the ThT molecule and its fragments in water will be in a range of 0.2 and 0.1 nsec respectively. Thus, in the polar solvent with low viscosity, there is a fast process that brings the excited ThT molecules to the configuration with φ close to 90 and 270°, from which the ground state is achieved in the radiationless manner. However, the reorganization of the ThT solvation shell leading to the equilibrium between the excited ThT molecule and the solvent occurs even faster. Let us consider two extreme cases. Figure 6A illustrates the first case when fluorescence is due to the transition from the nonequilibrium Franck-Condon state to the equilibrium ground Journal of Proteome Research • Vol. 6, No. 4, 2007 1397

research articles

Maskevich et al.

Figure 6. Model illustrating the reasons for the weak dependence of the fluorescence spectrum position on the solvent polarity. (A) Emission occurs from the nonequilibrium Franck-Condon state. The position of the fluorescence spectrum is mostly determined by the stabilization energy of the ground state due to the interaction of the charged ThT molecule with the polar solvent molecules (W0). (B) Emission occurs from the equilibrium excited state. The position of the fluorescence spectrum is mostly determined by the energy of interaction of the ThT molecules in excited-state with the polar solvent molecules (W1). (C) Emission occurs from the partially equilibrium excited state. Rotational oscillations of the ThT molecules relative to each other prevent establishment of the equilibrium with the solvent. Fluorescence is due to the transitions from the partially equilibrium excited-state to the partially equilibrium ground state. Changes in the solvent polarity (which change the energy of interaction between the positively charged ThT molecule with the polar solvent molecules) do not affect to a significant degree the position of the fluorescence maximum (C(I) and C(II)).

state. Under these circumstances, the positions of the fluorescence and absorption spectra should depend on the solvent polarity similarly, i.e., both spectra should shift to the shorter wavelengths with the increase in solvent polarity. Another case is when the fluorescence is determined by the transition from the equilibrium excited-state to the nonequilibrium ground state (see Figure 6B). In this case, the position of fluorescence maximum will experience a longer-wavelength shift in response to the increase in solvent polarity. However, in the excited state, besides the solvent adjustment aiming the establishment of the equilibrium between the ThT and solvent molecules, the rotational oscillations of the ThT fragments relative to each other take place. These rotational oscillations hinder the approaching of the equilibrium with solvent. As a result of the superposition of these two processes, the transition will occur from the partially nonequilibrium excited state to the partially nonequilibrium ground state, which can give rise to observed weak dependence of the ThT fluorescence spectrum position on the solvent polarity (see Figure 6C). ThT Fluorescence Excitation Spectra. Figure 5D shows that the fibril-incorporated ThT is characterized by the fluorescence excitation spectrum with λex,max ) 450 nm. This observation supports that the long-wavelength band of the absorption spectrum of the fibril-incorporated ThT has a maximum at 450 nm. Figure 5A-C shows that in aqueous and alcohol solutions, the ThT fluorescence excitation spectra monitored at 480 nm are rather strange. They possess maxima in the vicinity of 336 and 358 in water and ethanol, respectively (i.e., in the regions where corresponding absorption spectra have minima), and shoulders in the vicinity of the corresponding long-wavelength absorption bands (cf. Figure 5C,D). Furthermore, in both water and ethanol, ThT is characterized by a very low quantum yield (0.1%) upon excitation at the long-wavelength absorption band. This could be due to the distortion of the system of the π-conjugated bonds caused by the rotation of the benzthiazole and aminobenzene rings in the excited state.76 In the highly viscous solvents, the mentioned ring reorientation is slow and does not occur during the excited-state lifetime. As a result, the ThT fluorescence intensity under such conditions is higher in comparison with that measured in water and alcohols.76 Figure 5A-C shows that at T ) 298 K in 99% glycerol, ThT fluorescence is characterized by the quantum yield of 14%. Importantly, the fluorescence excitation spectrum has a maxi1398

Journal of Proteome Research • Vol. 6, No. 4, 2007

mum at 421 nm and possesses only a small shoulder in the vicinity of 310-320 nm. The shape and position of the ThT fluorescence excitation spectrum in glycerol are close to those of the long-wavelength absorption band. Excitation at the maximum of the ThT fluorescence excitation spectra in water and ethanol, as well as in the vicinity of the 310-320 nm shoulder in glycerol produces fluorescence with λfl,max of 435, 418, and 446 nm for ThT in water, ethanol, and glycerol. These results are in a good agreement with the literature data,53,54 according to which the aqueous ThT solution possesses fluorescence with the λfl,max of 438 nm (i.e., in the region of the long-wavelength absorption band) and the fluorescence excitation spectrum with maximum at 350 nm (i.e., in the region corresponding to the minimum of ThT absorption). Comparable spectral parameters are described in subsequent publications.55-57,60 It is also stated that the fluorescence excitation and emission spectra are long-wavelength shifted as a result of the ThT incorporation into the amyloid fibrils.55-57,60 In our previous work, we suggested that ThT was responsible only for the long-wavelength fluorescence emission and excitation bands, whereas the short-wavelength spectral components in water and alcohols were explained by the presence of some admixtures in the ThT samples, which possessed a maximal absorption at 320-360 nm and were characterized by a high quantum yield in the vicinity of 420450 nm.76 Presented in the current paper, results of the quantum-chemistry calculations provide an alternative explanation for the observed phenomenon. We have already emphasized that the ThT conformers corresponding to the energy mimima at φ ) 37 and 145°, and φ )217 and 325° are separated by relatively low barriers (∼1000 cm-1, see Figure 2B) located at φ ) 90 and 270°, respectively. Under these circumstances, the unified π-electron system is separated into the individual π-conjugated systems of the benzthiazole and aminobenzene rings, which now may serve as independent chromophores. The increase in the size of the system of π-conjugated bonds is known to be accompanied by the spectral shift toward the longer wavelengths. Therefore, any isolated ThT fragment (e.g., benzthiazole ring) should have shorter wavelength positions of the absorption and fluorescence spectra in comparison with those for the whole ThT molecule. As the internal rotation barrier is relatively low, some ThT molecules will be in a conformation characterized by the

research articles

Spectral Properties of Thioflavin T

independent chromophores of benzthiazole and aminobenzene rings. Computational analysis revealed that at room temperature the fraction of ThT molecules with φ ) 90 ( 30° and 270 ( 30° was ∼10% for the 700 cm-1 barriers, and ∼2% for the 1500 cm-1 barrier, whereas the fractions of ThT molecules with torsion angles φ ) 90 ( 20° and 270 ( 20° were 4 and 0.3% for the 700 and 1500 cm-1 barriers, respectively. The major reason for the low barrier separating conformers with minimum energy is the presence of the methyl group bound to the N5 atom of the benzthiazole ring. We have established that substitution of this methyl group by the hydrogen atom is accompanied by a dramatic increase in the energy barrier values at φ ) 90 and 270° (from ∼700 to 4000 cm-1) (Figure 2B). In this case, the fraction of the ThT molecules with torsion angles φ ) 90 ( 30° and 270 ( 30° accounts only for 0.001%. Therefore, the presence of the significant fraction of the ThT molecules with the violated system of the π-conjugated bonds in the ground state allows us to suggest that the shortwavelength fluorescence excited at 340 nm might be due to the emission of one of these isolated ThT chromophores, likely due to the fluorescence of benzthiazole ring. The mentioned conjugation breakdown could also explain the complex nature of the fluorescence excitation spectrum registered at 480 nm. Overall, the short-wavelength bands in the fluorescence excitation and emission spectra of ThT in aqueous and alcohol solutions might be either due to the highly fluorescent admixtures (including physically separated fragments of ThT molecules, such as benzthiazole or aminobenzene) or due to the existence of specific ThT conformations in which the torsion angle φ between the benzthiazole and aminobenzene rings is close to 90 or 270°. In such conformations, benzthiazole and aminobenzene rings will behave as independent chromophores characterized by the fluorescence emission and excitation spectra shifted to the shorter wavelength in comparison with the ThT molecule possessing integrated system of the π-conjugated bonds. We think that one of these factors, rather than unusual spectral properties of the ThT cation (as it was suggested before53-57,60), determines the existence of the shortwavelength excitation and emission bands in the ThT spectra measured in aqueous and alcohol solutions. It has been already emphasized76 that some erroneous conceptions related to the ThT spectral properties were due to the poor choice of the experimental conditions (e.g., high optical density of the studied solutions). For example, it has been shown that fluorescence excitation spectrum of ThT in glycerol represents a very broad band with two peaks.60 As these measurements were performed for the solution with high dye concentration (30 µM), we believe that this behavior is determined by the dependence of the fluorescence intensity on the optical density of the solution. In fact, the fluorescence intensity (Ifl) is known to be proportional to the exciting light intensity (I0), the fraction of the light absorbed by the sample (1 - T) and the fluorescence quantum yield (q): Ifl ) kI0(1 - T)q

(3)

where T ≡ I/I0 ) 10-D is the fraction of light passing through the sample (transmission), k is the coefficient, D is the optical density of the solution (D ) Cl), l is the optical path-length,  is the molecular extinction coefficient, and C is the chromophore concentration. Obviously, Ifl ) klI0Cq when D f 0 and Ifl ) kI0q when D f ∞. Therefore, the fluorescence intensity is proportional to the chromophore concentration only when

optical density of the studied solution is low. However, when optical density is high (in the presence of high fluorophore concentration) the fluorescence intensity is independent from the fluorophore concentration. In practice, the fluorescence intensity of the solutions with high optical density is not constant, but decreases after the optical density reaches particular level. This is due to the inner filter effect, where the exciting light does not reach the center of the cell, being absorbed by the layers adjacent to the cell surface. The consideration of the inner filter effect is especially crucial in the fluorescence excitation measurements. In fact, the optical density changes within the fluorescence excitation spectrum, approaching maximal values at the maximum of the absorption spectrum. This results in the broadening of the fluorescence excitation spectra and potentially leads to the appearance of the minima in the fluorescence excitation spectra. We believe that the described in literature minimum located at 430 nm in the ThT fluorescence excitation spectrum in glycerol60 can be explained by this phenomenon.

Conclusions Analysis of the ThT spectral properties in solvents with different dielectric properties and in fibril-incorporated form revealed that the absorption spectrum but not the fluorescence spectrum depends significantly on the solvent polarity. The most short- (412 nm) and long-wavelength shifted (450 nm) absorption spectra were observed for free ThT in water and for a dye bound to the amyloid fibril in water, respectively. Significant short-wavelength shift of the absorption spectrum position accompanying the increase in the solvent polarity is due to fact that the ground state is stabilized by the orientational interaction of the solvent molecules with the positively charged ThT fragments, whereas the excited Franck-Condon state is preferentially nonequilibrium. This nonequilibrium is determined by the charge re-distribution: quantum-chemical calculations revealed that the charge of the benzthiazole fragment decreases from +0.7e to +0.3e as a result of the transition form the ground to the excited state. Fluorescence maximum position is not so sensitive to the solvent polarity. This may be due to the fact that fluorescence is determined by the transitions from the partially equilibrium excited-state to the partially equilibrium ground state. Being excited at the long-wavelength absorption band (440 nm), ThT in water emits at λfl,max of 493 nm, whereas fibril-incorporated dye has λfl,max of 478 nm. Fluorescence excitation spectrum of the fibril-incorporated ThT possesses a maximum at 450 nm, which corresponds to the maximum of the long-wavelength absorption band. Fluorescence excitation spectra of ThT in water, alcohols, and other solvents possess rather strange shapes. For example, in water and ethanol, respectively, they have maxima at 336 and 358 nm (i.e., in the absorption minima) and show only a shoulder at the long-wavelength absorption band. Furthermore, the excitation at the λex,max in water and ethanol or in the vicinity of the 310-320 nm shoulder in glycerol leads to the fluorescence with λfl,max at 435, 418, and 446 nm (i.e., around the longwavelength absorption band) in water, ethanol, and glycerol, respectively. Appearance of the short-wavelength bands in the fluorescence excitation and emission spectra of ThT in water and some alcohols53-57,60 has been attributed to the highly fluorescent admixtures in the ThT samples.76 On the basis of the quantum-chemistry calculations for the ThT molecule in the ground state, we are providing here an Journal of Proteome Research • Vol. 6, No. 4, 2007 1399

research articles alternative explanation for this phenomenon. Computational analysis revealed that the dependence of the ThT energy on the torsion angle φ between benzthiazole or aminobenzene rings has four minima separated by relatively low-energy barriers, 700 cm-1. This might mean that at the room temperature some fraction of the ThT molecules in the ground state might have configuration with φ values close to 90 and 270°. These ThT molecules are characterized by the violated system of the π-conjugated bonds, i.e., their benzthiazole or aminobenzene rings behave as independent chromophors, which are responsible for the appearance of the short-wavelength excitation and emission bands. It is also possible that these benzthiazole or aminobenzene rings also exist in ThT solvents as independent entities (admixtures) due to the disruption of the chemical bond between the rings.

Acknowledgment. This work was supported in part by Belorussian Foundation of Basic Research (Grants F06-351 and X06P-115), Russian Foundation of Basic Research (Grants 0604-81033 and 07-04-01454), Federal Agency of Science and Innovation (Contract #02.445.11.7338), Program “Leading Scientific Schools of Russia” (Grant #9396.2006.4), Program “Molecular and Cell Biology” RAS, using equipment of the Joint Research Center “Material science and characterization in high technology” References (1) Kelly, J. W. The alternative conformations of amyloidogenic proteins and their multi-step assembly pathways. Curr. Opin. Struct. Biol. 1998, 8, 101-106. (2) Kelly, J. W. Alternative conformations of amyloidogenic proteins govern their behavior. Curr. Opin. Struct. Biol. 1996, 6, 11-17. (3) Dobson, C. M. Protein misfolding, evolution and disease. Trends Biochem. Sci. 1999, 24, 329-332. (4) Bellotti, V.; Mangione, P.; Stoppini, M. Biological activity and pathological implications of misfolded proteins. Cell. Mol. Life Sci. 1999, 55, 977-991. (5) Uversky, V. N.; Talapatra, A.; Gillespie, J. R.; Fink, A. L. Protein deposits as the molecular basis of amyloidosis. Part I. Systemic amyloidoses. Med. Sci. Monitor 1999, 5, 1001-1012. (6) Uversky, V. N.; Talapatra, A.; Gillespie, J. R.; Fink, A. L. Protein deposits as the molecular basis of amyloidosis. II. Localized amyloidosis and neurodegenerative disorders. Med. Sci. Monitor 1999, 5, 1238-1254. (7) Rochet, J. C.; Lansbury, P. T., Jr. Amyloid fibrillogenesis: themes and variations. Curr. Opin. Struct. Biol. 2000, 10, 60-68. (8) Uversky, V. N. Protein folding revisited. A polypeptide chain at the folding-misfolding-nonfolding cross-roads: which way to go? Cell Mol. Life Sci. 2003, 60, 1852-1871. (9) Zerovnik, E. Amyloid-fibril formation. Proposed mechanisms and relevance to conformational disease. Eur. J. Biochem. 2002, 269, 3362-3371. (10) Uversky, V. N.; Fink, A. L. Conformational constraints for amyloid fibrillation: the importance of being unfolded. Biochim. Biophys. Acta. 2004, 1698, 131-153. (11) Carrell, R. W.; Gooptu, B. Conformational changes and diseaseserpins, prions and Alzheimer’s. Curr. Opin. Struct. Biol. 1998, 8, 799-809. (12) Hashimoto, M.; Masliah, E. Alpha-synuclein in Lewy body disease and Alzheimer’s disease. Brain Pathol. 1999, 9, 707-720. (13) Koo, E. H.; Lansbury, P. T., Jr.; Kelly, J. W. Amyloid diseases: abnormal protein aggregation in neurodegeneration. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 9989-9990. (14) Chiti, F.; Dobson, C. M. Protein misfolding, functional amyloid, and human disease. Annu. Rev. Biochem. 2006, 75, 333-366. (15) Gazit, E. Mechanisms of amyloid fibril self-assembly and inhibition. Model short peptides as a key research tool. Febs. J. 2005, 272, 5971-5978. (16) Sunde, M.; Blake, C. C. From the globular to the fibrous state: protein structure and structural conversion in amyloid formation. Q. Rev. Biophys. 1998, 31, 1-39. (17) Sunde, M.; Serpell, L. C.; Bartlam, M.; Fraser, P. E.; Pepys, M. B.; Blake, C. C. Common core structure of amyloid fibrils by synchrotron X-ray diffraction. J. Mol. Biol. 1997, 273, 729-739.

1400

Journal of Proteome Research • Vol. 6, No. 4, 2007

Maskevich et al. (18) Goldsbury, C. S.; Cooper, G. J.; Goldie, K. N.; Muller, S. A.; Saafi, E. L.; Gruijters, W. T.; Misur, M. P.; Engel, A.; Aebi, U.; Kistler, J. Polymorphic fibrillar assembly of human amylin. J. Struct. Biol. 1997, 119, 17-27. (19) Harper, J. D.; Lieber, C. M.; Lansbury, P. T., Jr. Atomic force microscopic imaging of seeded fibril formation and fibril branching by the Alzheimer’s disease amyloid-beta protein. Chem. Biol. 1997, 4, 951-959. (20) Conway, K. A.; Harper, J. D.; Lansbury, P. T. Accelerated in vitro fibril formation by a mutant alpha-synuclein linked to early-onset Parkinson disease. Nat. Med. 1998, 4, 1318-1320. (21) Serpell, L. C.; Sunde, M.; Benson, M. D.; Tennent, G. A.; Pepys, M. B.; Fraser, P. E. The protofilament substructure of amyloid fibrils. J. Mol. Biol. 2000, 300, 1033-1039. (22) Uversky, V. N.; Li, J.; Fink, A. L. Evidence for a partially folded intermediate in alpha-synuclein fibril formation. J. Biol. Chem. 2001, 276, 10737-10744. (23) Nielsen, L.; Frokjaer, S.; Brange, J.; Uversky, V. N.; Fink, A. L. Probing the mechanism of insulin fibril formation with insulin mutants. Biochemistry 2001, 40, 8397-8409. (24) Souillac, P. O.; Uversky, V. N.; Millett, I. S.; Khurana, R.; Doniach, S.; Fink, A. L. Elucidation of the molecular mechanism during the early events in immunoglobulin light chain amyloid fibrillation. Evidence for an off-pathway oligomer at acidic pH. J. Biol. Chem. 2002, 277, 12666-12679. (25) Souillac, P. O.; Uversky, V. N.; Millett, I. S.; Khurana, R.; Doniach, S.; Fink, A. L. Effect of association state and conformational stability on the kinetics of immunoglobulin light chain amyloid fibril formation at physiological pH. J. Biol. Chem. 2002, 277, 12657-12665. (26) Guijarro, J. I.; Sunde, M.; Jones, J. A.; Campbell, I. D.; Dobson, C. M. Amyloid fibril formation by an SH3 domain. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 4224-4228. (27) Litvinovich, S. V.; Brew, S. A.; Aota, S.; Akiyama, S. K.; Haudenschild, C.; Ingham, K. C. Formation of amyloid-like fibrils by selfassociation of a partially unfolded fibronectin type III module. J. Mol. Biol. 1998, 280, 245-258. (28) Konno, T.; Murata, K.; Nagayama, K. Amyloid-like aggregates of a plant protein: a case of a sweet-tasting protein, monellin. FEBS Lett 1999, 454, 122-126. (29) Schuler, B.; Rachel, R.; Seckler, R. Formation of fibrous aggregates from a non-native intermediate: the isolated P22 tailspike betahelix domain. J. Biol. Chem. 1999, 274, 18589-18596. (30) Gross, M.; Wilkins, D. K.; Pitkeathly, M. C.; Chung, E. W.; Higham, C.; Clark, A.; Dobson, C. M. Formation of amyloid fibrils by peptides derived from the bacterial cold shock protein CspB. Protein Sci. 1999, 8, 1350-1357. (31) Chiti, F.; Webster, P.; Taddei, N.; Clark, A.; Stefani, M.; Ramponi, G.; Dobson, C. M. Designing conditions for in vitro formation of amyloid protofilaments and fibrils. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 3590-3594. (32) Krebs, M. R.; Wilkins, D. K.; Chung, E. W.; Pitkeathly, M. C.; Chamberlain, A. K.; Zurdo, J.; Robinson, C. V.; Dobson, C. M. Formation and seeding of amyloid fibrils from wild-type hen lysozyme and a peptide fragment from the beta-domain. J. Mol. Biol. 2000, 300, 541-549. (33) Damaschun, G.; Damaschun, H.; Fabian, H.; Gast, K.; Krober, R.; Wieske, M.; Zirwer, D. Conversion of yeast phosphoglycerate kinase into amyloid-like structure. Proteins 2000, 39, 204-211. (34) Yutani, K.; Takayama, G.; Goda, S.; Yamagata, Y.; Maki, S.; Namba, K.; Tsunasawa, S.; Ogasahara, K. The process of amyloid-like fibril formation by methionine aminopeptidase from a hyperthermophile, Pyrococcus furiosus. Biochemistry 2000, 39, 2769-2777. (35) Ohnishi, S.; Koide, A.; Koide, S. Solution conformation and amyloid-like fibril formation of a polar peptide derived from a beta-hairpin in the OspA single-layer beta-sheet. J. Mol. Biol. 2000, 301, 477-489. (36) Fezoui, Y.; Hartley, D. M.; Walsh, D. M.; Selkoe, D. J.; Osterhout, J. J.; Teplow, D. B. A de novo designed helix-turn-helix peptide forms nontoxic amyloid fibrils. Nat. Struct. Biol. 2000, 7, 10951099. (37) Fandrich, M.; Fletcher, M. A.; Dobson, C. M. Amyloid fibrils from muscle myoglobin. Nature 2001, 410, 165-166. (38) Pertinhez, T. A.; Bouchard, M.; Tomlinson, E. J.; Wain, R.; Ferguson, S. J.; Dobson, C. M.; Smith, L. J. Amyloid fibril formation by a helical cytochrome. FEBS Lett 2001, 495, 184186. (39) Goers, J.; Permyakov, S. E.; Permyakov, E. A.; Uversky, V. N.; Fink, A. L. Conformational prerequisites for alpha-lactalbumin fibrillation. Biochemistry 2002, 41, 12546-12551.

research articles

Spectral Properties of Thioflavin T (40) Pavlov, N. A.; Cherny, D. I.; Heim, G.; Jovin, T. M.; Subramaniam, V. Amyloid fibrils from the mammalian protein prothymosin alpha. FEBS Lett 2002, 517, 37-40. (41) Munishkina, L. A.; Fink, A. L.; Uversky, V. N. Conformational prerequisites for formation of amyloid fibrils from histones. J. Mol. Biol. 2004, 342, 1305-1324. (42) Xu, M.; Ermolenkov, V. V.; He, W.; Uversky, V. N.; Fredriksen, L.; Lednev, I. K. Lysozyme fibrillation: deep UV Raman spectroscopic characterization of protein structural transformation. Biopolymers 2005, 79, 58-61. (43) Morozova-Roche, L. A.; Zamotin, V.; Malisauskas, M.; Ohman, A.; Chertkova, R.; Lavrikova, M. A.; Kostanyan, I. A.; Dolgikh, D. A.; Kirpichnikov, M. P. Fibrillation of carrier protein albebetin and its biologically active constructs. Multiple oligomeric intermediates and pathways. Biochemistry 2004, 43, 9610-9619. (44) Sambashivan, S.; Liu, Y.; Sawaya, M. R.; Gingery, M.; Eisenberg, D. Amyloid-like fibrils of ribonuclease A with three-dimensional domain-swapped and native-like structure. Nature 2005, 437, 266-269. (45) Sipe, J. D.; Cohen, A. S. Review: history of the amyloid fibril. J. Struct. Biol. 2000, 130, 88-98. (46) Nelson, R.; Eisenberg, D. Recent atomic models of amyloid fibril structure. Curr. Opin. Struct. Biol. 2006, 16, 260-265. (47) Wickner, R. B. [URE3] as an altered URE2 protein: evidence for a prion analog in Saccharomyces cerevisiae. Science 1994, 264, 566-569. (48) Patino, M. M.; Liu, J. J.; Glover, J. R.; Lindquist, S. Support for the prion hypothesis for inheritance of a phenotypic trait in yeast. Science 1996, 273, 622-626. (49) Serio, T. R.; Cashikar, A. G.; Kowal, A. S.; Sawicki, G. J.; Moslehi, J. J.; Serpell, L.; Arnsdorf, M. F.; Lindquist, S. L. Nucleated conformational conversion and the replication of conformational information by a prion determinant. Science 2000, 289, 13171321. (50) Serio, T. R.; Lindquist, S. L. Protein-only inheritance in yeast: something to get [PSI+]-ched about. Trends Cell Biol. 2000, 10, 98-105. (51) King, C. Y.; Diaz-Avalos, R. Protein-only transmission of three yeast prion strains. Nature 2004, 428, 319-323. (52) Nelson, R.; Sawaya, M. R.; Balbirnie, M.; Madsen, A. O.; Riekel, C.; Grothe, R.; Eisenberg, D. Structure of the cross-beta spine of amyloid-like fibrils. Nature 2005, 435, 773-778. (53) Naiki, H.; Higuchi, K.; Hosokawa, M.; Takeda, T. Fluorometric determination of amyloid fibrils in vitro using the fluorescent dye, thioflavin T1. Anal. Biochem. 1989, 177, 244-249. (54) Naiki, H.; Higuchi, K.; Matsushima, K.; Shimada, A.; Chen, W. H.; Hosokawa, M.; Takeda, T. Fluorometric examination of tissue amyloid fibrils in murine senile amyloidosis: use of the fluorescent indicator, thioflavine T. Lab. Invest. 1990, 62, 768-773. (55) LeVine, H., 3rd. Thioflavine T interaction with synthetic Alzheimer’s disease beta-amyloid peptides: detection of amyloid aggregation in solution. Protein Sci. 1993, 2, 404-410. (56) LeVine, H., 3rd. Stopped-flow kinetics reveal multiple phases of thioflavin T binding to Alzheimer beta (1-40) amyloid fibrils. Arch. Biochem. Biophys. 1997, 342, 306-316. (57) LeVine, H., 3rd. Quantification of beta-sheet amyloid fibril structures with thioflavin T. Methods Enzymol. 1999, 309, 274284. (58) Yoshiike, Y.; Chui, D. H.; Akagi, T.; Tanaka, N.; Takashima, A. Specific compositions of amyloid-beta peptides as the determinant of toxic beta-aggregation. J. Biol. Chem. 2003, 278, 2364823655. (59) Allsop, D.; Swanson, L.; Moore, S.; Davies, Y.; York, A.; El-Agnaf, O. M.; Soutar, I. Fluorescence anisotropy: a method for early detection of Alzheimer beta-peptide (Abeta) aggregation. Biochem. Biophys. Res. Commun. 2001, 285, 58-63. (60) LeVine, H., 3rd. Thioflavine T interaction with amyloid -sheet structures. Amyloid. Int. J. Exp. Clin. Invest. 1995, 2, 1-6.

(61) Hobbs, J. R.; Morgan, A. D. Fluorescence Microscopy with Thioflavine-T in the Diagnosis of Amyloid. J. Pathol. Bacteriol. 1963, 86, 437-442. (62) Kumita, J. R.; Weston, C. J.; Choo-Smith, L. P.; Woolley, G. A.; Smart, O. S. Prevention of peptide fibril formation in an aqueous environment by mutation of a single residue to Aib. Biochemistry 2003, 42, 4492-4498. (63) Zhu, L.; Zhang, X. J.; Wang, L. Y.; Zhou, J. M.; Perrett, S. Relationship between stability of folding intermediates and amyloid formation for the yeast prion Ure2p: a quantitative analysis of the effects of pH and buffer system. J. Mol. Biol. 2003, 328, 235-254. (64) Ban, T.; Hamada, D.; Hasegawa, K.; Naiki, H.; Goto, Y. Direct observation of amyloid fibril growth monitored by thioflavin T fluorescence. J. Biol. Chem. 2003, 278, 16462-16465. (65) Turoverov, K. K.; Biktashev, A. G.; Dorofeiuk, A. V.; Kuznetsova, I. M. A. complex of apparatus and programs for the measurement of spectral, polarization and kinetic characteristics of fluorescence in solution. Tsitologiia 1998, 40, 806-817. (66) Dewar, M. J. S.; Zoebisch, E. G.; Healy, E. F.; Stewart, J. J. P. Development and use of quantum mechanical molecular models. 76. AM1: a new general purpose quantum mechanical molecular model. J. Am. Chem. Soc. 1985, 107, 3902-3909. (67) Stewart, J. J. P. Optimization of parameters for semiempirical methods II. Applications. J. Comput. Chem. 1989, 10, 221-264. (68) Binkley, J. S.; Pople, J. A.; Hehre, W. J. Self-consistent molecular orbital methods. 21. Small split-valence basis sets for first-row elements. J. Am. Chem. Soc. 1980, 102, 939-947. (69) Hehre, W. J.; Ditchfield, R.; Pople, J. A. Self-Consistent Molecular Orbital Methods. XII. Further Extensions of Gaussian-Type Basis Sets for Use in Molecular Orbital Studies of Organic Molecules. J. Chem. Phys. 1972, 56, 2257-2261. (70) Hariharan, P. C.; Pople, J. A. The influence of polarization functions on molecular orbital hydrogenation energies. Theoret. Chim. Acta. 1973, 28, 213-222. (71) Zerner, M. C.; Loew, G. H.; Kirchner, R. F.; Mueller-Westerhoff, U. T. An intermediate neglect of differential overlap technique for spectroscopy of transition-metal complexes. Ferrocene. J. Am. Chem. Soc. 1980, 102, 589-599. (72) Nemukhin, A. V.; Grigorenko, B. L.; Granovsky, A. A. Molecular modeling by using the PC GAMESS program: From diatomic molecules to enzymes. Moscow Univ. Chem. Bull. 2004, 45, 75. (73) Schmidt, M. W.; Baldridge, K. K.; Boatz, J. A.; Elbert, S. T.; Gordon, M. S.; Jensen, J. J.; Koseki, S.; Matsunaga, N.; Nguyen, K. A.; Su, S.; Windus, T. L.; Dupuis, M.; Montgomery, J. A. General atomic and molecular electronic structure system. J. Comput. Chem. 1993, 14, 1347-1363. (74) Portmann, S.; Luthi, H. P. MOLEKEL: An Interactive Molecular Graphics Tool. CHIMIA 2000, 54, 766-770. (75) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 2nd ed.; Kluwer Academic/Plenum Publishers: New York, 1999. (76) Voropay, E. S.; Samtsov, M. P.; Kaplevsky, K. N.; Maskevich, A. A.; Stepuro, V. I.; Povarova, O. I.; Kuznetsova, I. M.; Tutoverov, K. K.; Fink, A. L.; Uversky, V. N. Spectral properties of Thioflavine T and its complexes with amyloid fibrils. J. Appl. Spectrosc. 2003, 70, 868-874. (77) Muller, M. G.; Hardy, E. H.; Vogt, P. S.; Bratschi, C.; Kirchner, B.; Huber, H.; Searles, D. J. Calculation of the deuteron quadrupole relaxation rate in a mixture of water and dimethyl sulfoxide. J. Am. Chem. Soc. 2004, 126, 4704-4710. (78) Omta, A. W.; Kropman, M. F.; Woutersen, S.; Bakker, H. J. Negligible effect of ions on the hydrogen-bond structure in liquid water. Science 2003, 301, 347-349.

PR0605567

Journal of Proteome Research • Vol. 6, No. 4, 2007 1401