Near-Field Scanning Optical Microscopy Measurements of

Aug 7, 2009 - Near-Field Scanning Optical Microscopy Measurements of Fluorescent Molecular Probes. Binding to Insulin Amyloid Fibrils. Catherine C. Ki...
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J. Phys. Chem. B 2009, 113, 12090–12095

Near-Field Scanning Optical Microscopy Measurements of Fluorescent Molecular Probes Binding to Insulin Amyloid Fibrils Catherine C. Kitts† and David A. Vanden Bout* Department of Chemistry and Biochemistry, Center for Nano and Molecular Science and Technology, and Texas Materials Institute, UniVersity of Texas at Austin, 1 UniVersity Station A5300, Austin, Texas 78712 ReceiVed: April 16, 2009; ReVised Manuscript ReceiVed: June 15, 2009

The binding of two amyloid fibril stain dyes, thioflavin T (ThT) and its neutral analog 2-[4′-(dimethylamino)phenyl]-benzothiazole (BTA-2), are measured using near-field scanning optical microscopy (NSOM), which is able to image individual amyloid fibrils. Polarized NSOM images reveal that both dyes bind to the fibrils with the long axis of the molecule aligned parallel to the long axis of the fibrils. This indicates that the dyes bind along the surface of the β-sheet within the grooves of the fibril that run parallel to the fibril axis. The similarity in the binding motifs of the two dyes shows that electrostatic interaction of the charged amine group on the ThT dye plays a minimal role in the affinity of the dyes for the amyloids. The polarized NSOM images confirm that the enhanced fluorescence of the ThT and BTA-2 result from binding of the monomeric dye rather than micelles or excimer species. Introduction Amyloid fibrils have become an area of increasing interest due to their association with over 20 different neurodegenerative diseases (e.g., Alzheimer’s disease and variant CreutzfeldtJakob).1-6 The ability to detect fibrils and small protofibrils is critical to monitoring the progression of these diseases as well as to understanding the mechanism of fibril formation. Although the proteins associated with neurodegenerative diseases form amyloid fibrils, researchers have found that other proteins will also form amyloid fibrils under certain conditions.7,8 Such proteins offer a model system in which to study amyloid formation. The amyloid structure consists of antiparallel β-sheets that are perpendicular to the fibril axis9 and have been extensively studied using a variety of spectroscopy and microscopy techniques.2,9-17 One key method of amyloid structure identification is the use of molecular dyes that have specific interactions with the fibril structure. Two specific dyes that have been used extensively to characterize amyloid fibrils are Congo red (CR) and thioflavin T (ThT). These dyes are especially useful since they exhibit changes in their spectral properties when bound to the fibrils. When CR is bound, it exhibits a red-shift in its absorption,18,19 and a characteristic apple-green birefringence is observed when the fibrils are viewed through cross-polarizers.19-24 ThT is a fluorescent stain that shows a red-shift in excitation25,26 and enhanced fluorescence when bound to amyloid fibrils.7,10,25-29 Although CR and ThT have been extensively researched, they still have limitations for in vivo studies because they are both ionic, which limits their ability to pass through the blood-brain barrier that is critical to in vivo neurological studies.20,30,31 Alternative dyes that are neutral but still have a similar structure to either CR or ThT have been synthesized to overcome this challenge. One relatively new dye is 2-[4′-(dimethylamino)phenyl]-benzothiazole (BTA-2), which is a neutral derivative of * Corresponding author. Phone: 512-232-2824. Fax: 512-232-7316. E-mail: [email protected]. † Current address: CNR-ISMN, Via P. Gobetti, 101, Bologna, 40129, Italy. E-mail: [email protected].

ThT. When BTA-2 is bound to amyloid fibrils, it not only has enhanced fluorescence but also a blue-shifted emission,32 which allows for distinguishing between the free dye and bound dye. Another benefit of BTA-2 is that other research has shown BTA-2 to have higher binding affinity to amyloid fibrils than ThT.33 Despite its extensive use as an amyloid stain, the binding mechanism of ThT (and BTA-2) is still a matter of debate. Several models have been proposed for the binding of ThT to amyloid fibrils. Krebs et al. proposed that the dye binds in a monomeric form in the β-sheet grooves of the fibril. This model was based on polarized confocal microscopy studies of ThT bound to insulin spherulites that contained large amounts of amyloid.7 The emission of the dye indicated the molecule was aligned parallel to the long axis of the fibril. This model has recently gained support from molecular dynamics (MD) simulations by Wu et al. that found a similar binding motif.34 A different binding model was proposed by Khurana et al., who used atomic force microscopy (AFM) to examine the topography of amyloid fibrils in the presence of ThT.10 AFM images of the fibrils in the presence of 20 µM ThT, which is above their reported critical micelle concentration (CMC), exhibited characteristic “bumps” that were interpreted as micelles bound to the surface of the fibrils.10 However, this mechanism has recently been called into question by another study that found the CMC to be 31 µM rather than the 4 µM found by Khurana et al.10,35 Last, Groenning et al. have also proposed that the dye binds in the β-sheet groove,36,37 but some of their data suggests that excimers formed by two dyes bound in the same groove may be responsible for the enhanced emission upon binding.37 Extensive research has been done on the binding of ThT to amyloid fibrils, and therefore, it is important to compare BTA2’s binding to that of ThT. Because BTA-2 is a structurally similar dye, it may bind in a fashion similar to that of ThT. However, the lack of the charged group could alter the binding motif of BTA-2. Isothermal titration calorimetry experiments of ThT binding to fibrils by Groenning et al. found that the free energy of binding was dominated by the entropy.36 This

10.1021/jp903509u CCC: $40.75  2009 American Chemical Society Published on Web 08/07/2009

Measurements of Fluorescent Molecular Probes suggests that electrostatic interactions do not dominate the binding, and ThT and, thus, BTA-2 should have a similar binding mechanism. This idea is supported by the recent MD simulation from Wu et al. that found a similar binding motif for ThT and BTA-1.34 In this work, near-field scanning optical microscopy (NSOM) was used to probe individual amyloid fibrils stained with ThT or BTA-2. NSOM is a combined scanning probe microscopy and optical microscopy technique that is capable of imaging with resolution beyond the diffraction limit of traditional microscopes.38,39 This allows for simultaneous mapping of a sample’s topography along with correlated high spatial resolution optical imaging. By imaging individual amyloid fibrils and collecting the polarized fluorescence, the orientation of the dye bound to the fibrils can be measured directly to distinguish between the different binding models. The orientation of the dye can be determined from the transition dipole. Recent quantum mechanical calculations of ThT and BTA-2 have shown the transition dipole moment to be oriented along the long axis of the molecule.40 The individual fibrils containing either ThT or BTA-2 that are addressed have been adhered to a substrate and have a fixed orientation. Therefore, the polarization of the fluorescence reflects the orientation of the molecular probe, and the relative angle between the dye and the fibril can be measured directly. Specifically, we will distinguish between monomeric emission, excimer emission, and emission from bound micelles, each of which would have unique spectroscopic signatures. The experiments were done using solutions containing ThT or BTA-2 above their CMCs to ensure micelles are present prior to imaging to accurately determine which binding model is correct. The proposed model for monomeric binding is that the dye binds in the β-sheet grooves that run parallel to the long fibril axis. This would result in emission polarized parallel to the fibril axis. The excimer model assumes a similar molecular binding geometry; however, the excimer state would have an emission that is significantly red-shifted from the monomeric dye as well as a longer fluorescence lifetime than the monomer. Finally, bound micelles would have an isotropic polarization relative to the fibril axis because they would contain chromophores at all orientations. The current work definitively demonstrates that both ThT and BTA-2 bind in the monomeric form with emission polarized parallel to the fibril axis. Experimental Section Gold seal glass coverslips were purchased through Fisher Scientific. Insulin from a bovine pancreas, Thioflavin T, and a 0.1% poly(lysine) solution in water were used as purchased from Sigma-Aldrich with no further purification. Previous studies have attributed some fluorescence of ThT in aqueous solutions to impurities in the dye, but ThT is used in the present study only in the presence of amyloid fibrils when impurities are not an issue.40 2-[4′-(Dimethylamino)phenyl]-benzothiazole (BTA2) was synthesized as described in the literature.41 Additional purification was performed by dissolving the solid BTA-2 in ethanol and then filtering to remove any undissolved particles. Deionized water was added to the BTA-2 and ethanol solution until a pale yellow solid precipitated, and then the precipitate was filtered and allowed to air-dry. The structure of the solid BTA-2 was confirmed by 1H NMR and high resolution mass spectrometry. A stock fibril solution was prepared by dissolving insulin in pH 2 water (0.5 mg/mL). The solution was then heated at 60 °C for 24 h. After heating, the solution was cooled to room temperature and then centrifuged (Eppendorf 5415R) at 3000

J. Phys. Chem. B, Vol. 113, No. 35, 2009 12091 rpm for 2.5 min to remove any globular insulin. The supernatant containing the fibrils was then used as a fibril stock solution. Dilute fibril solutions were made using an aliquot of the stock fibril solution (100 µL), diluted with deionized water (880 µL), and an aliquot (20 µL) of 0.8 mg/mL ThT solution in water, with a final ThT concentration of 50.2 µM. Fibril solutions containing BTA-2 were made with an aliquot of the stock fibril solution (100 µL) and were diluted with pH 2 water (880 µL) and then an aliquot of 0.8 mg/mL BTA-2 solution in CH3CN (20 µL). The final BTA-2 concentration was 63.0 µM. In both cases, this should be above the CMC to examine if micelles in solution bind to the fibrils. For glycerin solutions, ThT was diluted until an absorbance of 0.1 was reached. Coverslips were cleaned using a March Plasma CS1701F RIE etching system at 200 W and 35 sccm O2 for 10 min; after cleaning, they were used immediately. An aliquot of the 0.1% poly(lysine) (PLys) solution was pipetted onto the coverslip to coat the entire surface. The coverslips were then covered and allowed to air-dry for 10 min. Once the PLys dried, the fibril solution containing either ThT or BTA-2 (as described above) was pipetted onto the surface of the coverslip. The samples were covered for 10 min to allow the fibrils to bind to the substrate. The samples were rinsed in deionized water to remove any unbound fibrils. Finally, the samples were covered and allowed to air-dry. Excitation and fluorescence spectra were collected on a Photon Technologies International Quanta Master model C cuvette-based scanning fluorometer. Bandpass filters were utilized to guarantee that the excitation beam was monochromatic. Long-pass filters were used to remove any scattered excitation light. Time-correlated single photon counting (TCSPC) lifetimes were collected with a home-built system consisting of a microchannel plate photomultiplier tube and standard TCSPC electronics. The fibril solutions and ThT in glycerin were excited at 440 nm with a frequency-doubled Ti: sapphire laser. The fluorescence was collected through a 500 nm bandpass filter, and a polarizer was used to collect fluorescence at the magic angle (54.7°). NSOM images were collected using a modified Aurora system (Thermomicroscopes/Veeco).42-44 A frequency-doubled Tisapphire laser was used to excite the sample at 400 or 440 nm. Near-field probes were manufactured in-house and mounted on piezo-electric tuning forks for shear-force feedback. Samples containing ThT were excited at 440 nm, and the fluorescence was collected through a 455 nm long-pass filter. Samples containing BTA-2 were excited at 400 nm, and the fluorescence was collected through a 418 nm long-pass filter. The fluorescence from the samples was then split into orthogonal polarizations with a polarizing beam splitter and imaged onto an avalanche photodiode and a micro photon detector. Results and Discussion Fluorescence spectra were acquired for a solution of ThT and a solution BTA-2 in the presence of amyloid fibrils, and both solutions contained the same dye concentration. Figure 1 shows the normalized excitation and emission spectra for ThT and BTA-2 bound to the amyloid fibrils. The excitation spectrum was collected for fibril solutions containing ThT for emission at 500 nm, and the excitation spectrum was collected for fibril solutions containing BTA-2 for emission at 425 nm. The ThT spectrum has an excitation peak at 460 nm; the BTA-2 has a maximum excitation at 386 nm. Emission spectra were collected for each dye near its peak excitation. An emission spectrum collected for ThT and fibril with 440 nm excitation had a

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Figure 1. Normalized excitation and emission spectra of BTA-2 (dashed lines) and ThT (solid lines) in the presence of amyloid fibrils. Inset shows the emission intensities of identical solutions of the BTA-2 and ThT bound to amyloid fibrils.

Figure 2. Time-resolved fluorescence decays of ThT in glycerol (blue) and bound to fibrils (black). Also shown are the biexponential fits (red) for each decay and the instrument response function (gray).

maximum peak at ∼482 nm. The emission spectrum acquired for the BTA-2 solution with 380 nm excitation revealed an emission maximum at 426 nm. Noticeable differences between ThT and BTA-2 emission are revealed when the spectra are compared. The non-normalized data, inset to Figure 1, shows that for solutions with similar dye and fibril concentrations, the solution of BTA-2 bound to fibrils is almost twice as fluorescent as when ThT is bound. In addition to being more fluorescent, the BTA-2 had a slightly larger Stokes shift of 40 nm, as compared to a 22 nm Stokes shift for ThT. BTA-2’s larger Stokes shift makes it easier to filter the excitation without absorbing fluorescence, making detection of the BTA-2 easier. The two dyes show one similarity in that the emission spectrum is a mirror image of the absorption spectrum. Although the two dyes have different Stokes shifts, both are less than 2000 cm-1, and excimer emission is generally red-shifted from the absorption peak by several thousand wavenumbers.45,46 This makes excimer formation unlikely to be the cause of the enhanced fluorescence upon binding because one would expect a larger shift in the emission spectrum. The possibility of excimer formation was also investigated by measuring the fluorescence lifetime of the ThT. Figure 2 shows the time-resolved fluorescence decay for ThT in a solution of glycerol and in water bound to fibrils. The decays are wellrepresented by double exponential decays in both cases. The ThT in glycerin has a slow component of 0.526 ns (A1 ) 9410) and a fast component of 0.173 ns (A2 ) 9890 counts). This yields an average lifetime of 0.44 ns, which is in excellent agreement with published results. The lifetime of the ThT bound to fibrils had a longer lifetime than in glycerin. The slow

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Figure 3. A 5 µm × 5 µm AFM image of dry amyloid fibrils on poly(lysine)-coated glass.

component of the ThT bound to fibrils had a lifetime of 1.73 ns (A1 ) 6544), and the fast component had a lifetime of 0.515 ns (A2 ) 4180), with an average lifetime of 1.5 ns. This suggests the increase in fluorescence when bound results from the rigidity of the dye when bound and not from the formation of excimers or emissive dimers. Increasing the rigidity should lead to a decrease in the nonradiative decay rate, resulting in both a longer lifetime and a high quantum yield. For excimers or dimers, one would expect a much longer fluorescence lifetime. Previous reports have attributed lifetime components in the 2-12 ns range to ThT dimers.47 Given the short lifetimes observed, they are consistent with the radiative rate of the monomer. As such, excimers can be ruled out as the source of the increased emission upon amyloid binding. Amyloid fibrils were immobilized onto solid substrates to investigate the orientation of the dye bound to the fibrils. Figure 3 shows an AFM image of the fibrils bound to a PLys-coated substrate. The image shows high aspect ratio features that run across the substrate surface which are the amyloid fibrils. The fibrils exhibit typical heights of 5-10 nm that are consistent with previous measurements.48-50 The image demonstrates that the fibrils can be isolated on the surface at a sufficiently low density that individual fibrils can be observed. Most importantly, the fibrils are bound to the surface with a well-defined orientation that extends for several micrometers. This allowed the orientation of each fibril to be uniquely determined. The interaction of the dye with fibrils was determined using NSOM imaging. Figure 4 shows the NSOM images of ThT bound to the amyloid fibrils. The sample was excited at 440 nm, which is near the peak of the excitation spectrum in solution. The resulting fluorescence was split into two orthogonal polarizations for imaging. The topography image (Figure 4A) shows the fibrils present on the surface and is similar to the AFM results. Because the NSOM tip is significantly larger than the AFM probe, the fibrils appear larger in the NSOM image. In addition, the shear-force feedback is not as robust as tapping mode AFM, leading to lower signal-to-noise images. However, the fibrils are still clearly visible in the images. Figure 4B is the fluorescence image polarized in the vertical direction, and Figure 4C is the horizontally polarized fluorescence image. To improve the signal-to-noise ratio, the image was processed with a 3 × 3 pixel median filter. From the images, it is clear that not all the fibrils are uniformly labeled with dye because some of the fibrils that can be observed in the topography do not appear in either of the fluorescence image. It is also apparent that individual fibrils have a very low fluorescence signal. Under the current conditions, the full scale of the image ranges from a minimum of 90 counts to a maximum of 125 counts with a

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Figure 5. NSOM images (5 µm × 5 µm) of fibrils with BTA-2. (A) The topography image of the fibrils. (B) The fluorescence image in the vertical polarization. (C) The fluorescence image in the horizontal polarization. The lower left quadrant of the fluorescence images shows the raw unprocessed images. (D) Linescan of the fluorescence intensity of the region indicated in the two images.

Figure 4. NSOM images (5 µm × 5 µm) of fibrils with ThT. (A) Topography image of the fibrils. (B) Fluorescence image in the vertical polarization. (C) Fluorescence image in the horizontal polarization. Boxes in the image highlight fibrils oriented in the vertical and horizontal directions.

dwell time of 40 ms. The low fluorescence signal is the result of several factors. The laser used for excitation was limited to 440 nm rather than the peak of the excitation spectrum. In addition, the small Stokes shift of the ThT led to a loss of fluorescence due to the long pass filters used to eliminate the excitation light. Despite the low signal, the fibrils are clearly visible in the fluorescence images. In the vertically polarized fluorescence image, most of the fibrils that can be seen are

oriented with their axis in the vertical direction. Similarly, the horizontal image shows mostly fibrils that are horizontally oriented. The small rectangular boxes in the images highlight two different fibrils that are oriented nearly parallel to the vertical and horizontal directions. The vertical fibril shows a strong intensity in the vertically polarized image, but no fluorescence is detected in the horizontal channel. The opposite is true for the horizontally oriented fibril. This would indicate that the ThT is bound such that its transition dipole is perpendicular to the long axis of the fibrils, which is consistent with Krebs et al.7 However, the low fluorescence signal of the ThT and the background noise made it difficult to quantify this effect. The fact that the emission is polarized along the same direction of fibril axis indicates that the dye is not binding as a micelle, which would yield an isotropic polarization. Samples containing BTA-2 were imaged using polarized NSOM and demonstrated an improved fluorescence over the ThT. Figure 5 shows the NSOM images of BTA-2 bound to the amyloid fibrils where the improved contrast is readily apparent. As discussed above, BTA-2 has an excitation maximum at ∼386 nm and an emission maximum at ∼426 nm when bound to fibrils. This produces a slightly larger Stokes shift that makes it easier to filter out the excitation light without diminishing the fluorescence. As reported by Klunk et al., BTA-2 had a binding affinity that was 6-fold greater than ThT’s binding affinity to amyloid fibrils.31 With a higher binding affinity, more BTA-2 dye molecules would bind and lead to the larger fluorescence signal seen in the NSOM images. Finally, the BTA-2 excitation peak falls perfectly in the optimal lasing region of the frequency-doubled Ti:sapphire laser used as the excitation source. NSOM images of the fibrils were collected by exciting the sample at ∼400 nm and collecting the polarized fluorescence through a 418 nm long-pass filter using the same procedure as for the ThT/fibril samples. Figure 5A shows the topography, and Figure 5B and C shows the polarized fluorescence images

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of BTA-2 bound to amyloid fibrils. The emission images have been put through a 3 × 3 pixel median filter; however, for clarity, the lower left quadrant of the two images is left in its raw form. The vertical image is dominated by fibrils that are vertically oriented in the image frame. Conversely, the horizontal image is dominated by fibrils that run in the horizontal direction. Figure 5D highlights these differences by plotting an intensity linescan acquired from each image. The intensity is plotted along the vertical line shown in each image. This line is perpendicular to an amyloid fibril that is horizontally oriented. The blue line shows the intensity in the horizontal polarization, and the red line is the intensity in the vertical polarization. The peak in the fluorescence is clear in the horizontal channel, and the amyloid is essentially the same as the background in the vertical channel. Similarly, fibrils oriented in the vertical direction have greater intensity in the vertical polarization and are virtually zero in the horizontal direction. Any fibrils oriented at 45° with respect to the image have nearly identical intensity in both images. The polarization reflects the orientation of the bound dye, not the excitation polarization. The excitation polarization will affect the signal-to-noise ratio of one fiber as compared to the next because the dot product of the excitation field and the absorption dipole will determine the excitation rate. But since the dye molecules have a fixed orientation, if they are excited, their emission will be the same, regardless of the excitation polarization. In an effort to balance the two directions from a signalto-noise perspective, the excitation polarization of the NSOM field was chosen to be equal for the two detectors. Ideally this should have been circularly polarized, but is more likely an elliptical polarization around 45°. Because the fibrils are fixed in space, their orientation can be measured from the topography image relative to the x-axis of the image (horizontal). The orientation of the fibril can then be compared to the polarization of the emission, which reflects the orientation of bound dye molecules. Assuming the dye has a single transition moment, the intensity, I, in each channel will be given by

Ix ) Itotal cos2 θ Iy ) Itotal sin2 θ

(1)

where θ is the angle between the transition dipole moment of the molecule and the x axis of the image frame. The transition moment for ThT and, presumably, BTA-2 lies predominately parallel to the molecular axis.7 Assuming the dye binds parallel to the fibril axis, the orientation of the fibril would be the same as the orientation of the dye molecule and the transition moment. The polarization could then be measured as a function of the fibril orientation. The polarization is quantified using the emission dichroism (D),

D)

Ix - Iy Itotal(cos2 θ - sin2 θ) ) ) cos(2θ) Ix + Iy Itotal(cos2 θ + sin2 θ)

(2)

Therefore, if the dye is oriented parallel to the fibril axis, a plot of the dichroism as a function of orientation should exhibit a cos(2θ) dependence. A plot of the dichroism of individual fibrils measured as a function of their orientation clearly shows the expected angular dependence (Figure 6). It is peaked at 0° and has a minimum in the perpendicular direction with a dichroism of zero at 45°. The orientation of each fibril was measured from the topography image. The dichroism was

Figure 6. Plot of the emission dichroism of BTA-2 bound to the amyloid fibrils as a function of the orientation of the fibril with respect to the horizontal axis of the image frame. The line is a fit to the expected cos(2φ) and angular dependence of the fluorescence.

measured by taking linescans perpendicular to the fibrils in each polarization image, as shown in Figure 5D. For the dichroism analysis, linescans were collected on images that had been background-corrected but not filtered. Since the images are collected simultaneously, they can be rigorously compared on a pixel-by-pixel basis. The intensity in each channel was obtained by fitting the linescans to a Gaussian profile. The same Gaussian was used to fit the linescan from each image, and the dichroism was determined from the amplitudes of the Gaussian fits. The error in the measured orientation is the result of uncertainty that arises from the length of the segment that is sampled. The uncertainty in the dichroism was calculated by propagating the shot noise in each channel into the measured dichroism. Although the fit yields the expected angular dependence, the amplitude is not exactly 1. If the dipole were perfectly orientated along the fibril axis, the dichroism would vary between +1 and -1. However, the measured dichroism yields a fit with a maximum of 0.85. There are a number of reasons for this discrepancy. First, it is possible that the measured polarization is affected by the finite numerical aperture of the collection lens.51 However, this affect can be estimated for the N.A. of 0.6 used in the experiments and reduces the maximum dichroism to 0.99 rather than 1.0. Another problem is the large background in the images. The images in Figure 5 have a background signal that is approximately 90 counts per pixel. This has been subtracted from each image, but due to the shot noise, it cannot be corrected for exactly. This leaves a residual signal in both channels that biases the dichroism toward zero and away from the extremes. Fitting the linescans rather than simply measuring the counts should minimize this affect, but cannot eliminate it. Finally, it is possible that the dichroism is not (1 because the dye is not perfectly oriented parallel to the fibrils. If there is a slight variation in the angle, the measured dichroism will be slightly less. Assuming the angle between the dye molecule and the fibril is given by ε, the maximum dichroism is given by

Dmax ) 〈cos(2ε)〉

(3)

where the brackets denote an angular average over all possible angles ε. Assuming the angle is normally distributed around zero, the maximum dichroism is given by

Measurements of Fluorescent Molecular Probes

Dmax ) 〈cos(2ε)〉 ) exp(-2σ2)

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(4)

where σ is the standard deviation of the angular distribution. When the maximum is set to the measured value of 0.85, it yields a standard deviation for the angle of 16°. This represents a maximum value, since the measure Dmax may be slightly reduced due to the experimental errors previously discussed. In addition, this dispersion in the angle may result from variation of the dye with respect to the normal axis of the fibril. Because the dye is binding on all sides of the fibril, any tilt of the dipole relative to the fibril axis will lower the polarization for molecules that are bound on the sides of the fibrils. Thus, 16° is an upper bound on the range of the angles, and the binding geometry could be closer to perfectly parallel. Conclusions The polarized NSOM images conclusively show that both the ThT and BTA-2 bind with their molecular axis parallel to the axis of the amyloid fibrils. This confirms the binding scheme for both charged and uncharged dyes that is proposed by Krebs et al. wherein the dye binds in the β-sheet grooves running along the fibrils. There is no evidence for binding of micelles, as has been previously proposed;10 neither is there any indication that either the ThT or BTA-2 exhibit excimer emission.37 BTA-2 has also been found to form micelles in aqueous solutions and most likely forms a micellular structure similar to ThT. However, the polarization data and uniform fluorescence rules out this binding mechanism. The dye may bind with two adjacent dye molecules in one groove, but the emission shows none of the characteristics expected for excimer emission. In addition, the micelle emission spectrum and the spectrum of BTA-2-bound fibrils have been shown to be distinct.32 The BTA-2 images show that it is a more effective fluorescent dye than the ThT and yields higher-contrast fluorescence images. The higher signal-to-noise of the BTA-2 images allows the orientation to be quantitatively analyzed. The dye is found to be aligned parallel to the fibril axis with a dispersion in orientation of no more than (16°. This angle assumes a transition dipole moment for the molecule that is along the long axis of the molecule. Any tilt of this transition dipole moment in the plane of the molecule will lead to a slight depolarization. As such, the molecules may be bound with a narrower angular distribution. Finally, the similarity of the binding for these two dyes suggests that the binding of the probes is driven by the size and shape of the molecules rather than specific electrostatic interactions with any charged groups, since BTA-2 is a neutral molecule. As such, synthesis of other dye probes should focus on molecules of similar shape whose properties are distinct when they are rigidly confined. Acknowledgment. We thank the Welch Foundation (F-1377 and through equipment in the Center for Nano- and Molecular Science and Technology). References and Notes (1) Kelly, J. W. Curr. Opin. Struct. Biol. 1996, 6, 11. (2) Allsop, D.; Swanson, L.; Moore, S.; Davies, Y.; York, A.; El-Agnaf, O. M. A.; Soutar, I. Biochem. Bioph. Res. Co. 2001, 285, 58. (3) Dobson, C. M. Nature 2003, 426, 884. (4) Sipe, J. D. Annu. ReV. Biochem. 1992, 61, 947. (5) Murphy, R. M. Annu. ReV. Biochem. 2002, 4, 155. (6) Harper, J. D.; Lansbury, P. T. J. Annu. ReV. Biochem. 1997, 66, 385. (7) Krebs, M. R. H.; Bromley, E. H. C.; Donald, A. M. J. Struct. Biol. 2005, 149, 30.

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