Infrared Linear Dichroism Spectroscopy on Amyloid Fibrils Aligned by

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Infrared Linear Dichroism Spectroscopy on Amyloid Fibrils Aligned by Molecular Combing Jose C. Rodríguez-Perez, Ian W. Hamley, and Adam M. Squires* Department of Chemistry, University of Reading, Reading, RG6 6AD, United Kingdom ABSTRACT: We report the use of molecular combing as an alignment method to obtain macroscopically oriented amyloid fibrils on planar surfaces. The aligned fibrils are studied by polarized infrared spectroscopy. This gives structural information that cannot be definitively obtained from standard infrared experiments on isotropic samples, for example, confirmation of the characteristic cross-β amyloid core structure, the side-chain orientation from specific amino acids, and the arrangement of the strands within the fibrils, as we demonstrate here. We employed amyloid fibrils from hen egg white lysozyme (HEWL) and from a model octapeptide. Our results demonstrate molecular combing as a straightforward method to align amyloid fibrils, producing highly anisotropic infrared linear dichroism (IRLD) spectra.

’ INTRODUCTION Self-assembled “amyloid” fibrils form from a range of proteins and peptides. In many cases, their presence is linked to a disease.1,2 However, they have recently become the focus of wider study to shed light on fundamental physicochemical aspects of protein folding and assembly3 and as potential nanomaterials.4,5 In this Article, we describe methods for reproducibly producing aligned films of amyloid fibrils onto solid surfaces based on the “molecular combing” technique. Whereas the production of aligned fibril samples has its own potential technological benefits, as we shall discuss, here we develop sample orientation primarily as an analytical tool. We first demonstrate the use of oriented samples in combination with polarized infrared spectroscopy. This gives structural information on the fibrils that cannot be obtained with conventional infrared spectroscopic methods. We then describe an investigation of the effect of certain parameters on the levels of orientation produced by the molecular combing technique. Amyloid Structure. Proteinaceous aggregates known as amyloid fibrils consist of long, unbranched filaments of lengths up to several micrometers and typically 40 to 120 Å wide,6,7 usually formed by the association of thinner protofilaments.8 Different arrangements of protofibrils have been observed and result in distinct fibril morphologies.8,9 Despite these differences, amyloid fibrils share a common core structure, giving rise to a characteristic cross-β X-ray diffraction pattern.10 The difficulty in obtaining crystals from amyloid fibrils means that high-resolution structural data for these systems is limited to a small number of cases.1113 As result, a wealth of different techniques has been employed to study the structure of amyloid fibrils, including electron microscopy, X-ray fiber diffraction, UV circular dichroism, infrared spectroscopy, solid-state nuclear magnetic resonance, and electron paramagnetic resonance.2,6,8 r 2011 American Chemical Society

Infrared Linear Dichroism. Infrared linear dichroism (IRLD) spectroscopy provides orientational information on the vibrating dipole moments present in the molecules.14 This technique has been employed as a source of structural information in a wide range of systems, from polymers to liquid crystals and biological materials.14 Previous examples of the use of polarized infrared spectroscopy to study protein fibres include the work of Ambrose and Elliot15 and Burke and Rougvie,16 who investigated films of fibrous insulin samples with this technique. Polarized ATR-FTIR methods have been used for proteins deposited on flat substrates to determine the orientation of certain bands relative to the substrate normal.17 However, the samples had uniaxial symmetry about this normal axis and were not oriented within the plane of the substrate. Information about the hydrogen-bonding orientation in peptide fibres can also be obtained from a combination of grazing angle and transmission IR spectroscopy, although at a qualitative level.18,19 Hiramatsu and coworkers have studied amyloid fibrils formed from the protein β2-microglobulin and a number of synthetic peptides based on its sequence, using IRLD microscopy on oriented microscopic particles within pellets.2022 Alignment Methods on Surfaces. There are a number of reasons why it is desirable to have some means of aligning molecules. Certain useful properties or types of behavior of a material may be present or exploitable only when samples show a degree of orientation. For example, an increased ability to bind cells is achieved when collagen fibres are arranged over their supporting surfaces in an ordered manner.23 Amyloid fibrils have also been the subject of investigation for similar applications.24 Mechanical properties would also be expected to depend on orientation, which is of relevance to the potential of amyloid to be Received: February 7, 2011 Revised: March 14, 2011 Published: March 29, 2011 1810

dx.doi.org/10.1021/bm200167n | Biomacromolecules 2011, 12, 1810–1821

Biomacromolecules used as biodegradable materials.25 In a recent publication in this area, free-standing films of lysozyme amyloid fibrils have been prepared, which show partial alignment.4 An understanding of the mechanical and biological properties of such systems requires methodologies for controlling, characterizing, and understanding this alignment. A second reason for wishing to align molecules is for analysis, to increase the information available on a system. Certain properties invaluable to the study of molecular structure are exhibited only at a macroscopic level when a sufficient degree of anisotropy is generated, such as birefringence or polarized spectroscopies. This latter forms the focus of this Article. An array of different techniques has been used to induce orientation in what would otherwise be isotropic bulk samples of fiber-like molecules. There is a large body of literature on a wide range of systems including DNA,26 protein fibers,2729 and carbon nanotubes,30 in addition to synthetic polymers.31 Among the methods reported, we find fiber stretching devices,10 airblowing,32 evaporation-driven fixation,33 spin-stretching,34 and fluid flow alignment.27,29 A number of these techniques are based on the molecular combing procedure described by Bensimon et al. to align and elongate DNA strands over a surface.35 The principle behind molecular combing resides in the forces exerted by the moving meniscus of a droplet of solvent containing the molecules in solution. Molecular combing approaches mainly differ from one another in the way that the movement of the solution upon the supporting surface is produced. Variations of the method include the inmersion of a flat surface into a solution and slowly pulling it out,36 forced flow of droplets over suitable surfaces by air blowing,32,37 mechanical means35,3840 or flow under gravity,41 as well as more complex setups such as peeling of solution-wetted micropatterned stamps in contact with a hard surface.42 We note that molecular combing, as with many of these techniques, produces a dried oriented sample. The issue of whether drying affects fibril structure is still a controversial one.43,44 In the case of both the lysozyme and octapeptide fibril systems studied here, separate data indicating no differences between hydrated and dried samples have already been published.29,43 To our knowledge, only one research group has used the molecular combing technique to align amyloid-like systems: a combination of molecular combing and transfer printing allowed Herland et al. to align partially insulin fibrils complexed with conjugated polymers, obtaining information about the orientation of the polymer with respect to the fibrils by means of singlemolecule fluorescence spectroscopy.45,46 However, to avoid the complications related to performing the measurements on carefully selected regions of the sample, or the use of extremely sensitive techniques, a sufficient alignment is required at a macroscopic scale. Our results demonstrate the successful orientation of significant amounts of fibrillar material, which can be studied by simple transmission infrared spectroscopy or X-ray diffraction techniques, as shown in this work.

’ EXPERIMENTAL METHODS Lysozyme. Hen egg white lysozyme, purchased from Sigma (L6876, 95% purity) was used with no further purification. Samples were weighed, dissolved in 0.01 M HCl, and pH-corrected to 2.0 ( 0.2 with 0.1 M HCl. Target concentrations were 75 mg/mL. Under gentle agitation, lysozyme quickly dissolves, and a clear solution is obtained. Stock solutions were incubated in an incubating block at 65 C for 2 weeks. After incubation, the samples consisted of a viscous gel, indicating that extensive fibrillization had taken place and a dense network of fibrils

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had formed. Before use, all samples were freezethawed by immersion in liquid nitrogen to increase their fluidity by shortening the fibrils (Corrigan et al.,47 Supporting Information). This step was necessary to break the gels and allow the samples to flow when using the molecular combing technique. To test the effect of concentration on alignment, dilutions of the lysozyme fibrils stock were prepared by the addition of 0.01 M HCl to obtain solutions at 50, 25, and 12.5 mg/mL. YYKLVFFC. NH2YYKLVFFCCOOH, referred to as YYKLVFFC, was custom synthesized by C. S. Bio Company and was used as received as a TFA salt. Purity was 96.87%, as tested by HPLC in water/acetonitrile (0.1% TFA). Electrospray ionization mass spectroscopy (Bruker microTOF, BioCentre, University of Reading) confirmed a molar mass 1082.54 consistent with the expected formula C56H75N9O11S. No evidence of disulfide or dityrosine cross-linking was observed under the electrospray ionization conditions employed for the mass spectroscopy. YYKLVFFC readily forms fibrils at low concentration, as detailed elsewhere.48 Without further treatment, a 1% w/w solution was prepared in Millipore-filtered water and left incubating for ∼3 h at room temperature. The pH of the YYKLVFFC solution under these conditions is 4.7.48 No dilutions or freezethawing steps were carried out on this sample. X-ray Diffraction. Silicon wafer containing films of aligned YYKLVFFC fibrils were mounted vertically onto the four axis goniometer of a RAXIS IVþþ X-ray diffractometer (Rigaku), equipped with a rotating anode generator and a Saturn 992 CCD camera (Biocenter, University of Reading). With the help of a video camera displaying the image of the sample in a video monitor, the position of the X-ray beam was adjusted such as to hit the center of the silicon wafers. This ensured that approximately the same region of the aligned film was analyzed with both X-ray and IR spectroscopy. ULTRATHIN polished silicon wafers (Virginia Semiconductor), 50 μm thick, were used as the supporting material for aligned samples for tandem analysis by X-ray diffraction and IR spectroscopy. The low atomic mass of silicon and the low thickness of the wafers make them suitable for X-ray diffraction experiments. Special undoped substrates were used so they are not electrically conductive and transparent to infrared radiation. Fibril Alignment. Aligned samples of amyloid fibrils were obtained by air-blowing molecular combing with a custom-built mechanical device. CaF2 windows were washed with detergent (Decon 90) to remove any protein remaining from previous experiments, rinsed with acetone, and dried with tissue, and any possible dust present in the surface was blown off with compressed air. Droplets of 10 μL of sample solution were deposited onto individual CaF2 windows. The glass tip that directed the air-jet was positioned at an approximate distance of 5 mm from the CaF2 window and tilted 45 from its normal in the plane of platform displacement. The air flow rate was set to 4.5 L/min, and the speed of the platform was set to ∼0.13 cm/s. Except for the cleaning stage, identical procedures were followed in the case of molecular combing onto silicon wafers. Polarized Infrared Spectroscopy. Data were collected in transmission mode on a Thermo Nicolet 8700 FTIR instrument (Thermo Scientific) fitted with a DTGS (deuterated triglycine) detector, working with a constant purge of water and carbon dioxide. For polarization measurements, a wire grid ZnSe polarizer (Specac, Slough, U.K.) was mounted on a special holder that allows rotation and placed in the infrared path after the samples. Sample-containing substrates were positioned perpendicularly to the IR beam with the combing direction along the vertical axis. Spectra were taken with the polarizer grid at 0 and 90 relative to the direction of fibril alignment. Individual background spectra for each orientation were taken to compensate for the different transmission efficiency of the polarizer at different orientations. A minimum of 128 spectra per sample were collected at 4 cm1 resolution. Unless stated otherwise, no further data treatment was performed except for baseline subtraction. Area and height measurements were 1811

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Figure 1. Schematics of air-blow molecular combing. Droplets of solution containing the fibrils are forced to move along a surface by the pushing action of a gas jet.

Figure 3. Polarized IR spectra of aligned lysozyme amyloid fibrils. Amide and II regions. Blue trace: parallel polarization; red trace: perpendicular polarization; bottom: difference spectrum, parallel minus perpendicular. Figure 2. Molecular combing device used in our laboratory for aligning amyloid fibril samples onto surfaces. The air flow is regulated with a variable flow meter (left-hand side of the Figure) and directed toward the sample through a glass tube that stays in a fixed position. The platform holding the sample moves at a controlled speed toward the air jet, which pushes the deposited droplet of solution along the supporting surface. performed with Octave49 scripts written in-house for spectra batch processing. We observed that the dichroic ratio values did not change substantially by measuring the intensities as peak heights or peak areas. The order parameter ÆP2æ for different vibrations was calculated from the dichroic ratios, as explained in the main text. Molecular Combing. The molecular combing method we developed resembles some of those previously reported. It is essentially an air-blowing procedure (Figure 1), similar to the methods described by Li et al.32 for DNA alignment or Herland et al.45 for conjugated polymers onto amyloid-like fibrils. However, there are important differences between our setup and those employed by others. First, we have built a mechanical device that ensures a greater repeatability between successive experiments. Second, the surfaces used were not pretreated in any way save for a thorough cleaning. Finally, the concentration range used was in general much higher than any of the previously mentioned methods to enable transmission FTIR experiments. A picture of our setup is shown in Figure 2. A horizontal platform that acts as sample holder is mounted on a screw cylinder connected to the shaft of a stepper motor. An electronics board is used to control the direction and the speed settings of the moving platform. A metallic arm attached to the structure holding the platform and the stepper motor holds the gas tubing, which is connected to a compressed air supply. The volumetric flow can be measured and regulated with a variable area flow meter inserted in the gas circuit. A rectangular-shaped hollowed glass tip is attached to the gas tubing and is used to direct the air over the sample droplets. The rectangular shape was adopted to project a wide and even air jet over the sample; its dimensions at the edge are 8 1 mm. The maximum horizontal displacement of the platform is 11 cm, and the speed settings can be varied from 0 to ∼2.5 cm/s. In terms of air volumetric flow, the fitted flow meter has a range of 05 L/min, although this is not a hard limit of our device. The distance from the air jet to the surface supporting the sample can be adjusted as well as its tilt angle in the vertical plane. This device was built by the workshop in the department of Chemistry, University of Reading.

Figure 4. Representation of three of the most polarized vibrational modes in the infrared spectra of polypeptides. From top to bottom: amide A, amide I, and amide II modes. The arrows indicate the directions of atom displacement that most contribute to each vibration.51 In β-sheets, the amide A and amide I have a resultant dipole moment perpendicular to the backbone, whereas in the case of the amide II, it is parallel to it.

The molecular combing device allowed us to prepare our samples in a repeatable way and to find quickly a set of experimental conditions that successfully obtained oriented films of the samples studied. With it, we were able to keep the physical variables of the experiment constant at the desired values, that is, the speed of the sample with respect to the air jet and the direction, position, and flow rate of the incoming gas. A range of other experimental variables such as concentration, pH, different sample treatments, or use of different surfaces can then be introduced to test the effect they have in sample alignment. 1812


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’ RESULTS AND DISCUSSION IRLD Spectra of Aligned Lysozyme Fibrils. The IRLD spectra of a sample of lysozyme fibrils aligned by molecular combing are shown in Figure 3. The two traces in the Figure correspond to the data collected with the polarizer positioned at parallel and perpendicular orientations relative to the direction of alignment. Also shown is the difference spectrum that results from subtracting the perpendicular spectrum from the parallel one. Disordered regions, or protein monomers that are not attached to the fibril will not orient and therefore do not contribute to the difference spectrum. Bands caused by normal modes whose transition dipole moments are preferentially aligned parallel to the alignment direction appear as positive peaks in the difference spectrum. The amide A (∼3285 cm1) and amide I (∼1631 cm1) bands show a higher intensity in the spectrum collected with the polarizer parallel to the direction of alignment (positive bands in the difference spectrum), whereas the amide II (∼1540 cm1) is more intense when light is collected in the perpendicular direction. For a given molecular structure, the direction of the transition dipole moments with respect to the peptide chain for these vibrations determines the sign of the observed dichroism. In sheet structures, the dipole moments of the amide A (NH stretching) and amide I (mainly CO stretching) vibrations are perpendicular to the polypeptide backbone, as seen in Figure 4. The amide II band, originated mainly by a combination of inplane NH bending and CN stretching vibrations, has a dipole moment oriented parallel to the peptide chain.50 Cross-β Structure. The observed dichroism is consistent with the expected cross-β structure commonly accepted for amyloid fibrils. In this classical structure, the extended β-sheet strands are hydrogen bonded in the direction of the fibril axis, forming sheets that stack side by side.10 The difference IRLD spectrum shows that the amide I changes between orthogonal polarizations occur mostly at ∼1631 cm1, that is, the signal arising from β-sheet regions of the protein. The transition dipole moment for this normal mode is oriented parallel to the carbonyl bonds; therefore, we can determine that the strands of the sheets are oriented perpendicular to the direction of alignment. The same argument explains the polarization of the amide A band (NH stretch vibration), whose transition dipole moment is oriented in the same direction as that of the carbonyl bonds (amide I). The amide II band, having a perpendicularly polarized transition dipole moment, presents a negative peak in the difference spectrum. Therefore, the IR data confirm the cross-β structure deduced from X-ray diffraction experiments on dried stalk samples. We highlight in this stage the possibility of polarized IR as a definitive structural test for amyloid fibrils, as an alternative to fiber X-ray diffraction. Both techniques demonstrate the existence of fibrillar species (without which no alignment would be obtained), that these fibrils are predominantly β-sheet, and furthermore that the β-sheet structure is oriented in the cross-β conformation. This final point is in contrast with different β-sheet structures that can potentially be adopted in fibrous structures, such as those of βkeratin and some silks,52 where the β-strands lie parallel to the fiber axis. FTIR instrumentation is less expensive and in general more widely available than that required for fiber X-ray diffraction, the need for an IR polarizer notwithstanding. Other Assignments. The high sensitivity of the amide I to the secondary structure of the polypeptide chain enables us to extract more detailed information from these spectra.

Figure 5. Top: polarized infrared spectra of a YYKLVFFC amyloid fibrils film aligned by molecular combing. Blue trace: spectrum collected with light polarized parallel to the alignment direction; red: light polarized perpendicular to the director; bottom: difference spectrum, parallel minus perpendicular.

First, it appears that the fibrils are not exclusively formed by βsheet structures. A significant intensity contribution to the amide I envelope appears at frequencies not commonly associated with extended chains in the β-conformation, as reflected by the broad shape of the band (Figure 3). This indicates the presence of polypeptide regions involved in other types of secondary structure. However, the difference spectrum in the amide I region is dominated by the positive peak centered at 1631 cm1, assigned to β-sheets, indicating that most of the structures giving rise to spectral features at other wavenumbers do not orient, either because they are in disordered parts of the fibril or because they belong to nonfibrillized monomers. Second, a low intensity, distinctive peak with positive dichroism appears in the difference spectrum at ∼1674 cm1 (Figure 3, bottom trace). By virtue of the polarization of this band, we can discard the possibility that it arises from antiparallel sheets, whose high frequency component has a net dipole moment perpendicular to the carbonyl bonds50 and therefore would show as a negative peak in the difference spectrum. We cannot discard the possibility that it arises from a side-chain vibration. However, the peak shows a strong positive dichroism associated with a vibration parallel to the fibril axis, and we think it is more likely that this would arise from a vibrational mode more tightly linked to the backbone than from a side chain. The spectral region at which this peak appears, 16601690 cm1, is also assigned to turn structures on the basis of empirical correlations and model calculations.53 The characterization of turns in proteins by IR spectroscopy is problematic owing to the number of different possible turn geometries,54 the presence of overlapping signals,53,55 and their lack of long-range order, which precludes the existence of spectral features as well-defined as those arising from coupled modes such as those of β-sheets.56 However, with the polarization sensitivity achieved with IRLD, these features may be better resolved and become visible in the difference spectra. The presence of turns within the amyloid core has been reported for amyloid-β57 and R-synuclein amyloid fibrils.58 The structural findings for these systems evidence an in-register arrangement of parallel β-sheets, with a central region forming a loop that folds two strands upon one another. It has been suggested 1813


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Figure 6. Amide I region of spectra shown in Figure 5. Blue trace: parallel polarization; red trace: perpendicular polarization; dotted trace: difference spectrum. The opposite polarization of the peaks at 1627 and 1690 cm1 becomes evident in the difference spectrum.

from DFT calculations that the spectral marker for these loop regions should appear in the high-frequency amide I region (∼1670 cm1), arising from a ν(0, 0) mode.59 The direction of the transition dipole moment for this vibration is oriented in the direction of the carbonyl bonds, which would give rise to positive peaks in the difference IRLD spectra and thus be consistent with our data. We therefore tentatively assign the 16601690 cm1 peak to a turn region. IRLD Spectra of Aligned YYKLVFFC Fibrils. The polarized IR spectra of YYKLVFFC fibrils (Figure 5) are similar to those previously shown for lysozyme amyloid fibrils. In this case, the amide A (3270 cm1) is sharper and more symmetric compared with the case of lysozyme amyloid fibrils. The same is observed for the amide I band (16001700 cm1), with a very sharp and well-defined peak at 1627 cm1. Owing to the lower number of residues, there is an increased relative intensity for individual amino acids. Therefore, in the amide II region, a characteristic peak at 1516 cm1 assigned to tyrosine is visible without the need for band enhancement techniques. Cross-β Structure. These spectra indicate a high degree of alignment of the fibrils within the samples. There is a great intensity difference between the spectra collected at parallel and perpendicular orientations for the amide A, amide B, amide I, and amide II bands. At lower wavenumbers, the amide III band (12001350 cm1) and other contributions possibly arising from amino acid side chains also present dichroism, although of a lower magnitude. Again, the amide A and main amide I bands appear as positive peaks in the difference spectrum (Figure 5). The amide II and the peak appearing at 1690 cm1 in the amide I region present the opposite polarization. As explained before, these observations are consistent with the cross-β structure expected from amyloid fibrils, where the interstrand hydrogen bonds are oriented parallel to the fibrils (amide I and amide A). With the strands perpendicular to the fibril axis, vibrations whose transition dipoles lie parallel to them exhibit negative dichroism (amide II). Antiparallel Strand Conformation. The polarization of the amide I vibrations allows for the characterization of the strand arrangement with high certainty, which in this case is identified as

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antiparallel β-sheets. In the amide I band, two peaks with opposite dichroism in the difference spectrum are visible, at 1627 and 1690 cm1. (See Figure 6.) The high intensity peak at 1627 cm1 corresponds to the ν(π,0) vibration of the amide I,60 where the carbonyl units oscillate out-of-phase in the same strand and in-phase with the units in the adjacent strands, resulting in a strong net transition dipole perpendicular to the strands. This peak appears in both parallel and antiparallel β-sheets and presents the same polarization in both cases, with the resultant transition dipole moment oriented perpendicular to the strands and parallel to the fibril axis.50,60 The high-frequency peak at 1690 cm1, termed ν(0,π), is polarized in the opposite direction.60 The polarization of this band was predicted from theoretical calculations and confirmed by experiments on model systems.61 In an ideal antiparallel β-sheet, this band arises from the oscillators in the unit cell vibrating with 0 phase angle relative to the adjacent oscillators and with a phase angle π relative to the oscillators in the adjacent strand. As a result, the (smaller) components of the transition dipole moments in the direction of the strands add together, whereas the (larger) components in the direction of the fibril axis cancel out. This gives rise to a net transition dipole moment that is smaller than that of the ν(π,0) vibration and with the opposite polarization. Therefore, the dichroism observed in our spectra is in agreement to what would be expected for an antiparallel β-sheet arrangement, in terms of both frequency and polarization of the amide I vibrations.21 Other Possible Assignments. There are other possible structures that could in principle give rise to this high-frequency component of the amide I band, such as turns and β-hairpins. We will briefly consider them now and explain why they can be discounted in this case, partially on the basis of the polarization of the observed modes. As explained in previous sections, turn structures also present components in the amide I high-frequency region. Recently, 2D IR spectroscopy studies and normal mode calculations suggested that the vibrations arising from the turn region of islet amyloid peptide are oriented close to perpendicular to the fibril axis.62 Therefore, in principle, we could not rule out this possibility. However, the short length of the YYKLVFFC peptide makes the formation of a similar long loop implausible. Hairpin structures are accessible to peptides as short as four residues long.55 In β-hairpin structures, the polypeptide chain folds into itself, forming a two-stranded sheet. Both high- and low-frequency components are observed in the amide I region of their IR spectra, with the high components assigned to the turn portion.55 Nevertheless, according to calculations, the transition dipole moment of the high-frequency component is oriented at ∼49 relative to the perpendicular of the strands.63 Thus, taking into account axial symmetry, the expected dichroism for a peak caused by the high-frequency vibration of a turn in a β-hairpin would be slightly positive, and therefore incompatible with our results. The same argument prohibits the assignment of this peak to loops similar to those discussed in the case of lysozyme, where the strands are arranged in a parallel fashion with the carbonyl groups pointing in the same direction, which are expected to give rise to a high-frequency peak with positive dichroism. Therefore, it must be stressed that there are several structures giving rise to similar frequency patterns in the high-frequency amide I region of polypeptides. Without polarization information of the transition dipole moments, accessible through IRLD or 2D IR spectroscopies, assignments made solely on the basis of frequencies can be misleading. Tyrosine Residues. At 1516 cm1, in the amide II region, a distinctive peak caused by a tyrosine vibration is observed. Owing 1814


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Table 1. Dichroic Ratio Measurements, P2 Values, and Calculated Angles with Respect to the Fibril Axis for Several Bands of the Polarized IR Spectra of YYKLVFFC band

)

~ (cm1) ν D /^

ÆP2æ

angleb,c

amide A

amide I

amide I

amide II

tyrosine

aa b

3270 4.11 ( 0.01 4.85 ( 0.05

1627 4.35 ( 0.09 5.40 ( 0.10

1690 0.31 ( 0.05 0.30 ( 0.05

1535 0.32 ( 0.01 0.32 ( 0.01

1516 0.77 ( 0.01 0.79 ( 0.01

c

4.45 ( 0.03

4.71 ( 0.09

0.25 ( 0.03

0.32 ( 0.01

0.81 ( 0.02

a

0.51 ( 0.01

0.53 ( 0.01

0.30 ( 0.03

0.29 ( 0.01

0.09 ( 0.01

b

0.56 ( 0.01

0.60 ( 0.01

0.30 ( 0.03

0.29 ( 0.01

0.08 ( 0.01

c

0.54 ( 0.01

0.55 ( 0.01

0.34 ( 0.01

0.29 ( 0.01

0.07 ( 0.01

a

8 ( 2

0

b

12 ( 3

0

(>81)d

(>82)

60 ( 1

c

8 ( 1

0

62 ( 1 60 ( 1

Values from three different samples, indicated with a letter code, are shown. b Amide I ν(π,0) vibration is assumed to be parallel to the fibril axis. c Angular values left in blank correspond to inconsistent combinations of ÆP2æ values. d Minimum value for the angle between these dipole moments within experimental error. a

to the overlap with the broad and more intense amide II band, it may be difficult to ascertain the polarization of this signal from the difference spectrum alone (Figure 6). The difference spectrum shown in Figure 6 suggests a negative sign at 1516 cm1, although area measurements are required to verify this point and extract meaningful orientation information for these residues, as discussed in the next section. Quantitative IRLD Measurements. Quantitative information about the orientation of some molecular features can be obtained by means of IRLD measurements. We have calculated the order parameter ÆP2æ for the transition dipole moments of the amide A, I, and II vibrations as well as for the tyrosine peak observed at 1516 cm1. To this end, the dichroic ratio R for these bands, that is, the ratio between the absorption intensities at parallel and perpendicular orientations, was measured and related to the order parameter using the expression14,64 ÆP2 ðcos θÞæ ¼

R1 Rþ2

ð1Þ

where θ is the angle between the transition moment and the axis of the combing direction. Because the orientation of the transition dipole moments may not be coincident with the molecular geometry, the Legendre addition theorem can be employed in cases of axial symmetry14,64 ÆPl ðcos βÞæ ¼ Pl ðcos ξÞÆPl ðcos θÞæ

ð2Þ

where Pl(cos ξ) depends on the angle ξ of the transition moment with respect to the fibril axis65 and β is the angle between the fibril axis and the combing direction. The calculated order parameters were used to estimate the average angles the transition dipole moments from each band make with the fibril axis, assuming that the distribution of orientations of the fibrils in the samples is identical to the distribution of orientations of the transition moment of the main β-sheet peak. Because the resultant transition moment for the 1627 cm1 vibration is perpendicular to the strands, this assumption is equivalent to considering that the strands within the sheets forming the fibrils are not significantly tilted with respect to the fibril axis normal. The dichroic ratio values, order parameters, and angles obtained from three aligned samples are detailed in Table 1.

Amide A. The values obtained for the NH angle relative to the fibril axis (average ∼10) suggest a β-sheet structure close to the canonical coordinates. The ideal antiparallel β-sheet geometry allows for linear NH 3 3 3 O hydrogen bonds.66 However, real protein structures often deviate from the ideal geometry. Statistical analysis of protein crystal structures reveals that the angle in β-strands is ∼16067 and ranges from 137 to average close to 180.68 The hydrogen bond distance and therefore hydrogen bond strength is correlated with the angle among the donor, proton, and acceptor.67 We conclude from these data that the fibrils formed by YYKLVFFC are very regular structures, with a strand organization that allows an optimal geometry to maximize the hydrogen bonding interactions. Amide I and II. ν(0,π) vibrations of both amide I and amide II bands (1690 and ∼1535 cm1, respectively) are oriented parallel to the strands; therefore, the order parameters ÆP2æ adopt negative values. For samples a and c, the ÆP2æ values for the bands at 1690 and 1535 cm1 are even more negative than 0.5ÆP2(1627)æ, the value we would expect for transition dipoles exactly perpendicular to the fibril axis (as represented by the transition moment for the 1627 cm1 band). Such cases lead to average angular solutions with complex components, and we could not therefore calculate explicit angular values for these samples. In the case of sample b, the angles for the transition moments of the 1690 and 1535 cm1 vibrations calculated from the average ÆP2æ values are 81 and 82, respectively. (Note that all values in Table 1 are rounded up to two decimal places.) For other values of P2, the angular solutions can, within experimental uncertainty, adopt values up to 90 or be undetermined. Despite the problems associated with the calculation mentioned above, these Figures show that the transition moments of the 1690 and 1535 cm1 vibrations are oriented nearly perpendicular to the fibril axis, in agreement with the theoretical values for antiparallel β-sheets. We note that the order parameters are very similar to each other across samples, even in those cases where a definite angle could not be calculated. We attribute the small differences and inconsistencies to errors associated with the intensity measurements. Indeed, precise measurements of the intensity of these bands are complicated for two different reasons. In the case of the 1690 cm1 peak, its low extinction coefficient69 makes its intensity in the parallel spectrum very low. Errors in baseline handling (Figure 7) can therefore distort significantly the dichroic ratio for this band, translating eventually 1815


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Figure 7. Intensity measurements on the amide I peaks. A linear baseline (16001710 cm1) was subtracted from the whole band, and heights at 1627 cm1 were taken as the intensity for the main β-sheet peak. A cubic spline from 1683 to 1703 cm1 was used as the baseline for the 1690 cm1 ν(0,π) peaks. The baseline limits were chosen such as to obtain a symmetric shape in the subtracted peaks in both the parallel and perpendicular spectra. Top: original spectra. Bottom: 1690 cm1 peak after baseline subtraction.

Figure 8. Amide II region of the IRLD spectra in YYKLVFFC. To measure the intensity of the tyrosine vibration at 1516 cm1 at each polarization geometry, we traced a linear baseline from the edges of this peak.

to an incorrect order parameter: changing the baseline boundaries by four wavenumbers for the 1690 cm1 peak results in up to 20% differences in the peak height values. The other bands, with a higher intensity, are less affected by shifts in the baseline. In these cases, band overlapping is probably the main source of error, affecting each band in a different measure. As an example, the broad and complex shape of the amide II ν(0,π) vibration at 1535 cm1 reveals that more vibrational modes are present in that region, making precise intensity measurements problematic. The uncertainties detailed in Table 1 reflect the errors associated with baseline handling and peak integration, which can be quantified. Tyrosine Residues. The intense and narrow peak at 1516 cm1 (Figure 8) is a characteristic marker in the IR spectra of proteins and polypeptides containing tyrosine. This peak is particularly well suited to study the orientation of tyrosine side chains by IRLD because its orientation is readily interpretable in terms of the orientation of the phenolic ring. It corresponds to a stretching vibration of the Cβ and Cγ atoms (ν(CC)), as shown in Figure 9.70 The resultant transition moment is oriented in the direction of this bond because other atom displacements involved in this vibration

Figure 9. Displacement vectors for the normal mode originating the 1516 cm1 infrared peak. The transition dipole moment is colinear with the axis defined by Cβ and Cγ. Adapted from ref 70.

are symmetric about the plane perpendicular to the tyrosine ring passing through atoms Cγ and Cζ. The dichroism data collected result in negative values for the order parameter (average ÆP2æ = 0.08) and therefore in an average angle for the dipole moments >4.7 away from the fibril 1816


Biomacromolecules axis (the magic angle). The presence of two tyrosine residues in YYKLVFFC limits our analysis; because the measured absorption is the average from both residues, it is impossible to differentiate between their individual orientations. Still, we can rule out some geometries involving both residues oriented in a particular direction. Given the obtained average polar angle of 61 for the CβCγ axes, we can discard the possibility of an orientation of both tyrosine rings parallel to the fibril axis as well as the case where both residues are perfectly perpendicular to it. If only one tyrosine residue was present, then the angular value obtained could be assigned unequivocally, although these data would not suffice to determine completely its orientation: the azimuthal and tilt angles would remain undetermined. Although the axial symmetry of the system makes it impossible to establish the azimuthal orientation, the tilt could, in principle, be determined by using a vibration whose transition moment is perpendicular to the tyrosine ring. We performed a normal modes analysis on a single tyrosine molecule in search for possible vibrations that could be used with this purpose. Calculations were carried out with the Gaussian suite of programs with the help of Dr. Bravo Perez (University of Reading). Unfortunately, there are few normal modes with the required transition moment orientation; their intensity is rather low, and they mostly appear in the fingerprint region of the spectrum, where band overlapping is maximal. Comparison with Previous Results. On the basis of frequency structure correlations it has been previously concluded that the strands forming this peptide are arranged in an antiparallel manner.48 As discussed above, this kind of analysis has its shortcomings because different vibrations may arise in the same region. Specifically, there is an overlap between the region assigned to turns (16621686 cm1) and that assigned to the high-frequency β-sheet peak (16741695 cm1).51 In the present study, we arrived at the same conclusion regarding the arrangement of the β-strands in the fibrils formed by this system. However, the use of polarized spectroscopy on aligned samples provides directional information about the transition moments causing the observed bands, helping to determine the underlying structure and allowing us to make more solid assignments. Complementary information obtained from UV-LD and polarized Raman spectroscopy experiments performed on this system is detailed in a recent publication.29 The average orientation of the tyrosine rings determined here by IRLD, found to be >57 from the fibril axis, as well as the confirmation of cross-β structure by spectroscopic means, is consistent with the conclusions drawn in that study. UV-LD and polarized Raman measurements on aligned samples determined that the carbonyl bonds of the strands are preferentially oriented parallel to the direction of fibril alignment. UV dichroism from transitions associated with the tyrosine residues as well as data derived from the tyrosine Raman scattering showed the ring normals to be aligned perpendicular to the fibril axis. Molecular Combing Performance. The results detailed above demonstrate the use of molecular combing as a useful sample preparation method to induce sample orientation. Focusing on the alignment technique, the most readily accessible parameters that can potentially affect the outcome of the molecular combing experiments are: speed of sample holder, height and tilt angle of air jet, shape and dimensions of air tubing tip, and volumetric flow. Keeping these variables constant, we studied the effect of the concentration of lysozyme amyloid fibrils on the alignment achieved and explored the effect of applying multiple layers of material over the same substrate.

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Effect of Concentration. We performed a comparison of the degree of alignment achieved by molecular combing at different concentrations of lysozyme fibrils. Typical TEM pictures of mature fibrils show a great degree of entanglement (not shown), which is expected to increase with concentration and could be thought to have an impact on the alignment obtained. The initial concentration of the protein solution was not changed for these experiments because the kinetics of selfaggregation and possibly the morphology of the resultant fibrils would be affected.9 Instead, we incubated all lysozyme samples under the same standard conditions, freezethawed to allow the gel to flow,47 and prepared different dilutions from this stock sample of mature fibrils to final concentrations of 12.5, 25, 50, and 75 mg/mL. We relied on the dichroic ratio of the amide I band (β-sheets) to quantify the extent of alignment for each sample. The structural or isotropic absorbance was used to obtain information about the relative film thickness between samples of different concentration, which gives a measure of the amount of material deposited onto the substrate. As shown in Figure 10, the dichroic ratio measurements indicate that in the range studied the alignment increases linearly with the concentration. We interpret these data under the light of the formation of nematic phases observed for this system.47 Polarized optical microscopy revealed the formation of liquid crystal domains in lysozyme fibril solutions (from 9 mg/mL for the full-length fibrils) of an increased content with concentration.47 We suggest that at this concentration range flow alignment of the nematic phase domains is the main working principle of molecular combing for amyloid systems and that other factors such as the nature of the substrate are secondary. The driving mechanism of alignment in other implementations of the molecular combing technique (usually for DNA studies) seemingly depended on the interactions between the solute and the supporting surface. The single instance of a previously reported use of molecular combing to align amyloid-like fibrils that we are aware of, by Herland et al.,45 employed concentrations of ∼5 μM of fibrillated insulin. As a result, in these works, individual aligned molecules could be observed by atomic force microscopy (AFM) or fluorescence microscopy (examples can be found in Chan et al.,71 Herland et al.45 or Nakao et al.41). In the case of DNA, it has been explicitly suggested that DNA molecules anchor to certain modified surfaces because of their high binding specificity for the ends of these molecules.35 In our case, the films deposited onto the CaF2 windows are on the order of hundreds of nanometers thick, as estimated by AFM (results not shown). The width of lysozyme fibrils, as measured by TEM, is 7.8 ( 1.4 nm, implying that a large number of fibrils are deposited on top of each other. As shown in Figure 10, polarized FTIR measurements confirm alignment at every concentration tried, with higher dichroic ratios at higher concentrations. Therefore, we conclude that surfacesolute interactions can play a minor role only in fibril alignment in this case. As further evidence, we demonstrate later in this Article that the same dichroic ratio is obtained on two surfaces that are chemically very different. The formation of liquid crystal phases has been reported in amyloid fibril solutions, such as those prepared from lysozyme47 and YYKLVFFC.48 Corrigan et al. observed that the formation of liquid crystal nematic phases in lysozyme fibrils was lower for freezethawed samples, as expected from their shorter fibril length. They reported noticeable birefringence at lysozyme concentrations above ∼17 mg/mL,47 which is close to the lowest 1817


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Figure 10. Effect of concentration on the degree of alignment of lysozyme amyloid fibrils (a) and on film thickness as measured by the isotropic absorbance of the amide I band (b). The dichroic ratio was calculated for the 1630 cm1 peak in the amide I region. The alignment can be modeled with a linear fit. The absorbance increases with concentration in a seemingly exponential manner.

concentration we studied. According to the Onsanger criterion, nematic phase formation is expected at volume fractions above 4.5D/L, where D and L are the diameter and length of the fibrils.29 We assume that our samples contain a distribution of lengths, whose lower and upper bounds we estimated from TEM micrographs taken on freezethawed lysozyme fibrils.47 Using our TEM width measurements (7.8 nm) in combination with these length values (250 nm to 1.25 μm), we found that nematic phase formation is expected for the longer fibrils above ∼30 mg/ mL. Whereas these figures are only approximate estimates, they indicate that our system may be forming a nematic phase to a certain extent. Recently, a theoretical study on the isotropic nematic phase transition in lysozyme amyloid fibrils predicted the concentration limits for phase separation.72 The specific values from that study are not applicable to our work because it focused on full length fibrils, that is, not shortened by freezethawing. However, the prediction of a linear relation between concentration and nematic volume fraction could potentially explain the increase in alignment we observe with concentration. Figure 10b provides information about the film thickness of the aligned samples at different concentrations. Surprisingly, the amount of material deposited onto the surface appears to increase exponentially with the concentration. In principle, all other parameters being equal, we would expect a simple linear relation between these two variables. However, these results imply that a higher volume of fibril-containing solution is deposited onto the supporting surface as concentration increases. During our experiments, it was observed that at higher concentrations the droplets of solution tend to flow slower than the diluted ones for the same molecular combing experimental parameters. It is possible that the increased time concentrated solutions spend over the surface during a combing run increases significantly solvent evaporation. At any rate, we do not have a quantitative explanation for this behavior, and the exponential fit in Figure 10 should only be understood as a visual aid for the data trend. Multilayer Molecular Combing. The effect that multiple molecular combing runs have on the quality of the alignment was tested empirically for lysozyme fibrils. Molecular combing can be used to prepare aligned samples that can be analyzed with different techniques, such as FTIR and X-ray diffraction. However, the specific sample preparation requirements needed for techniques that rely on different physical principles make this a

Figure 11. Plots of the 1630 cm1 β-sheet dichroic ratio (a) and amide I structural absorbance (b) against the number of layers applied by molecular combing. The concentration was 75 mg/mL, and the fibrils were freezethawed before the combing experiments.

complicated task. As an example, the typical thickness of the samples prepared by molecular combing is insufficient to obtain a good scattering signal with X-ray diffraction techniques. By aligning the samples multiple times over the substrates, we can increase the amount of fibrils present and thus achieve a better signal-to-noise ratio. We prepared samples of lysozyme fibrils with one, two, three, and five layers applied consecutively. As in the previous section, the dichroic ratio of the 1630 cm1 β-sheet peak was used to monitor alignment, and the structural absorbance of the amide I band was used as a measure of the amount of material deposited onto the substrate. The plot of the dichroic ratio (Figure 11 a) shows that the degree of alignment of the fibrils decreases with each new layer applied. The absorbance plot (Figure 11 b) shows the expected linear increase in film thickness with each new molecular combing run. It is worth noting that the first layer deposits more material onto the CaF2 windows than the subsequent ones, indicating some preference of the fibrils for the clean substrate over the already aligned film. The degraded performance of the molecular combing technique as the number of layers increases is explained by the existence of some practical problems associated with our implementation. The first and perhaps the most limiting one is the difficulty to keep the same direction of droplet displacement between runs: although molecular combing is performed in a controlled fashion with the mechanical device described in the Experimental Methods section, the protein films already deposited 1818


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Figure 13. X-ray diffraction pattern of a multilayered YYKLVFFC film onto a silicon window. The drops of fibrils solution were flown in the vertical direction. The visible arcs correspond to the meridional 4.7 Å reflection caused by the spacing of the β-strands.

Figure 12. Top: polarized infrared spectra of a YYKLVFFC amyloid fibrils film aligned by molecular combing onto a silicon wafer. Blue trace: spectrum collected with light polarized parallel to the alignment direction; red: light polarized perpendicular to the director. Bottom: difference spectrum, parallel minus perpendicular. A low-pass filter was applied to the data to minimize distortions caused by interference fringing, although there are still some noticeable artifacts.

)

onto the surface are not perfectly flat, often causing the new droplets to spread sideways. The second problem arises from the narrower central film area left with each layer. The trail of material deposited onto the surface by the droplets of solution normally has a wide central area and two distinctive edges. Usually, the edges of the droplets flow at a lesser pace than the central part, leaving small amounts of solution that evaporate later. When the direction of the droplets is kept correctly, these dried edges tend to accumulate toward the central region of the film, reducing the total number of clean layers that can be applied. In summary, multilayer molecular combing can be useful to increase the amount of aligned material deposited onto a suitable surface. However, there is a compromise to be made between sample alignment and signal-to-noise ratio, which has to be gauged depending on the technique used to perform the measurements. Effect of Substrate. To demonstrate the versatility of this alignment technique, we prepared samples on a different substrate, namely, silicon wafers. This material was chosen for its transmission properties, which potentially enable its use for X-ray as well as FTIR applications. No significant decline in fibril orientation was noticed when using this substrate to produce aligned fibrils of YYKLVFFC. The spectral bands shown in Figure 12 have a similar dichroic ratio to those observed for CaF2 surfaces (D /^ ≈ 4.9 for the amide I band). Unfortunately, the spectra obtained when using silicon surfaces present strong interference fringing artifacts, which obliged us to filter the data, resulting in a degraded spectral quality. In any case, these data further suggest that the mechanism of alignment, under the experimental conditions employed here, does not depend strongly on the interactions between the fibrils and the surface. Therefore, this alignment technique has the potential to be used with a wide range of substrate materials to suit the requirements of the application considered. Wide-angle X-ray diffraction patterns were also obtained from YYKLVFFC aligned onto silicon wafers. To achieve a sufficient signal-to-noise ratio, we applied multiple layers of fibril solution

onto the surfaces. The resultant image (shown in Figure 13) presents a meridional reflection at 4.7 Å, caused by the interstrand spacing within the fibrils. The expected reflection at ∼10 Å is barely visible as a very broad and diffuse ring. The presence of two arcs for the 4.7 Å reflection instead of a full circle shows that the fibrils are preferentially oriented in the intended direction. This is an example where the molecular combing method was employed to produce samples that can be analyzed by two different techniques. Orientation information collected by IR spectroscopy can be used to probe the overall arrangement of different chemical groups by virtue of their specific vibrations, regardless of whether such groups are part of a crystal or other regularly ordered structure. X-ray diffraction can be employed to determine with great accuracy the arrangement of highly regular structures present in the system regardless of the chemical nature of the groups that they contain. As an example, orientation studies on samples such as silk, which contain crystalline and amorphous domains, could possibly benefit from the combination of these two techniques by making use of the molecular combing preparation method. Comparison of the Two Fibril Systems. As our data show, YYKLVFFC fibril samples give spectra showing much greater dichroism than those from lysozyme. This probably reflects the greater ease with which YYKLVFFC fibrils can be aligned by the combing technique. We would expect the extent to which combing aligns different fibril systems to depend on a number of factors, including fibril length, interfibrillar interactions, and the straightness and stiffness of the fibrils. TEM images indicate that YYKLVFFC48 forms straighter fibrils than lysozyme,73 which would cause an increase in degree of orientation.

’ FINAL REMARKS In this Article, we have demonstrated the potential of infrared linear dichroism on oriented films of amyloid fibrils to obtain structural information that cannot be obtained from conventional infrared spectroscopy. The possibility of making assignments based on the polarization as well as the frequency of bands offers information that is invaluable to resolving spectral features that cannot be assigned on the basis of frequency alone, as in conventional spectroscopy on unaligned samples. For example, it can be used to assign the cross-β configuration, to resolve structures such as turns and antiparallel sheet arrangements, and to measure the average angles of specific side chain groups. Our further experiments have investigated the effect of different parameters in the combing procedure. These demonstrate that the molecular combing methodology can be used with different substrates, indicating the potential of its use with a much wider range of techniques and applications. 1819


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’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected].

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Infrared Linear Dichroism Spectroscopy on Amyloid Fibrils Aligned by

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