Article pubs.acs.org/JPCB

Characterization of Intermolecular Structure of β2‑Microglobulin Core Fragments in Amyloid Fibrils by Vacuum-Ultraviolet Circular Dichroism Spectroscopy and Circular Dichroism Theory Koichi Matsuo,*,†,‡ Hirotsugu Hiramatsu,§ Kunihiko Gekko,∥ Hirofumi Namatame,† Masaki Taniguchi,† and Robert W. Woody‡ †

Hiroshima Synchrotron Radiation Center, Hiroshima University, Higashi-Hiroshima 739-0046, Japan Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado 80523, United States § Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai 980-8578, Japan ∥ Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima 739-8526, Japan ‡

S Supporting Information *

ABSTRACT: Intermolecular structures are important factors for understanding the conformational properties of amyloid fibrils. In this study, vacuum-ultraviolet circular dichroism (VUVCD) spectroscopy and circular dichroism (CD) theory were used for characterizing the intermolecular structures of β2-microglobulin (β2m) core fragments in the amyloid fibrils. The VUVCD spectra of β2m20−41, β2m21−31, and β2m21−29 fragments in the amyloid fibrils exhibited characteristic features, but they were affected not only by the backbone conformations but also by the aromatic side-chain conformations. To estimate the contributions of aromatic side-chains to the spectra, the theoretical spectra were calculated from the simulated structures of β2m21−29 amyloid fibrils with various types of β-sheet stacking (parallel or antiparallel) using CD theory. We found that the experimental spectrum of β2m21−29 fibrils is largely affected by aromatic-backbone couplings, which are induced by the interaction between transitions within the aromatic and backbone chromophores, and these couplings are sensitive to the type of stacking among the β-sheets of the fibrils. Further theoretical analyses of simulated structures incorporating mutated aromatic residues suggested that the β2m21−29 fibrils are composed of amyloid accumulations in which the parallel β-sheets stack in an antiparallel manner and that the characteristic Phe−Tyr interactions among the β-sheet stacks affect the aromatic-backbone coupling. These findings indicate that the coupling components, which depend on the characteristic intermolecular structures, induce the spectral differences among three fragments in the amyloid fibrils. These advanced spectral analyses using CD theory provide a useful method for characterizing the intermolecular structures of protein and peptide fragment complexes.

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INTRODUCTION

is crucial for understanding the amyloidogenesis mechanisms, and the conformations and functions of amyloid fibrils.14 β2-microglobulin (β2m) is one of the most typical proteins that form amyloid fibrils. The deposition of β2m amyloid fibrils in vivo causes dialysis-related amyloidosis.11,12 To elucidate the basic amyloidogenesis mechanism of β2m, Hasegawa et al. synthesized six peptide fragments corresponding to the βstrand regions of β 2 m and reported that the β2 m 21−31 (NFLNCYVSGFH) and β2m21−29 (NFLNCYVSG) fragments successfully formed the amyloid fibrils under physiological conditions, suggesting that both peptide fragments are core regions for fibril formation of β2m.11 Recently, Hiramatsu et al.

Amyloid fibrils are highly ordered aggregates of proteins and peptide fragments, and are known to cause pathologic disorders related to more than 20 human diseases.1,2 These fibrils are thought to have some common properties, such as an elongated morphology with a diameter between 5 and 13 nm, an ability to bind dyes like Congo red, and a cross-β structure with some protofilaments composed of the β-sheet layers.3−6 However, their size and toxicity, and the rate of fibril formation often vary depending on the amino-acid7−9 and on the solvent conditions (pH, cosolvents, etc.).10−13 One of the conclusions from these reports is that the characteristics of amyloid fibrils can be largely affected by the differences in intermolecular structures formed by disulfide bonds, hydrophobic interactions, including the π−π interactions, and electrostatic interactions. Thus the structural information related to the intermolecular interactions © 2014 American Chemical Society

Received: September 26, 2013 Revised: February 9, 2014 Published: February 10, 2014 2785

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characterized the amyloid fibrils of β2m21−31 at pH 7.5 and β2m21−29 at pH 8.5 using Fourier transform infrared (FTIR) and Raman spectroscopy, and suggested that both fibrils form parallel β-sheets and contain 55−65% β-strand.15−18 However, the nature of the intermolecular interactions (disulfide bonds, hydrophobic interactions, and electrostatic interactions) between the parallel β-sheets remains unclear because the amyloid fibril conformations are difficult targets for X-ray and NMR analyses, although Raman spectroscopy showed the existence of disulfide bonds between the parallel β-sheets in the β2m21−31 fibrils.16 Circular dichroism (CD) spectroscopy is one of the most useful methods for monitoring and characterizing the structures of proteins in the native state and non-native state, such as amyloid fibrils in aqueous solution, because CD spectra can be measured for proteins at a low concentration under various solvent conditions and are sensitive to the conformations of the peptide backbone (far-UV region) and aromatic side chains (near-UV region) (secondary and tertiary structures).19,20 In the past two decades, two great advances have occurred in protein structural analysis using CD. One is the construction of vacuum-ultraviolet circular dichroism (VUVCD) spectrophotometers, which use synchrotron radiation as a light source.21,22 These instruments allow us to easily obtain the protein CD spectrum in the wavelength region from the near UV to the VUV region (∼160 nm), giving a wide range of structural information, based on the chromophores of proteins.23−26 Second is the development of CD theory using a matrix method in which the CD components of backbone and aromatic side chains of proteins can be separately calculated from the three-dimensional coordinates of the X-ray or NMR structures.27−29 Thus, this theory can be used for evaluating the backbone conformations of proteins obtained from molecular dynamics (MD) simulations, by the comparison of the theoretical CD spectrum calculated from the simulated structures with the experimentally observed one.30 Recently, the matrix method was successfully extended to take account of mixing of n−π* and π−π* transitions with transitions in the deep UV, using ab initio-derived bond polarizability tensors to calculate the contributions of the high-energy transitions.31 Further, the CD of the aromatic side chains obtained by the matrix method can consider the components of the exciton coupling generated by the interactions among aromatic rings.32 Hence, wide-range spectral analysis (near-UV to VUV regions) using VUVCD spectroscopy and CD theory could be a powerful method for characterizing the conformations, not only of the backbones, but also of aromatic side chains, including the intermolecular structures that are formed in amyloid fibrils. In the present study, we measured the CD spectra of three β2m fragments (β2m20−41, β2m21−31, and β2m21−29) in the amyloid fibrils from the near-UV to VUV region (300 to 178 nm) and showed that these fibril spectra are largely affected by the aromatic side-chain conformations. Further, we constructed six structurally different models of β2m21−29 amyloid fibrils and calculated their theoretical CD spectra using CD theory and MD simulations. From the comparisons of these theoretically calculated spectra with the experimentally observed spectrum, we discussed the intermolecular structures of the β2m21−29 amyloid fibrils based on the stacking among β-sheets and the aromatic-ring interactions. This is the first time that CD theory has been applied to the CD spectral analysis of amyloid fibrils in aqueous solution.

Article

MATERIALS AND METHODS

Materials. Three peptide fragments (β2m20−41 [20SNFLNCYVSGFHPSDIEVDLLK41], β2m21−31 [21NFLNCYVSGFH31], and β2m21−29 [21NFLNCYVSG29]) were obtained by solidphase synthesis at the Center for Analytical Instruments, National Institutes for Basic Biology (Okazaki, Japan). These fragments were purified (>99%) by an HPLC method, and a stock solution (2%) in dimethyl sulfoxide (DMSO) was prepared. The molecular weights were verified by mass spectrometry. Sample Preparations. The fibrils of these fragments were prepared as described previously.16,18 β2m20−41 fragments were dissolved in 50 mM citrate buffer at pH 2.5, including 100 mM NaCl. The fibrils were obtained spontaneously by incubation at 37 °C for 10 h. β2m21−31 and β2m21−29 fibrils were prepared in the same way, using 50 mM sodium phosphate buffer at pH 7.5 and pH 8.5, respectively, both with 100 mM NaCl. The absorbance of DMSO significantly complicates the CD measurements in the VUV region. Therefore, to remove the DMSO solvent, these fibril solutions were centrifuged at 14 000 rpm for 50 min, and the pellets were resuspended in each corresponding buffer. The fibril solutions were sonicated with a probe sonicator (VP-5S, Taitec) to near optical transparency, and then they were placed at 37 °C for 10 h again (elongation conditions). The final concentration of fragments was adjusted to 0.1−0.05%. Fibril formation was checked by observing the Thioflavin T fluorescence at 485 nm upon excitation at 455 nm. CD Measurements. The VUVCD spectra of amyloid fibrils in the wavelength region from 260 to 178 nm were measured using the VUVCD spectrophotometer at Hiroshima Synchrotron Radiation Center (HiSOR) and an assembled-type optical cell with 50-μm path length at 25 °C. The details of the spectrophotometer are available elsewhere.22−24 All of the VUVCD spectra were recorded with a 16-s time constant, a 4nm min−1 scan speed, and 9 accumulations. The ellipticity was reproducible within an error of 5%, with this error being mainly attributable to noise and to inaccuracy in the length of the light path. The CD spectrum of β2m21−29 amyloid fibrils from 300 to 240 nm was measured using a conventional CD spectrophotometer (J-720W, Jasco) and the assembled-type optical cell with 100-μm path length at 25 °C. The spectrum was recorded with a 4-s time constant, a 50-nm min−1 scan speed, and 16 accumulations. Initial Structures of β2m21−29 Amyloid Fibrils. The three-dimensional structure of amyloid protofilaments of β2m20−41 fragments has already been determined by a solidstate NMR analysis, in which four β2m20−41 fragments formed a β-strand−loop−β-strand structure and stacked in a parallel and staggered manner.33 In the present study, the β2m21−29 region of this NMR structure (which is one parallel β-sheet composed of four peptide fragments) was adopted as a basic structure of β2m21−29 amyloid fibrils (Figure S1), because recent FTIR analysis suggested that the β2m21−29 fibrils at pH 8.5 are composed of parallel β-sheets.16,18 Further, the Raman spectrum of the β2m21−29 amyloid fibrils exhibited the S−S stretching band, ν(S−S), at 534 cm−1 and no S−H stretching band around 2570 cm−1 (Figure S2), showing that all cysteine residues form disulfide bridges. The presence of the ν(S−S) band agrees with the finding of Hasegawa et al.,11 who suggested the predominance of the dimeric species in the β2m21−29 fibrils. These spectroscopic data imply that the β2m21−29 fragment exists in the dimer linked with disulfide 2786

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Figure 1. Six initial structures of β2m21−29 amyloid fibrils. (a) model 1: two parallel β-sheets stacked in a parallel manner and connected with disulfide bonds between the cysteine side chains. (b) model 2: two parallel β-sheets stacked in an antiparallel manner and connected with disulfide bonds between the cysteine side chains. (c) model 3: three clusters of model 1 lined up so their sheet surfaces stack in a parallel manner. (d) model 4: three clusters of model 1 lined up so their sheet surfaces stack in an antiparallel manner. (e) model 5: three clusters of model 2 lined up so their sheet surfaces stack in a parallel manner. (f) model 6: three clusters of model 2 lined up so their sheet surfaces stack in an antiparallel manner. The short solid lines between cysteine residues indicate the disulfide bonds.

bridges in the fibrils, and two fibril conformations are possible for the fragment, i.e., a parallel dimer in which two fragments orient in the same direction with each other, and an antiparallel dimer in which two fragments orient in the opposite direction. On the basis of these considerations, we constructed six initial structures of β2m21−29 amyloid fibrils (two out of six are composed of two parallel β-sheets and the remaining four contain six parallel β-sheets) as described below. Two parallel βsheets: Two parallel β-sheets were placed so their surfaces with the cysteine side-chain face each other in a parallel or an antiparallel manner at the distance of 5 Å. Then, the two sheets were linked by disulfide bonds through the cysteine side chains of each fragment. The constructed β-sheet pairs are designated as model 1 when facing in the parallel manner and as model 2 when facing in the antiparallel manner. Six parallel β-sheets: Three clusters of model 1 were lined up so their sheet surfaces stack in a parallel (model 3) or an antiparallel (model 4) manner at a distance of 10 Å. In a similar way, three clusters of model 2 were lined up so their sheet surfaces stack in a parallel (model 5) or an antiparallel (model 6) manner. The movements and rotations of the basic structure of β2m21−29 amyloid fibrils were performed with Euler’s formula or rotation matrix, and the disulfide bonds between cysteine side chains were formed on Molstudio software. These six initial structures are shown in Figure 1. Molecular Dynamics Simulation. All MD simulations were carried out for six initial structures of β2m21−29 amyloid fibrils using the GROMACS (Groningen Machine for Chemical

Simulation) package34−36 in conjunction with the OPLS-AA/L all-atom force field.37 The temperature and the pressure during the simulations were maintained at 298 K and 1 bar by coupling to an external heat and an isotropic pressure bath,34 with time constants of 0.1 and 0.5 ps, respectively. The compressibility was set to 4.5 × 105 bar−1 in all box dimensions. Coulomb interactions were treated using the fast particle-mesh Ewald PME method, and nonbonded interactions were generated using a twin-range method.38,39 Cutoffs of both interactions were 1.4 nm and updated every 5 steps. The LINCS algorithm was used to constrain bond lengths.40 The simulation time-step was 1 fs and coordinates were saved for analysis every 2 ps. All simulations were performed on an HPC 3000 computer (HPC Systems, Japan). The six initial structures were placed in the center of a periodic truncated cubic box, which was subsequently filled with simple point-charge water molecules.41 The distances between any atom of peptide fragments and the wall of the periodic box were at least 1.4 nm. The number of water molecules was about 8000 for models 1 and 2, and about 30 000 for models 3 to 6. Sodium and chloride counterions were added to the systems to yield a concentration of 100 mM. All carboxyl and amino termini of the peptide fragments were charged. Prior to the simulations, the potential energy of each system was minimized using a steepest-descent algorithm, followed by a 250-ps MD simulation with position restraints, to relax the water molecules. Unrestrained MD simulations for each system were performed for 20 ns. 2787

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DichroCalc. When the experimental spectrum of β2m20−41 fibrils was compared to the calculated ones from the three methods, the result of program PROTPOL showed the best agreement (Figure S4). These results suggest that the program PROTPOL is useful for proteins and fragments with a large amount of β-strand, although it is inferior to the programs PROTEIN and DichroCalc for proteins in general. The source code of program PROTPOL is available at the Web site http:// home.hiroshima-u.ac.jp/koichi/PROTPOL.html.

Analysis of Simulated Structures. The relative conformational changes and the conformational fluctuations during the unrestrained MD simulations of six models for 20 ns were estimated by the root-mean-square deviation (rmsd) and the root-mean-square fluctuations (rmsf), respectively, of α carbon atoms (Cα) with respect to the energy-minimized structure by the steepest-descent algorithm.42,43 The accurate definitions of the dihedral angle regions (ϕ and ψ) of each secondary structure on the Ramachandran map are difficult, although Hagarman et al. suggested the centers of distributions of βstrand structures: parallel β-strand (−90° > ϕ ≥ −130°, 140° > ψ ≥ 100°), antiparallel β-strand (−130° > ϕ ≥ −180°, 180° > ψ ≥ 100°), and transition region between antiparallel β-strand and polyproline type II (−90° > ϕ ≥ −130°, 180° > ψ ≥ 140°).44,45 However, these distributions are representative and too strict, hence, in this study, we roughly defined the dihedral angle region (−90° > ϕ ≥ −180°, 180° > ψ ≥ 100°) as the βsheet structure, which contains the three types of distributions described above. Calculation of CD Spectra. The computation of CD spectra from the simulated structures was performed using the matrix method taking account of the contributions of polarizabilities, as described in the work of Woody and coworkers.27,28,30−32 This method is implemented in the program PROTPOL, in which the CD spectra of the complete peptide and of two components (backbone and aromatic side chains) are calculated. In the program PROTPOL, n−π*, πo−π*, and π+−π* electronic transitions were used for peptide group, and four ππ* electronic transitions (La, Lb, Ba, and Bb) were considered for Tyr and Phe side chains.28 Although charge-transfer transitions46 were not considered in this study, the program PROTPOL includes contributions of the deep UV region to transitions in the accessible UV by considering the contributions of polarizabilities.31 The parameter set consists of a combination of experimental data and semiempirical MO calculation data using the intermediate neglect of differential overlap method.47,48 Bandwidths used were 10.5 nm for the n−π* transition, 11.3 nm for the πo−π* transition, 7.2 nm for the π+−π* transition, and 12.8 to 7.1 nm for the various aromatic transitions. These bandwidths were calculated from an empirical relationship between bandwidth and transition wavelength.28 The CD spectrum of each simulated structure is the average of calculated spectra of the 50 simulated structures, which were extracted at 400 ps intervals from the whole trajectory (20 ns). The hydrating water molecules around amyloid fibrils were ignored when calculating the CD spectra. The suitability of the program PROTPOL for the CD calculations was evaluated using typical proteins (two α-helixrich proteins and six β-strand-rich proteins) of known structure, and the calculated spectra were compared with those from the programs PROTEIN27,28 and DichroCalc,49 and the experimental spectra (Figure S3). The program PROTPOL worked well with β-strand-rich proteins such as β2m which contains the sequences of β2m20−41, β2m21−31, and β2m21−29, although it did not do well with the α-helix-rich proteins compared to the other two programs. Further, the program PROTPOL was applied to the CD calculations of 25 simulated structures of β2m20−41 fibrils extracted at 400-ps intervals from the whole trajectory (10 ns) by using the NMR conformation as initial structure.33 The fibril spectra were also calculated from the same simulated structures using the programs PROTEIN and

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RESULTS AND DISCUSSION VUVCD Spectra of β2m Fragments in the Amyloid Fibrils. The VUVCD spectra were successfully measured from 260 to 185 nm for β2m20−41 amyloid fibrils and down to 178 nm for β2m21−31 and β2m21−29 amyloid fibrils at 25 °C (Figure 2). The short-wavelength limitations on the VUVCD measure-

Figure 2. VUVCD spectra of β2m fragments in the amyloid fibrils at 25 °C: β2m20−41 at pH 2.5 (black), β2m21−31 at pH 7.5 (red), and β2m21−29 at pH 8.5 (blue). An optical cell with 50-μm path length was used for the measurements from 260 to 178 nm. All of the VUVCD spectra were recorded with a 16-s time constant, a 4-nm min−1 scan speed, and 9 accumulations.

ments depend on the buffer components (the citrate buffer has a larger absorption than the phosphate buffer in the VUV region).21 To minimize the artifact signals on the VUVCD spectra due to the light scattering of samples, the optical cell containing sample solution was placed adjacent to the detector (photomultiplier tube) of the VUVCD instrument. The effects of linear dichroism on the fibril CD spectra are small because the spectral changes due to rotation of the sample cell by 90 degrees were slight (Figure S5). The VUVCD spectra were constant during the data-acquisition period (about 2 h), indicating that the synchrotron radiation at HiSOR (electron energy: 0.7 GeV) did not damage the fibrils. The VUVCD spectrum of β 2 m 20−41 amyloid fibrils [20SNFLNCYVSGFHPSDIEVDLLK41] exhibited a negative peak around 220 nm and a positive peak around 198 nm, which was consistent with the spectrum down to 200 nm reported previously.50 The β2m21−31 amyloid fibrils [21NFLNCYVSGFH31], whose sequence is identical to the N-terminal region of β2m20−41 fragment, showed two negative CD peaks around 220 and 185 nm and a positive CD peak around 202 nm. This spectrum had red-shifted peak positions and depressed peak intensities around 220 and 195 nm, compared to those of β2m20−41 fibrils. The β2m21−29 fibrils [21NFLNCYVSG29] showed peak positions similar to those of the β2m21−31 fibrils, but the peak intensities around 202 and 185 nm were significantly different. Since the peak positions of the β2m20−41, β2m21−31, and β2m21−29 amyloid fibrils were mostly 2788

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Figure 3. Structural parameters obtained from the 20-ns simulations of models 1 and 2. (a) Plots of the Cα rmsd values against the simulation time. (b) Probabilities of β-strand formation for each residue of β2m21−29 fragment during simulations. Black and gray indicate model 1 and model 2, respectively.

Figure 4. Theoretical CD spectra of β2m21−29 amyloid fibrils from 300 to 178 nm calculated from the simulated structures of (a) model 1 and (b) model 2 using the program PROTPOL: total spectrum (black), backbone component (red), and aromatic side-chain component (blue). The green line indicates the experimentally observed spectrum. The insets show the theoretical and experimental spectra in the wavelength region from 300 to 240 nm. All theoretical CD spectra were the average of the calculated spectra of the 50 simulated structures, which were extracted at 400 ps intervals from the whole trajectory (20 ns). The hydrating water molecules around the amyloid fibrils were ignored when calculating the CD spectra.

consistent with those of the component spectrum of β-strand described in our previous paper24 and further FTIR analysis showed that these fibrils commonly formed the parallel β-sheets and contained 55−65% β-strand contents,16,18 we suggest that the β2m20−41, β2m21−31, and β2m21−29 fibrils take similar backbone conformations or secondary structures, although the differences in their CD intensities are significant. The VUVCD spectra are generally affected by the backbone conformation of proteins such as α-helix and β-strand structures. However, particularly in short peptides, the aromatic side chains might give a significant contribution to the CD spectra in the far-UV and VUV regions as well as the near UV, because of the exciton coupling generated by the aromatic− aromatic and/or the backbone-aromatic interactions.32,51 Since β2m20−41, β2m21−31, and β2m21−29 fragments contain three, three, and two aromatic residues, respectively, the different conformations of aromatic side chains may be reflected in the differences among their CD spectra. Further, the F22−V27 region has a high β-sheet-forming propensity associated with aliphatic and aromatic groups,18,52 and the aromatic residues (F22 and Y26) of the β2m21−29 fragment are directly involved in the fibril formation, so the spectral analysis of β2m21−29 fibrils would be useful for understanding the contributions of aromatic residues to the CD. The matrix method of CD theory can be used to calculate the CD contributions of backbone and aromatic side chains of a protein from its three-dimensional coordinates.29 Hence to clarify the contributions of aromatic side chains to the CD of the β2m21−29 amyloid fibrils, we calculated the theoretical CD

spectra (total, and backbone and aromatic side-chain components) from the simulated structures (GROMACS simulation software) of β2m21−29 amyloid fibrils using CD theory (program PROTPOL) and compared them with the experimentally observed CD spectrum. Simulations and CD calculations of β2m21−29 Amyloid Fibrils Composed of Two Parallel β-Sheets. First, we constructed the two simple models of the β2m21−29 amyloid fibrils based on the structural information from NMR, FTIR, and Raman analyses (model 1: two parallel β-sheets stacked in a parallel manner and connected with disulfide bonds; model 2, two parallel β-sheets stacked in an antiparallel manner and connected with disulfide bonds) (see Materials and Methods). The MD simulations of the two models were successfully performed for 20 ns, and the Cα rmsd values are plotted against the simulation time in Figure 3a. Evidently, the values converged within about 0.5 nm for model 1 and within about 0.3 nm for model 2. The rmsd of model 1 fluctuated during the 20 ns simulation time more than that of model 2. The extended simulation to 30 ns of model 1 also showed similar level of fluctuations. This indicates that the structure of model 1 might be different from that of β2m21−29 fibrils because the fibril conformations would be generally steady in solution. Further, the Cα rmsd values reflect the degree of the relative conformational changes of the simulated models with respect to the energy-minimized structure, and hence it is suggested that model 1 (stacked in a parallel manner) would have a different conformation compared with model 2 (stacked in an antiparallel manner). However, the differences would be most 2789

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Figure 5. Structural parameters obtained from the 20 ns simulations of models 3 to 6. (a) Plots of the Cα rmsd values against the simulation time. (b) Probabilities of β-strand formations on each residue of β2m21−29 fragment during simulations. The parameters are described in black for model 3, red for model 4, blue for model 5, and cyan for model 6.

significant in the N- and C-terminal regions of β2m21−29 fragments, judging from the Cα rmsf values of each residue (Figure S6). The probabilities of β-strand formations on each residue of β2m21−29 fragment during simulations were calculated for models 1 and 2, with results shown in Figure 3b. Evidently, the highest probabilities of β-strand structures were observed in the F22−V27 region. In a recent FTIR analysis combined with 13 C isotope-labeling, the F22−V27 region on the β2m21−29 fragment was assigned as the β-strand in the amyloid fibrils, and the remaining residues (N21, S28, and G29) were designated as random coil.18 Thus the results obtained by the simulations are consistent with those of FTIR analysis. The average percentages of β-strands for F22−V27 region were 66.5% for model 1 and 76.5% for model 2, suggesting that model 2 (stacked in an antiparallel manner) has a more stable β-sheet conformation than model 1 (stacked in a parallel manner). From the Cα rmsd values and the probabilities of β-strand formation, we suggest that the simulated structures of β2m21−29 amyloid fibrils are more plausible when the parallel β-sheets stack in the antiparallel manner. This suggestion is supported by the solid-state structure of β2m20−41 amyloid fibrils (NMR)33 and is consistent with the proposed structure of β2m21−31 amyloid fibrils (FTIR)16 because the fibril structures of both these fragments were composed of two parallel β-sheets stacked in an antiparallel manner. To evaluate the simulated structures of models 1 and 2, their theoretical CD spectra (total, and backbone and aromatic sidechain components) from 300 to 178 nm were calculated and are shown in Figure 4. The total CD spectrum of model 1 from 240 to 178 nm exhibited one positive peak around 200 nm and two negative ones around 222 and 188 nm (Figure 4a). Compared to the experimental spectrum, the peak positions were similar but the intensities were significantly different below 210 nm. The total spectrum of model 2 exhibited similar peak positions but with depressed intensities below 210 nm compared with that of model 1 (Figure 4b). The backbone components in both models exhibited similar peak positions (two negative peaks around 222 and 188 nm, and one positive peak around 202 nm) and intensities, while the aromatic sidechain spectra were significantly different from each other (positive peak around 195 nm and negative peak around 185 nm in model 1 and negative peak around 190 nm and positive peak around 182 nm in model 2). These theoretical results suggested that the differences in the total CD spectra of both models could be ascribed to the different contributions of aromatic side chains. However, interestingly, the total spectra of

each model cannot be reproduced by the addition of the spectra of backbone and aromatic side-chain components, showing that the aromatic-backbone couplings that are induced by the interactions between transitions of the aromatic and backbone chromophores are strong within the CD spectrum of amyloid fibrils in the far-UV and VUV regions. The theoretical spectra in the wavelength region from 300 to 240 nm are shown in the insets of Figure 4. The experimentally observed spectrum, which exhibited two negative peaks around 285 and 248 nm and a small positive peak around 240 nm, is also shown in the same insets for comparison. Since this region is generally affected by the aromatic side-chains such as Tyr and Trp, the backbone components showed mostly zero in both models. The total spectrum of model 1 showed two positive peaks around 280 and 245 nm, which are very different from the experimental one (inset of Figure 4a). In model 2, the spectrum, which had one negative peak around 278 nm and one positive peak around 245 nm, is also different from the experimental spectrum (inset of Figure 4b). These differences between theoretical and experimental spectra indicate that the conformations of aromatic side chains were not optimized well in the two simulations, probably because all aromatic side chains in both models were completely exposed to the solvent water, which is a different environment from the real condition of the amyloid fibrils. Hence, MD simulations of β2m21−29 amyloid fibrils considering the environments around aromatic side chains are necessary for obtaining more accurate simulated structures and theoretical spectra. Further, the differences between the total spectrum and the aromatic side-chain component showed that the aromatic-backbone couplings influence the CD spectrum in the near-UV region as well as the far-UV and VUV regions. Simulations and CD Calculations of β2m21−29 Amyloid Fibrils Composed of Six Parallel β-Sheets. We newly constructed four models of the β2m21−29 amyloid fibrils considering the environments around aromatic side chains. In these four models, the sheet surfaces with aromatic side chains (Phe and Tyr) of models 1 or 2 stack on each other in a parallel or antiparallel manner (see Figure 1). The fibril widths of these models are about 6.0 nm, which are within the range of experimental values observed by X-ray diffraction (5−15 nm).3−6,53 The MD simulations of the four models were successfully performed for 20 ns and the Cα rmsd values are plotted against the simulation time in Figure 5a. The converged values were 0.65 nm in model 3, 0.45 nm in model 4, 0.55 nm in model 5, and 0.35 nm in model 6. Models 3 and 5 showed larger rmsd values than models 4 and 6, and hence the models 2790

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Figure 6. Theoretical CD spectra of β2m21−29 amyloid fibrils from 300 to 178 nm calculated from the simulated structures of (a) model 3, (b) model 4, (c) model 5, and (d) model 6 using the program PROTPOL: total spectrum (black), backbone component (red), and aromatic side-chain component (blue). The green line indicates the experimentally observed spectrum. The insets show the theoretical and experimental spectra in the wavelength region from 300 to 240 nm. These spectra were calculated with the same methods as described in the caption of Figure 4.

istic CD depending on the types of the stacking of parallel βsheets (parallel or antiparallel manner) of model 1 and model 2. The backbone components showed similar spectral shapes among the four models, although the CD intensities in models 3 and 5 were slightly depressed compared to those of models 4 and 6. These low intensities in models 3 and 5 might correlate with their decreased β-strand contents as indicated in Figure 5b. Unlike the backbone components, the aromatic side-chain components exhibited diverse spectra among the four models, showing that the differences of total spectra of the four models are mainly due to the conformational differences of aromatic side chains, depending on the type of stacking of parallel βsheets. Further, large aromatic-backbone couplings also exist in these four models because the additivity of backbone and aromatic components for the total spectrum was not observed. These results suggested that the aromatic-backbone coupling calculated here should be very sensitive to the conformations of aromatic side chains among the parallel β-sheets. For the evaluation of the similarities between experimentally observed and theoretically calculated spectra, the comparisons of their CD peak positions, intensities, and signs would be important. Further, the rmsd values between the theoretical and experimental spectra in the wide-range wavelength region would be a quantitative measure of the agreement between theory and experiment. Hence we determine which model structure is most plausible by comparing the spectral characteristics and using the rmsd values between theoretical

whose parallel β-sheets stack in a parallel manner might have larger deviations in conformation from the fibril structures, as with the results obtained in models of two parallel β-sheets. The probabilities of β-strand formation on each residue of β2m21−29 fragments were calculated for the simulations of models 3 to 6 and depicted in Figure 5b. From this figure, evidently the F22−V27 region showed high probabilities for models 3 to 6 as well as the results of models 1 and 2. The average contents of β-strands for the F22−V27 region were 62.4% in model 3, 62.6% in model 4, 65.7% in model 5, and 75.3% in model 6. The β-strand contents of models 3 and 4 (which are based upon model 1) were mostly the same as that of model 1 (66.5%). Model 5 showed a decrement of about 10% for the β-strand content compared to model 2 (76.5%), while model 6 had a similar content with the constituent model. Further, the converged rmsd values of models 4 and 6 were similar to those of model 1 (0.5 nm) and model 2 (0.3 nm), respectively (Figure 3a and 5a). These structural parameters indicate that the stacking of model 1 and model 2 in an antiparallel manner does not largely affect the conformations of amyloid fibrils. These results could be also suggested from the similarities of Cα rmsf values between models 1 and 4 and between models 2 and 6 (Figure S6). Figure 6 shows the theoretical CD spectra (total, and backbone and aromatic side-chain components) from 300 to 178 nm of four models calculated from the respective simulated structures. Evidently, each total spectrum exhibited character2791

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calculated the potential of mean force of interacting aromatic amino acids (Phe−Phe, Phe−Tyr, and Tyr−Tyr) in water using MD simulations and suggested that the stable aromatic interactions were observed when the distances between centroids of the rings were in the range from 3.5 to 5.5 Å, although this distance range became narrower as the angles between the rings increased (e.g., when the angle was 80°, the stable interactions were observed in the range from about 5.5 to 5.0 Å).55 Figure 7 shows the plots of Phe−Tyr, Phe−Phe, and

and experimental CD. The rmsd values between experimental and theoretical total CD spectra were calculated in units of molar ellipticity (103·deg·cm2·dmol−1) and are listed in Table 1. Table 1. The rmsd Values between Experimental and Theoretical CD Spectra in Units of Molar Ellipticity (103· deg·cm2·dmol−1) wavelength region

model 3

model 4

model 5

model 6

240−178 nm 300−240 nm

9.9 1.22

13.9 0.43

16.2 0.66

10.6 0.23

From this table, models 3 and 6, respectively, showed smaller rmsd values of 9.9 and 10.6 in the far-UV and VUV regions (240 to 178 nm), implying that both theoretical spectra are closer to the experimental one compared to the other models (13.9 for model 4 and 16.2 for model 5). None of the available programs (PROTPOL, PROTEIN, and DichroCalc) are demonstrably superior for all of the proteins studied. However, PROTPOL gives the best agreement with β2m, the precursor of the fibrils under study (Figure 3S). In addition, as shown in Figure S4, the theoretical spectrum of β2m20−41 fibrils calculated by PROTPOL gives the best agreement with experiment, with an rmsd of 3.6. This indicates that the difference between an rmsd of 9.9 and 16.2 in Table 1 is statistically significant. The negative peak around 285 nm of the experimentally observed spectrum (insets of Figure 6) was roughly reproduced in the total spectra of models 4, 5, and 6, and the negative peak around 248 nm and the small positive inflection around 240 nm in the observed spectrum were reproduced well only in the spectrum of model 6. Then, as confirmed from the rmsd values in the near-UV region (300 to 240 nm) listed in Table 1 (1.22 in model 3, 0.43 in model 4, 0.66 in model 5, 0.23 in model 6), the theoretical CD of model 6 gives the spectrum closest to the experimental one. These findings allow us to propose that the β2m21−29 fibrils are composed of amyloid accumulations in which the parallel β-sheets stack in an antiparallel manner as described in model 6 (Figure 1). The simulated structures obtained from this model provide the most accurate theoretical CD spectra containing the components of backbone, aromatic side chain, and aromatic-backbone coupling. Aromatic−Aromatic Interactions in β2m21−29 Amyloid Fibrils. From the comparisons among the theoretically calculated total spectra of models 3−6, we suggest that the CD spectrum of β2m21−29 amyloid fibrils is significantly affected by aromatic-backbone coupling, which is very sensitive to the conformations of aromatic side chains among the parallel βsheets, and the simulated structures observed in model 6 (all parallel β-sheets stacked in an antiparallel manner) are the most reasonable conformations that reproduce the spectral characteristics of β2m21−29 amyloid fibrils. Then we could investigate the conformations of aromatic side chains of β2m21−29 fibrils from the simulated structures of model 6. The β2m21−29 fragment has two aromatic residues, Phe and Tyr. Hence, model 6 should have three types of aromatic side-chain interactions between the parallel β-sheets: Phe−Tyr, Phe−Phe, and Tyr−Tyr (see Figure 1). To characterize the side-chain interactions between the parallel β-sheets, we investigated the relationships between angles and distances between aromatic rings during 20 ns simulations. Here, the angles between rings were calculated using the normal vectors obtained from the coordinates of six carbon atoms in the ring, and the distances between rings were estimated using the centroids of the rings.54,55 Chelli et al.

Figure 7. Plots of the angles against the distances between aromatic rings obtained from the simulated structures of model 6; Phe−Tyr (black), Tyr−Tyr (red), and Phe−Phe (blue). The dotted line (green) indicates a distance of 5.5 Å.

Tyr−Tyr angles against their distances observed between the parallel β-sheets. From this figure, evidently distances between Phe−Phe and Tyr−Tyr were more than about 6.5 Å, indicating that both interactions are weak. On the other hand, the distances between Phe−Tyr were mainly concentrated in the range from 3.5 to 5.5 Å. The characteristic distribution of distances and angles of Figure 7 was very similar to those estimated by Chelli et al.,55 showing that the Phe−Tyr interactions should be favorable in the β2m21−29 amyloid fibrils. We searched the Phe and Tyr side-chain interactions from the simulated structures (for example, Figure S7 shows the simulated structures of model 6 at 10 and 20 ns) and found three typical types of interactions as shown in Figure 8. One is the normal stacking (Figure 8a), in which the Tyr side chains of the up and down fragments were located inside of the Phe side chains without Tyr−Tyr interaction. The second is the shifted stacking (Figure 8b), in which the Phe side chains of the up and down fragments were located inside of the two Tyr side-chains without Phe−Phe interaction. The third is the intermediate stacking (Figure 8c), in which the aromatic side chains of the up (or down) fragment were located at the similar positions with those of the down (or up) fragment. Further, we checked the relative populations of the three types of Phe−Tyr interactions and found that the normal stacking was 44% and the shifted stacking was 29%, and the intermediate stacking was 27%. The Phe−Tyr interactions were not observed in the βsheets completely exposed to the solvent, although there were some minor Phe−Phe and Tyr−Tyr interactions between the β-strands (Figure S8). Contributions of Phe−Tyr Interactions to Theoretical CD Spectrum. To confirm the existence of the Phe−Tyr sidechain interactions in the fibrils, we estimated the influences of Phe and Tyr side-chains on the theoretical CD spectrum using the mutation analysis of program PROTPOL, in which the Phe and Tyr residues were changed to Ala in silico. Figure 9 shows the contributions of Phe and Tyr side chains to the theoretical total CD spectrum of β2m21−29 amyloid fibrils in the 240 to 178 2792

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backbone component and F22A spectrum), and hence the differences between total and Y26A spectra should be ascribed as the effects of Phe−Tyr side-chain interactions. In Figure 9b, the negative CD peak around 275 nm of the theoretical total spectrum should be assigned as the peak around 285 nm of the experimentally observed spectrum. The Y26A spectrum did not show any CD around 280 nm because the 260 nm transition of Phe (Lb band) is weak and is forbidden both electrically and magnetically, implying that the contributions of Phe side-chain and Phe-backbone coupling to the CD are negligible in this region. The Tyr aromatic side chain has its Lb band around 280 nm,29 and hence the F22A spectrum has a negative CD band around 280 nm. This CD band becomes substantially more negative in the total spectrum. Since the direct contribution of the Phe side chain to the CD should be zero, the differences between the total and F22A spectra around 280 nm can be ascribed to the effects of Phe−Tyr side-chain interactions. The theoretical CD negative peak around 245 nm should be assigned as the peak around 248 nm of the experimental spectrum. The backbone component was similar to the Y26A spectrum. Further the total spectrum was different from that of F22A. Hence, in addition to the contributions of Phe−Tyr interactions to the CD around 285 nm, the peak around 248 nm also contains the contribution of Phe−Tyr interactions. Our models also consider the contribution of disulfide bonds between cysteine residues, which exhibit CD in the near-UV region. However, the contributions of the disulfide bonds to CD were small, as shown in Figure S9. From the program PROTPOL, we can definitely confirm the effect of Phe−Tyr interactions on the CD of β2m21−29 amyloid fibrils, which supports the existence of the three types of Phe and Tyr interactions observed in the simulated structures of model 6. The aromatic-backbone coupling is sensitive to the conformations of the aromatic side chains. Hence the characteristic conformations formed by the Phe and Tyr interactions should exhibit the unique CD from the near-UV to VUV regions. Further, these characteristic CD depending on the aromatic side-chain conformations would affect the spectral differences among β2m20−41, β2m21−31, and β2m21−29 amyloid fibrils (Figure 2) because the environments around aromatic rings would be significantly different in each fibril conformation because of the different numbers of aromatic residues and lengths of amino-acid sequences.

Figure 8. Three types of Phe−Tyr interactions observed in the simulation of model 6. (a) Tyr side chains of the up and down fragments are located inside of Phe side chains (normal stacking). (b) Phe side chains of the up and down fragments are located inside of Tyr side chains (shifted stacking). (c) Aromatic side chains of the up (or down) fragment were located at positions similar to those of the down (or up) fragment (intermediate stacking). Left panel is the typical conformation obtained from the simulated structures, and right panel is its schematic.

nm (Figure 9a) and in the 300 to 240 nm (Figure 9b). In Figure 9a, the F22A spectrum, in which only the Tyr residue and backbone were considered in the calculation, exhibited a spectrum similar to the backbone component, meaning that the effects of the Tyr side-chain and Tyr-backbone coupling on the CD are negligible, although the Tyr side-chain has absorption bands near 230 and 190 nm (La and B bands).29 The Y26A spectrum, in which only the Phe residue and backbone were considered in the calculation, exhibited blue-shifted peaks around 202 and 188 nm and increased intensities, compared to the total spectrum which included the contributions of Phe and Tyr residues. However, the contribution of Tyr side-chain to the CD should be very small (from the comparisons of

Figure 9. Theoretical CD spectra of mutated β2m21−29 amyloid fibrils (a) from 240 to 178 nm and (b) from 300 to 240 nm calculated from the simulated structures of model 6 using the program PROTPOL: total (black), F22A (red), and Y26A (blue), and backbone component (dotted black line). The green line indicates the experimentally observed spectrum. These spectra were calculated using the same methods as described for Figure 4. 2793

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The Journal of Physical Chemistry B The structures of β2m21−29 amyloid fibrils obtained by the simulations of model 6 (1) maintained a stable β-sheet conformation during the 20 ns simulation, (2) had the same positions of β-strand structures on the sequence level as found in FTIR analysis, (3) exhibited the theoretical spectrum similar to the experimentally observed one in the wavelength range from the near-UV to VUV region, and (4) had the three unique interactions between Phe and Tyr side-chains, which are necessary for explaining the experimentally observed and theoretically calculated spectra. These results allow us to propose that the conformations of β2m21−29 amyloid fibrils are composed of parallel β-sheets stacked in an antiparallel manner, and the Phe−Tyr interactions are formed among the parallel-βsheets.

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REFERENCES

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CONCLUSIONS At present, we can measure the CD spectrum of proteins and peptide fragments from the near-UV to VUV regions using VUVCD (and CD) spectrophotometers. Generally, the CD in the near-UV region is used for the tertiary-structure analysis and that in the far-UV and VUV regions is for the secondarystructure analysis. However, from the conformational analysis of amyloid fibrils in this study, we showed that a wide-range spectral analysis of CD spectrum from the near-UV to VUV regions using VUVCD (and CD) spectroscopy and CD theory can provide structural information not only on the tertiary and secondary structures but also the intermolecular structures between proteins and peptide fragments. This combination analysis should be a powerful tool for characterizing the conformation of amyloid fibrils and of proteins interacting with proteins, peptides, nucleic acids, and sugars, leading to the extension of the usefulness of CD in structural biology. ASSOCIATED CONTENT

S Supporting Information *

Solid-state NMR structure of amyloid fibrils composed of β2m20−41 fragments and the β2m21−29 fragment regions used for the MD simulation in this study (Figure S1), Raman spectrum of the β2m21−29 amyloid fibrils (Figure S2), theoretical CD spectra of eight proteins calculated by the programs PROTPOL, PROTEIN, and DichroCal (Figure S3), theoretical CD spectrum of β2m20−41 amyloid fibrils calculated by the programs PROTPOL, PROTEIN, and DichroCal (Figure S4), spectral changes of β2m21−29 amyloid fibrils depended on the rotation within the face of optical cell (Figure S5), Cα rmsf values of each individual residue with respect to the energyminimized structure in six models (Figure S6), simulated structures of model 6 at 10 and 20 ns (Figure S7), plots of the angles against the distances between aromatic rings observed in the β-sheets completely exposed to the solvent (model 6) (Figure S8), and contributions of the disulfide bonds to the theoretically calculated CD spectrum (Figure S9). These materials are available free of charge via the Internet at http://pubs.acs.org/.

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ACKNOWLEDGMENTS

This work was financially supported by a Research Fellowship of the Japan Society for the Promotion of Science for Young Scientists (Grant No. 19001913 to K.M.) and Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan (No. 22870021 to K.M.). We thank Professor Sergio Abbate for the cooperation of posting the program PROTPOL on the website.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +81-82-424-6293. Fax: +81-82-424-6294. Notes

The authors declare no competing financial interest. 2794

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