Two-Dimensional Infrared Spectra of Isotopically Diluted Amyloid Fibrils from a 40

Two-dimensional infrared spectra of isotopically diluted amyloid fibrils from A␤40 Yung Sam Kim†, Liu Liu‡, Paul H. Axelsen‡§, and Robin M. Hochstrasser†§ †Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104-6323; and ‡Departments of Pharmacology, Biochemistry and Biophysics, and Medicine/Infectious Diseases, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6084 Contributed by Robin M. Hochstrasser, March 26, 2008 (sent for review March 9, 2008) The 2D IR spectra of the amide-I vibrations of amyloid fibrils from ␤ A A 40 were obtained. The matured fibrils formed from strands F A E F 22 D V V 20 21 23 24 having isotopic substitution by 13CA18O at Gly-38, Gly-33, Gly-29, K L 18 19 G G Y H Q 16 17 25 E V H 15 or Ala-21 show vibrational exciton spectra having reduced dimen- 9 10 11 12 13 14 S 26 sionality. Indeed, linear chain excitons of amide units are seen, for N 27 G G V K V M 28 which the interamide vibrational coupling is measured in fibrils V 39 38 37 36 L G 40 35 34 G I I A 30 29 grown from 50% and 5% mixtures of labeled and unlabeled 33 32 31 I V strands. The data prove that the 1D excitons are formed from G A I G V 40 31 32 33 L G 29 30 34 M V G 38 39 parallel in-register sheets. The coupling constants show that for K 35 36 37 28 N each of the indicated residues the amide carbonyls in the chains are 27 S Q H H V E G ؎ separated by 0.5 0.05 nm. The isotope replacement of Gly-25 26 Y K 15 14 13 12 11 10 9 L 16 G V 17 does not reveal linear excitons, consistent with the region of the 25 F F 18 D E A 19 V 22 21 20 strand having a different structure distribution. The vibrational 24 23 frequencies of the amide-I modes, freed from effects of amide vibrational excitation exchange by 5% dilution experiments, point to there being a component of an electric field along the fibril axis B s(n-1) s(n+1) that increases through the sequence Gly-38, Gly-33, Gly-29. The O H O C N C field is dominated by side chains of neighboring residues. N C N H *O sn H O 12 16 : C = O ␤-amyloid 40 ͉ exciton ͉ two-dimensional infrared spectroscopy C O H O C N C N C N myloid fibrils are found in the brain tissue of persons with C 13 18 H *O H : C = O (s+1)n *O AAlzheimer’s disease, where they accumulate as plaques fibril axis surrounded by regions of neuronal death. Although it remains O H O unclear whether fibril formation is a cause of the neuronal loss C N C in Alzheimer’s disease, or a byproduct of another neurotoxic N C N H *O H process, the formation and structure of amyloid fibrils is of (s+2)n intense interest. Fibrils are composed primarily of ␤-amyloid (A␤) proteins Fig. 1. Diagrams of A␤40 fibrils. (A) A cross section of two laterally displaced that range in length from 39 to 42 residues. They diffract x-rays molecular layers of the A␤40 fibril according to Petkova et al. (6). Residues with a characteristic ‘‘cross-␤’’ pattern (1–3), indicating that the 9–40 are shown. (B) Idealized structure of a portion of the parallel ␤-sheet. polypeptide strands lie transverse to the fibril axis, with an The strands are indicated by s, and the residue number in a given strand is 13 A18 interstrand spacing that is typical of a ␤-sheet. Solid-state NMR indicated by n. The asterisk denotes the carbonyl group of a C O doubly labeled amide group. Dotted lines represent hydrogen bonds. (4) and EPR (5) studies have shown that the ␤-sheet configu- ration is parallel and in register. Based on information from NMR, supplemented with information from atomic force mi- to investigate excitations in solids by means of isotope dilution croscopy, electron microscopy, and cross-linking studies, Tycko or replacement (21) should be useful in characterizing amyloid and coworkers (6, 7) have developed detailed models of the fibrils. This approach is in contrast to isotope editing of smaller fibrils formed by 40-residue (A␤40) proteins. These models molecules where the goal is to isolate the spectra of individual feature parallel in-register ␤-sheets involving residues 9–24 and group vibrations. Amide-I IR spectra of parallel ␤-sheets are 30–40 and a loop region, 23–29, that can vary with fibrillization dominated by motions in backbone CAO groups, but they are conditions. A monofilament is formed by folding of each strongly influenced by intrastrand coupling and, to a lesser polypeptide chain such that the two sheets are apposed to each extent, interstrand coupling (14, 22–25). The coupling between the two apposing ␤-sheets of the fibril is expected to be relatively other, and two such monofilaments comprise the fibril (Fig. 1A). ␤ There is significant plasticity in the structural features of amyloid weak, rendering excitons in a -sheet to be effectively delocal- fibrils (8), and alternative models have been proposed (9). ized in only two dimensions. Their spatial extent is limited by site energy fluctuations and static imperfections (26–29). In the current work the structure and internal dynamics of amyloid fibrils are examined by 2D IR spectroscopy (10–12). There has been a considerable amount of experimental work Author contributions: P.H.A. and R.M.H. designed research; Y.S.K. and L.L. performed relating the 2D IR spectra of small peptide aggregates to their research; Y.S.K. analyzed data; and Y.S.K., P.H.A., and R.M.H. wrote the paper. structure (13–17), and the recent experimental and theoretical The authors declare no conflict of interest. applications of 2D IR have involved macromolecular systems §To whom correspondence may be addressed. E-mail: [email protected] or such as folding proteins (18) and fibrils (19, 20). [email protected] Vibrational states within fibrils would be expected to resemble This article contains supporting information online at www.pnas.org/cgi/content/full/ those of molecular crystals more so than small molecules or 0802993105/DCSupplemental. globular proteins. Accordingly, ideas introduced some time ago © 2008 by The National Academy of Sciences of the USA 7720–7725 ͉ PNAS ͉ June 3, 2008 ͉ vol. 105 ͉ no. 22 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0802993105 Downloaded by guest on October 1, 2021 30 ACG38* B G38* G38* 15 (mOD) absorb. 0 10 10 1680 6 days 12 days 19 days 10 6 6 5 ) 1640 -1 2 2 (cm 0 τ 1600 x4 -2 x4 -2 x4 ω 1600 1600 1600 1590 -6 1590 -6 1590 -5 1560 1580 1580 1580 1560 1580 1600 1560 1580 1600 1560 1580 1600 -10 -10 -10 30 DFG38*/G38 E G38*/G38 G38*/G38 15 (mOD) absorb. 0 10 10 1680 6 days 12 days 19 days 10 6 6 5 ) 1640 -1 2 2 (cm 0 τ 1600 x4 -2 x4 -2 x4 ω 1600 1600 1600 -5 1590 -6 1590 -6 1590 1560 1580 1580 1580 1560 1580 1600 1560 1580 1600 1560 1580 1600 -10 -10 -10 1560 1600 1640 1680 1560 1600 1640 1680 1560 1600 1640 1680 -1 -1 ωt (cm-1) ωt (cm ) ωt (cm ) Fig. 2. Linear and 2D IR spectra of A␤40 doubly labeled with 13CA18O at Gly-38 (G38*) and a 1:1 mixture of G38* and unlabeled A␤40 (G38*/G38) at different maturation times. (A–C) Linear IR spectra and 2D IR spectra at T ϭ 0 of G38* at maturation times of 6 days (A), 12 days (B), and 19 days (C). (D–F) Linear IR spectra and 2D IR spectra at T ϭ 0 of G38*/G38 at maturation times of 6 days (D), 12 days (E), and 19 days (F). The Inset in each 2D spectrum is an enlarged and four-times-intensified view of the area enclosed by the outlined region. The dotted circles in the linear spectra of A–C highlight the isotope-labeled amide-I transition regions. The magnitude of each 2D spectrum was scaled to have the same difference of maximum and minimum values. BIOPHYSICS The vibrational excitations can be radically altered when the information (SI) Figs. S1 and S2.] At pD 7.4, the fibrils that atoms of an amide unit in an extended system are isotopically formed were short and curvilinear. Fibrils formed at pD 7.4 also replaced. The energy shifts can result in the effective isolation of exhibited an additional absorbance band, attributed to amino arrays having reduced dimensionality (21). When an amyloid acid side chains, that overlapped with that of 13CA18O. This fibril is formed from A␤40 proteins in which one of the residues absorbance could be recorded in a background spectrum and involved in a ␤-sheet has a single 13CA18O substitution, the subtracted from the spectra of labeled proteins. However, its substituted groups form a linear array along the fibril axis (Fig. intensity was negligible at pD 2.0, and such manipulations were 1B). The substituted groups along this dimension have strong obviated. resonance coupling, whereas the interactions with unsubstituted FTIR and 2D IR spectra of fibrils formed from A␤40 labeled groups are reduced by the ratio of the coupling magnitude to the with 13CA18O at Gly-38 (G38*), and from a 1:1 mixture of isotope shift.

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