Published on Web 11/15/2010 Intermolecular Alignment in 2-Microglobulin Amyloid Fibrils Galia T. Debelouchina,† Geoffrey W. Platt,‡ Marvin J. Bayro,† Sheena E. Radford,‡ and Robert G. Grifﬁn*,† Department of Chemistry and Francis Bitter Magnet Laboratory, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States, and Astbury Centre for Structural Molecular Biology and Institute of Molecular and Cellular Biology, UniVersity of Leeds, Leeds LS2 9JT, U.K. Received September 4, 2010; E-mail: rgg@mit.edu Abstract: The deposition of amyloid-like ﬁbrils, composed pri- marily of the 99-residue protein 2-microglobulin ( 2m), is one of the characteristic symptoms of dialysis-related amyloidosis. Fibrils formed in vitro at low pH and low salt concentration share many properties with the disease related ﬁbrils and have been exten- sively studied by a number of biochemical and biophysical methods. These ﬁbrils contain a signiﬁcant -sheet core and have a complex cryoEM electron density proﬁle. Here, we investigate the intrasheet arrangement of the ﬁbrils by means of 15N-13C MAS NMR correlation spectroscopy. We utilize a ﬁbril sample grown from a 50:50 mixture of 15N,12C- and 14N,13C-labeled 2m monomers, the latter prepared using 2-13C glycerol as the carbon source. Together with the use of ZF-TEDOR mixing, this sample allowed us to observe intermolecular 15N-13C backbone-to- backbone contacts with excellent resolution and good sensitivity. The results are consistent with a parallel, in-register arrangement of the protein subunits in the ﬁbrils and suggest that a signiﬁcant structural reorganization occurs from the native to the ﬁbril state. 2-Microglobulin ( 2m) is a 99-residue protein that forms amyloid ﬁbril deposits associated with dialysis-related amyloidosis (DRA).1 Under acidic conditions (pH ) 2.5) and low salt concentration, the protein can also form amyloid ﬁbrils in Vitro through a nucleation-dependent mechanism.2,3 These ﬁbrils are long, straight, and unbranched in appearance (Figure S1) and share many properties with the ﬁbrils isolated from tissues of DRA patients, including the same characteristic amide I′ band in FTIR spectra.4 It has been shown that the ﬁbrils themselves, and not the preﬁbrillar oligomeric species formed in the lag phase of assembly, can disrupt model membranes and are toxic to cells.5 While an atomic structural model for these ﬁbrils is not yet available, structural details emerged ﬁrst through methods like limited proteolysis,6,7 hydrogen exchange,8,9 and more recently by magic angle spinning (MAS) NMR,10 electron paramagnetic resonance (EPR),11 and cryo-electron microscopy (cryoEM).12 In particular, analysis of the chemical shifts of 64 assigned residues of 2m ﬁbrils has shown that the protein contains a rigid ﬁbril core with substantially more -sheet character than the native protein.10 CryoEM maps revealed a complex picture of the ﬁbrils, where non-native globular 2m monomers pack in “dimer-of-dimers” building blocks that associate asymmetrically into crescent-shaped units.12 In addi- tion, site-directed EPR spin labeling suggested that the major building block consists of six 2m polypeptide chains, arranged in a parallel, in-register manner.11 In the experiments described here, we investigate the tertiary structure of 2m amyloid ﬁbrils with 15N-13C MAS NMR correla- † Francis Bitter Magnet Laboratory and Massachusetts Institute of Technology. ‡ Astbury Centre for Structural Molecular Biology and University of Leeds. 10.1021/ja107987f  2010 American Chemical Society Figure 1. (a) 13C CP spectrum of mixed 2- 2m ﬁbrils, 512 scans; (b) ZF- TEDOR spectrum obtained with τmix ) 1.76 ms, 512 scans;, (c) ZF-TEDOR with τmix ) 18 ms, 5120 scans. tion spectroscopy. MAS NMR has been successfully used to obtain information about the inter- and intramolecular interactions that form the -sheet core of amyloid ﬁbrils, including A (1-40),13 a 22-residue fragment of 2m,14 Het-s(218-289),15 and curli amy- loid.16 Various sample preparation techniques and experiments have been employed to achieve that end, including methods that rely on the incorporation of single labels17,18 or proton-mediated transfer.19 Here, we use ZF-TEDOR (z-ﬁltered transferred echo double resonance) mixing20,21 to obtain intermolecular 15N-13C correla- tions that establish that the protein subunits in long, straight 2m ﬁbrils formed at pH 2.5 are arranged as parallel, in-register -sheets. Our experiments utilize ﬁbrils formed from a 50:50 mixture of 15N,12C- and 14N,13C- labeled 2m monomers, the latter half being prepared using [2-13C]-glycerol as the carbon source. This sample, referred to as “mixed 2- 2m”, offers improved resolution in the 13C dimension22-24 (Figure 1a) as well as potential gains in experimental transfer efﬁciency due to the signiﬁcantly reduced number of directly bonded 13C atoms.25 The absence of 13C J-couplings and the elimination of strong (intramolecular) dipolar 15N-13C couplings as a result of the mixed nature of the sample improve the efﬁciency of ZF-TEDOR.20,26,27 In a 100% uniformly 15N,13C labeled 2m sample, the experi- mental one-bond 15N-13C transfer efﬁciency after 1.76 ms of ZF- TEDOR mixing is typically ∼20% of the 13C CP signal. In the mixed 2- 2m sample, after such a short mixing time, no signiﬁcant buildup of 13C polarization is observed, as shown in Figure 1b. This is due to the absence of 13C nuclei in the 15N,12C-labeled monomers, which were prepared using 13C-depleted glucose (99.9% purity) to eliminate contributions from natural abundance. In particular, signals from one-bond 15N-13C interactions are not detected. On the other hand, longer ZF-TEDOR mixing times lead to the buildup of 13C intensity, which reaches a maximum at 18 ms (Figure 1c and Figure S2) and is consistent with 15N-13C J. AM. CHEM. SOC. 2010, 132, 17077–17079 9 17077