Two different packing arrangements of antiparallel polyalanine.

Polyalanine (polyA) sequences are the simplest polypeptide sequence to naturally form antiparallel b sheets, and are a key element in the structure of silk fibers. Remarkably, there are no structures of antiparallel polyA longer than Ala3 that have been determined at atomic resolution. Therefore, we performed a systematic analysis of antiparallel polyA by using X-ray crystallography and solid-state NMR spectroscopy. PolyA packs into two different arrangements, depending on the length of the sequence. Short sequences (n = 6 or less) pack into a rectangular arrangement, as shown by the crystal structure of Ala4 (Figure 1). In contrast, longer sequences pack in a staggered arrangement, for which a structure was derived by using a combination of solid-state NMR spectroscopy and powder diffraction. Polymorphism is emerging as a common feature of amyloid fibers, and the polymorphism that is identified here for polyA demonstrates that amyloid is not unique in this respect. PolyA sequences of different lengths from Ala3 through Ala8 and Ala12 were synthesized and crystallized. As has been shown previously, Ala3 can be crystallized in both parallel and antiparallel b-sheet structures. However, the longer peptides form only antiparallel structures, as indicated by FTIR and C NMR spectra (Figure 2 and Figure S1 in the Supporting Information). Of these peptides, only Ala4 formed single crystals that were large enough for X-ray diffraction experiments. In the structure of Ala4, the molecules are aligned in head-to-tail rows with methyl groups arranged alternately above and below the plane of the sheets (Figure 1). Single water molecules bridge between adjacent N and C termini. The strands are packed into a rectangular lattice and form hydrogen bonds both side-to-side as well as end-to-end. The end-to-end interactions occur through the bridging water molecules. We note that this structure differs from an earlier crystal structure of antiparallel Ala3 [3] and from the standard and widely quoted model for polyalanine b sheets, which was derived from fiber diffraction data. In our structure, all of the Ala residues are in equivalent positions, whereas in the other structures, there are two alternative locations. Evidence from solid-state cross-polarization/magic-angle spinning (CP/MAS) C NMR spectra and X-ray powder diffraction patterns shows that the short, antiparallel polyA oligomers Ala3, Ala5, and Ala6 have similar crystal structures to that of Ala4 (Figure 2). In the C NMR spectra of Ala3, Ala5, and Ala6 there is a single central b-carbon signal at d = 20.4 ppm that is surrounded by smaller resonances. The smaller signals are generated by the carbon atoms at the two termini. In the spectrum of Ala6 there is also a broad signal at d = 17 ppm, which is assigned to disordered residues. The Xray powder diffraction spectra (Figure 2b) have two prominent peaks: one at 2q = 17.28, which is a result of the 5.16 spacing between the Ala planes, and one at 2q = 19.28, which is a result of the 4.62 spacing between adjacent chains, parallel to dimension b of the unit cell. These data demonstrate that the structure of the Ala4 crystal is also maintained in microcrystalline samples. This is an important finding, because it is now clear that amyloid crystals and fibrils can have different morphologies, even for identical sequences, which leads to problems in structural analysis. 11] For Ala7 and higher, both the C NMR spectra and X-ray diffraction data are markedly different from those for short polyA sequences (Figure 2). The C NMR spectra have two Figure 1. Crystal structure of antiparallel Ala4. Bridging water molecules are indicated by circles. In this structure the mean f and y dihedral angles are 1528 and + 1528, respectively. This is a low-energy region of the Ramachandran plot, but slightly above and to the left of the standard antiparallel b-sheet region in the conventional representation. This result is possibly a consequence of the straightness of the chain in this structure, relative to the twisted b sheet that is normally present in proteins.