Structure of polyglutamine

At least eight diseases (Huntington's disease (HD), spinobulbar muscular atrophy (SBMA or Kennedy's disease), dentatorubral-pallidoluysian atrophy (DRPLA), and spinocerebellar ataxias (SCAs) types 1, 2, 3, 6, and 7) are now known to be associated with a polyglutamine stretch which is greatly expanded in a¡ected individuals [1,2]. While the function of only two of these genes is known (the gene for SBMA is the androgen receptor, and the gene for SCA6 is a sodium channel), it is clear from the DNA and protein sequences that the genes are diverse and unrelated. Thus, an expanded polyglutamine stretch itself may somehow play a role in disease etiology. The study of the structure of polyglutamine has a long history. Apparently the ¢rst to study the structure of polyglutamine were Krull et al. in 1965 [3]. Using infrared, optical rotary dispersion and X-ray di¡raction techniques they found polyglutamine to be in a L-sheet conformation. In the abstract of their paper they note, ``These observations indicate that side-chain amides may associate with peptide amides in the same or other molecules through hydrogen bonding T''. They pointed out that their own molecular models, as well as those they had received in a personal communication from Elkan Blout, founding editor of Biopolymers, indicated that polyglutamine was a highly aggregating molecule in a L-sheet conformation. That polyglutamine forms aggregates and is in a L-sheet conformation was independently rediscovered by Max Perutz and colleagues [4]. They found the peptide Asp2Gln15Lys2 to be highly aggregating with a large circular dichroism (CD) peak at 197 nm. The structure of polyglutamine as a monomer, however, was still not known. We synthesized a peptide Lys2Gln15Lys2 [5]. This peptide was readily soluble in water at pH 7, and we got a similar CD spectra to that of Perutz. However, ultracentrifugation analysis showed that this peptide also formed large aggregates. We found that Robert Baldwin had made a monomeric short basic peptide Ac-(Ala4Lys)3Ala-NH2 which is 80% helical in aqueous solution [6]. We thought that by £anking a polyglutamine stretch by such peptides we might be able to get a water-soluble monomeric peptide. We synthesized the peptide Ac-(Ala4Lys)3Gln9LysAla4LysAla-NH2. Ultracentrifugation analysis showed that this peptide was monomeric. The CD spectra showed the peptide to be 59% random coil, and 41% K-helix. We synthesized another peptide AcTyrGlyAla2LysAla4LysGln17LysAla4LysAla-NH2. This peptide was also found to be a monomer, with some nascent aggregate formation. The CD spectra showed 67% random coil, 26% K-helix, and 7% L-sheet. As the percentage of random coil in the peptides increased with a larger number of glutamines, and as the alanine/lysine containing segment of our peptides were nearly identical to Baldwin's helical peptide, we concluded that polyglutamine was in a random coil conformation [5]. Furthermore, in previous work with poly[N5-(hydroxyalkyl-glutamine)] compounds it was found that poly-[N5-(hydroxybutyl-glutamine)] was strongly helical in aqueous solution, poly-[N5-(hydroxypropyl-glutamine)] mixed helix and coil, and poly-[N5-(hydroxyethyl-glutamine)] all coil [7,8]. Thus, we might expect polyglutamine itself to be in a random coil, rather than helical conformation. From recent modeling of a wheat protein containing a number of short polyglutamine stretches, Masci et al. propose that these polyglutamine stretches will be ``unordered and £exible'' [9]. Also, they note that glutamine residues being in a £exible con¢guration is supported by chain £exibility predictions of Karplus and Schulz [10]. Recently Sharma et al. [11] have studied peptides containing polyglutamine stretches. In their abstract, they suggest ``that all the polyglutamine peptides with and without interruptions(s) adopt a L-structure and not random coil''. Sharma et al. have studied four peptides Gln22, Gln8HisGlnHisGln8, Gln10HisGlnHisGln10, and Gln8HisGln4HisGln8. For Gln22 they found a spectrum qualitatively similar to the one found by Perutz. Their peak, however, is at 202 nm, rather than at 197 nm as in Perutz' spectrum. They note that the histidine containing compounds are more soluble in aqueous solution than pure polyglutamine. They suggest that histidine plays a `major role' in this solubility. However, we had already shown that a peptide, Lys2Gln15Lys2, with more consecutive glutamines than any of the peptides of Sharma et al., is soluble in aqueous solution at pH 7. Based on a blue-shifted positive CD band in the 193^195 nm range, Sharma et al. conclude that their Gln/His containing peptides are in intramolecular L-sheet conformation. However, details of CD spectra can be notoriously di¤cult to interpret. The suggestion that a peptide adopts an intramolecular L-sheet conformation requires demonstration that the peptide is a monomer. Sharma et al. suggest that histidine interruptions of polyglutamine stretches in the SCA1 protein promote protein solubility, and that loss of histidines in mutant proteins leads to protein aggregation and disease. However, all SCA1 patients have both a loss of histidines and an extended polyglutamine stretch [12], which does not prove the suggestion of Sharma et al. As all evidence required to show that the peptides studied by Sharma et al. are intramolecular L-sheets has not been given, we stand by our demonstration that polyglutamine stretches of moderate length, e.g. nine glutamines, are in a random coil conformation. Even if peptides containing polyglutamine and histidine are in intramolecular L-sheets, this does not imply that moderate length stretches of polyglutamine itself are in a L-sheet conformation. In summary, to support their claim that their peptides form intramolecular L-sheets, Sharma et al. would have to demonstrate that their peptides are monomers. Even if they did so, this does not imply that moderate length polyglutamine stretches are in a L-sheet conformation. Future work using NMR or other techniques may be able to study the structure of polyglutamine in more detail using our monomeric polyglutamine containing peptides [5].