The pathology of Huntington's disease is characterized by neuronal degeneration and inclusions containing N-terminal fragments of mutant huntingtin (htt). To study htt aggregation, we examined purified htt fragments in vitro, finding globular and protofibrillar intermediates participating in the genesis of mature fibrils. These intermediates were high in ␤-structure. Furthermore, Congo Red, a dye that stains amyloid fibrils, prevented the assembly of mutant htt into mature fibrils, but not the formation of protofibrils. Other proteins capable of forming ordered aggregates, such as amyloid ␤ and ␣-synuclein, form similar intermediates, suggesting that the mechanisms of mutant htt aggregation and possibly htt toxicity may overlap with other neurodegenerative disorders.Huntington's disease (HD) 1 is an inherited, neurodegenerative disorder resulting from an expanded polyglutamine (poly(Q)) region in the N terminus of huntingtin (htt) (1-4). Individuals affected with HD have a poly(Q) stretch of 36 or more glutamines and an age of disease onset inversely correlated with the length of the expanded poly(Q) region (5). HD post-mortem brain examination reveals intranuclear and cytoplasmic neuronal inclusions and other deposits, consisting of fibrillar huntingtin aggregates (6 -9). However, the presence of these large inclusions does not correlate well with neuronal death (10 -14), suggesting that they may not be the toxic species. In vitro, expanded htt forms fibrillar aggregates with morphological and biophysical properties similar to those formed by amyloid ␤ (A␤) peptide (15). The aggregation threshold in vitro mimics the glutamine length threshold for the disease phenotype (16), suggesting that an abnormal conformation of expanded htt plays a role in disease pathogenesis. Recently, Thompson and colleagues (17) have designed polypeptides that bind mutant htt, suppress aggregation in vivo, and reduce pathology in both cell and animal models of poly(Q) disease. It is possible that the pathway from soluble htt to fibrillar aggregate is a multistep process, with the toxic species formed before the mature fiber. To better understand this pathway, we have characterized the morphological and structural features of poly(Q)-mediated htt fibrilization using biochemical and biophysical techniques, including SDS-PAGE, transmission electron microscopy (TEM), Fourier transform infrared (FTIR) spectroscopy, and atomic force microscopy (AFM). EXPERIMENTAL PROCEDURESPlasmid Construction-Plasmids encoding huntingtin exon 1 (htt exon1) proteins with either 16 or 44 consecutive glutamine residues were prepared using a multistep PCR-based and synthetic DNA approach (18). The 5Ј and 3Ј regions of htt exon1 DNA were individually cloned into the Bluescript vector (Stratagene; 5Ј, SalI and XbaI sites; XbaI and NotI sites). The 3Ј htt exon1 cDNA fragment was then inserted into the 5Ј vector (XbaI and NotI sites) to yield a modified httt exon1 cDNA with a 42-base pair linker between the 5Ј and 3Ј regions. To generate the 16Q htt exon1 const...

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Fourier transform infrared spectroscopy is becoming an increasingly important method to study protein secondary structure. The amide I region of the protein infrared spectrum is the widely used region, whereas the amide III region has been comparatively neglected due to its low signal. Since there is no water interference in the amide III region and, more importantly, the different secondary structures of proteins have more resolved differences in their amide III spectra, it is quite promising to use the amide III region to determine protein secondary structure. In our current study, a partial least squares (PLS) method was used to predict protein secondary structures from the protein IR spectra. The IR spectra of aqueous solutions of 16 different proteins of known crystal structure have been recorded, and the amide I, the amide III, and the amide I combined with the amide III region of these proteins were used to set up the calibration set for the PLS algorithm. Our results correlate quite well with the data from X-ray studies, and the prediction from the amide III region is better than that from amide I or combined amide I and amide III regions.

Botulinum neurotoxin (BoNT), which cause the deadly neuroparalytic disease, botulism, is the most toxic substance known to man. BoNT can be used as potential bioterrorism agents, and therefore, pose great threat to national security and public health. Rapid and sensitive detection of BoNTs using molecular and biochemical techniques is an essential component in the diagnosis of botulism, and is yet to be achieved. The most sensitive and widely accepted assay method for BoNTs is mouse bioassay, which takes 4 days to complete. This clearly can not meet the need for clinical diagnosis of botulism, botulinum detection in field conditions, and screening of large scale samples. Consequently, the clinical diagnosis of botulism relies on the clinical symptom development, thus limiting the effectiveness of antitoxin treatment. In response to this critical need, many in vitro methods for BoNT detection are under development. This review is focused on recently developed in vitro detection methods for BoNTs, and emerging new technologies with potential for sensitive and rapid in vitro diagnostics for botulism.

Botulinum neurotoxins type A (BoNT/A), the most toxic substance known to man, is produced by Clostridium botulinum type A as a complex with a group of neurotoxin-associated proteins (NAPs), possibly through a polycistronic expression of a clustered group of genes. The botulinum neurotoxin complex is the only known example of a protein complex where a group of proteins (NAPs) protect another protein (BoNT) against acidity and proteases of the GI tract. We now report that NAPs also potentiate the Zn2+ endopeptidase activity of BoNT/A in both in vitro and in vivo assays against its known intracellular target protein, 25 kDa synaptosomal associated protein (SNAP-25). While BoNT/A exhibited no protease activity prior to reduction with dithiothreitol (DTT), the BoNT/A complex exhibited a high protease activity even in its nonreduced form. Our results suggest that the bacterial production of NAPs along with BoNT is designed for the NAPs to play an accessory role in the neurotoxin function, in contrast to their previously known limited role in protecting the neurotoxin in the GI tract and in the external environment. Structural features of BoNT/A change considerably upon disulfide reduction, as revealed by near-UV circular dichroism spectroscopy. BoNT/A in the reduced form adopts a more flexible structure than in the unreduced form, as also indicated by large differences in DeltaH values (155 vs 248 kJ mol-1) of temperature-induced unfolding of BoNT/A.