Keith A. Oberg

Tau filaments from human brain and from in vitro assembly of recombinant protein show cross-β structure

Abnormal filaments consisting of hyperphosphorylated microtubule-associated protein tau form in the brains of patients with Alzheimers disease, Downs syndrome, and various dementing tauopathies. In Alzheimers disease and Downs syndrome, the filaments have two characteristic morphologies referred to as paired helical and straight filaments, whereas in tauopathies, there is a wider range of morphologies. There has been controversy in the literature concerning the internal molecular fine structure of these filaments, with arguments for and against the cross-β structure demonstrated in many other amyloid fibers. The difficulty is to produce from brain pure preparations of filaments for analysis. One approach to avoid the need for a pure preparation is to use selected area electron diffraction from small groups of filaments of defined morphology. Alternatively, it is possible to assemble filaments in vitro from expressed tau protein to produce a homogeneous specimen suitable for analysis by electron diffraction, x-ray diffraction, and Fourier transform infrared spectroscopy. Using both these approaches, we show here that native filaments from brain and filaments assembled in vitro from expressed tau protein have a clear cross-β structure.

Distinct β-Sheet Structure in Protein Aggregates Determined by ATR–FTIR Spectroscopy

Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) was used to study the conformation of aggregated proteins in vivo and in vitro. Several different protein aggregates, including amyloid fibrils from several peptides and polypeptides, inclusion bodies, folding aggregates, soluble oligomers, and protein extracts from stressed cells, were examined in this study. All protein aggregates demonstrate a characteristic new β structure with lower-frequency band positions. All protein aggregates acquire this new β band following the aggregation process involving intermolecular interactions. The β sheets in some proteins arise from regions of the polypeptide that are helical or non β in the native conformation. For a given protein, all types of the aggregates (e.g., inclusion bodies, folding aggregates, and thermal aggregates) showed similar spectra, indicating that they arose from a common partially folded species. All of the aggregates have some nativelike secondary structure and nonperiodic structure as well as the specific new β structure. The new β could be most likely attributed to stronger hydrogen bonds in the intermolecular β-sheet structure present in the protein aggregates.

Methods for collecting and analyzing attenuated total reflectance FTIR spectra of proteins in solution

Publisher Summary Attenuated Total Reflectance FTIR (ATR-FTIR) is a method that has been applied for the study of protein conformation. This chapter demonstrates that ATR-FTIR spectra of proteins in solution can be collected and used successfully for secondary structural analysis. ATR has been used for monitoring adsorption of proteins or blood components to surfaces, and for the structural analysis of proteins dried onto an IRE (thin fihn). It has also been used for exploring the effects of solution conditions on the structure of proteins irreversibly adsorbed to an IRE, and has been shown to be useful for studying the secondary structure and ligand binding properties of membrane proteins. Structural analysis using both curve-fitting and multivariate statistical methods can be employed to ATR-FTIR spectra. However, because disordered and helix bands may not be well resolved, band assignments in curve-fitting analysis can be difficult. Partial least-squares analysis (PLS) analysis, on the other hand, can determine the secondary structure of proteins from H 2 O ATR-FTIR spectra with higher accuracy, because it is not necessary to assign bands using this method.