A conformational isoform of the mammalian prion protein (PrP Sc ) is the sole component of the infectious pathogen that causes the prion diseases. We have obtained X-ray fiber diffraction patterns from infectious prions that show cross-␤ diffraction: meridional intensity at 4.8 Å resolution, indicating the presence of ␤ strands running approximately at right angles to the filament axis and characteristic of amyloid structure. Some of the patterns also indicated the presence of a repeating unit along the fiber axis, corresponding to four ␤-strands. We found that recombinant (rec) PrP amyloid differs substantially from highly infectious brainderived prions, both in structure as demonstrated by the diffraction data, and in heterogeneity as shown by electron microscopy. In addition to the strong 4.8 Å meridional reflection, the recPrP amyloid diffraction is characterized by strong equatorial intensity at approximately 10.5 Å, absent from brain-derived prions, and indicating the presence of stacked ␤-sheets. Synthetic prions recovered from transgenic mice inoculated with recPrP amyloid displayed structural characteristics and homogeneity similar to those of naturally occurring prions. The relationship between the structural differences and prion infectivity is uncertain, but might be explained by any of several hypotheses: only a minority of recPrP amyloid possesses a replication-competent conformation, the majority of recPrP amyloid has to undergo a conformational maturation to acquire replication competency, or inhibitory forms of recPrP amyloid interfere with replication during the initial transmission.amyloid ͉ protein ͉ neurodegeneration ͉ PrP ͉ ␤-helix

Tauopathies are sporadic and genetic neurodegenerative diseases characterized by aggregation of the microtubule-associated protein Tau. Tau pathology occurs in over 20 phenotypically distinct neurodegenerative diseases, including Alzheimer disease and frontotemporal dementia. The molecular basis of this diversity among sporadic tauopathies is unknown, but distinct fibrillar wild-type (WT) Tau conformations could play a role. Using Fourier transform infrared spectroscopy, circular dichroism, and electron microscopy, we show that WT Tau fibrils and P301L/V337M Tau fibrils have distinct secondary structures, fragilities, and morphologies. Furthermore, P301L/ V337M fibrillar seeds induce WT Tau monomer to form a novel fibrillar conformation, termed WT*, that is maintained over multiple seeding reactions. WT* has secondary structure, fragility, and morphology that are similar to P301L/V337M fibrils and distinct from WT fibrils. WT Tau is thus capable of conformational diversity that arises via templated conformation change, as has been described for amyloid ␤, ␤ 2 -microglobulin, and prion proteins.Tau filament deposition in Alzheimer disease, frontotemporal dementia, and other tauopathies correlates closely with cognitive dysfunction and cell death (1). About 10% of tauopathies are due to dominant mutations in the Tau gene. These diseases are collectively termed frontotemporal dementia with parkinsonism linked to chromosome 17, FTDP-17 (2-4). Most of the mutations occur in the microtubule-binding region of the Tau protein, which is thought to be both its functional (5) and pathogenic (6) "core." Approximately 90% of tauopathies occur sporadically and involve only wild-type (WT) 2 Tau. Both familial and sporadic tauopathies vary by regional involvement, disease duration, age of onset, Tau isoform expression, and fibril morphology (7). It is unknown how the pathology of WT Tau might generate distinct disease phenotypes in sporadic tauopathies, and whether conformational diversity of the protein could potentially play a role in disease, as it does in prion disorders (8,9).Mutations in the Tau gene can generate conformationally distinct Tau species. Structural differences between in vitro prepared WT, G272V, N279K, P301L, V337M, and ⌬K280 Tau fibrils have been observed using Fourier transform infrared spectroscopy (FTIR) (10), and differential susceptibilities to protease cleavage in vitro have been described for WT and P301L Tau fibrils (11). Furthermore, Tau filaments extracted from diseased brain are often morphologically distinct, consisting of straight or paired helical filaments of various periodicities and widths (12). It is unknown whether WT Tau can assume self-propagating, structurally distinct fibrillar conformations, as has been described for amyloid ␤ peptide (13), ␤ 2 -microglobulin (14), and the prion protein (15). In this study, we have used biochemical and biophysical methods to test the hypothesis that WT Tau fibrils exhibit conformational diversity that is maintained by templated conformation change. ...

Fusion of biological membranes is mediated by distinct integral membrane proteins, e.g., soluble N-ethylmaleimide-sensitive factor attachment protein receptors and viral fusion proteins. Previous work has indicated that the transmembrane segments (TMSs) of such integral membrane proteins play an important role in fusion. Furthermore, peptide mimics of the transmembrane part can drive the fusion of liposomes, and evidence had been obtained that fusogenicity depends on their conformational flexibility. To test this hypothesis, we present a series of unnatural TMSs that were designed de novo based on the structural properties of hydrophobic residues. We find that the fusogenicity of these peptides depends on the ratio of ␣-helix-promoting Leu and ␤-sheet-promoting Val residues and is enhanced by helix-destabilizing Pro and Gly residues within their hydrophobic cores. The ability of these peptides to refold from an ␣-helical state to a ␤-sheet conformation and backwards was determined under different conditions. Membrane fusogenic peptides with mixed Leu͞ Val sequences tend to switch more readily between different conformations than a nonfusogenic peptide with an oligo-Leu core. We propose that structural flexibility of these TMSs is a prerequisite of fusogenicity.

The misfolding of the Amyloid-beta (Aβ) peptide into β-sheet enriched conformations was proposed as an early event in Alzheimer's Disease (AD). Here, the Aβ peptide secondary structure distribution in cerebrospinal fluid (CSF) and blood plasma of 141 patients was measured with an immuno-infrared-sensor. The sensor detected the amide I band, which reflects the overall secondary structure distribution of all Aβ peptides extracted from the body fluid. We observed a significant downshift of the amide I band frequency of Aβ peptides in Dementia Alzheimer type (DAT) patients, which indicated an overall shift to β-sheet. The secondary structure distribution of all Aβ peptides provides a better marker for DAT detection than a single Aβ misfold or the concentration of a specific oligomer. The discrimination between DAT and disease control patients according to the amide I frequency was in excellent agreement with the clinical diagnosis (accuracy 90% for CSF and 84% for blood). The amide I band maximum above or below the decisive marker frequency appears as a novel spectral biomarker candidate of AD. Additionally, a preliminary proof-of-concept study indicated an amide I band shift below the marker band already in patients with mild cognitive impairment due to AD. The presented immuno-IR-sensor method represents a promising, simple, robust, and label-free diagnostic tool for CSF and blood analysis.

The extraction of disease specific information from Fourier transform infrared (FTIR) spectra of human body fluids demands the highest standards of accuracy and reproducibility of measurements because the expected spectral differences between healthy and diseased subjects are very small in relation to a large background absorbance of the whole sample. Here, we demonstrate that with the increased sensitivity of modern FTIR spectrometers, automatisation of sample preparation and modern bioinformatics, it is possible to identify and validate spectral biomarker candidates for distinguishing between urinary bladder cancer (UBC) and inflammation in suspected bladder cancer patients. The current dataset contains spectra of blood serum and plasma samples of 135 patients. All patients underwent cytology and pathological biopsy characterization to distinguish between patients without UBC (46) and confirmed UBC cases (89). A minimally invasive blood test could spare control patients a repeated cystoscopy including a transurethral biopsy, and three-day stationary hospitalisation. Blood serum, EDTA and citrate plasma were collected from each patient and processed following predefined strict standard operating procedures. Highly reproducible dry films were obtained by spotting sub-nanoliter biofluid droplets in defined patterns, which were compared and optimized. Particular attention was paid to the automatisation of sample preparation and spectral preprocessing to exclude errors by manual handling. Spectral biomarker candidates were identified from absorbance spectra and their 1(st) and 2(nd) derivative spectra using an advanced Random Forest (RF) approach. It turned out that the 2(nd) derivative spectra were most useful for classification. Repeat validation on 21% of the dataset not included in predictor training with Linear Discriminant Analysis (LDA) classifiers and Random Forests (RFs) yielded a sensitivity of 93 ± 10% and a specificity of 46 ± 18% for bladder cancer. The low specificity can be most likely attributed to the unbalanced and small number of control samples. Using this approach, spectral biomarker candidates in blood-derived biofluids were identified, which allow us to distinguish between cancer and inflammation, but the observed differences were tiny. Obviously, a much larger sample number has to be investigated to reliably validate such candidates.

Misfolding and subsequent aggregation of endogeneous proteins constitute essential steps in many human disorders, including Alzheimer and prion diseases. In most prion protein-folding studies, the posttranslational modifications, the lipid anchor in particular, were lacking. Here, we studied a fully posttranslationally modified cellular prion protein, carrying two N-glycosylations and the natural GPI anchor. We used time-resolved FTIR to study the prion protein secondary structure changes when binding to a raft-like lipid membrane via its GPI anchor. We observed that membrane anchoring above a threshold concentration induced refolding of the prion protein to intermolecular ␤-sheets. Such transition is not observed in solution and is membrane specific. Excessive membrane anchoring, analyzed with molecular sensitivity, is thought to be a crucial event in the development of prion diseases.FTIR ͉ membrane anchoring ͉ prion protein ͉ protein aggregation ͉ secondary structure

The secondary structure change of the Abeta peptide to beta‐sheet was proposed as an early event in Alzheimer's disease. The transition may be used for diagnostics of this disease in an early state. We present an Attenuated Total Reflection (ATR) sensor modified with a specific antibody to extract minute amounts of Abeta peptide out of a complex fluid. Thereby, the Abeta peptide secondary structure was determined in its physiological aqueous environment by FTIR‐difference‐spectroscopy. The presented results open the door for label‐free Alzheimer diagnostics in cerebrospinal fluid or blood. It can be extended to further neurodegenerative diseases.

Blood samples of urinary bladder cancer (UBC) patients and patients with urinary tract infection were analysed with advanced automated high throughput Fourier transform infrared (HT-FTIR)-spectroscopy. Thin dried film samples were robotically prepared on multi-well titer plates (MTP) for absorbance measurements in transmission mode. Within the absorbance, 1st and 2nd derivative spectra of serum and two plasma preparations, discriminative patterns were identified and validated using bioinformatic tools. The optimal spectral resolution for data acquisition was determined. An accurate discrimination of the patient groups was achieved with three different independent spectral variable sets. The HT-FTIR blood test may support future clinical diagnostics.