Disulfide Bridges Remain Intact while Native Insulin Converts into Amyloid Fibrils

Amyloid fibrils are β-sheet-rich protein aggregates commonly found in the organs and tissues of patients with various amyloid-associated diseases. Understanding the structural organization of amyloid fibrils can be beneficial for the search of drugs to successfully treat diseases associated with protein misfolding. The structure of insulin fibrils was characterized by deep ultraviolet resonance Raman (DUVRR) and Nuclear Magnetic Resonance (NMR) spectroscopy combined with hydrogen-deuterium exchange. The compositions of the fibril core and unordered parts were determined at single amino acid residue resolution. All three disulfide bonds of native insulin remained intact during the aggregation process, withstanding scrambling. Three out of four tyrosine residues were packed into the fibril core, and another aromatic amino acid, phenylalanine, was located in the unordered parts of insulin fibrils. In addition, using all-atom MD simulations, the disulfide bonds were confirmed to remain intact in the insulin dimer, which mimics the fibrillar form of insulin

Defining the Conformational Features of Anchorless, Poorly Neuroinvasive Prions

Infectious prions cause diverse clinical signs and form an extraordinary range of structures, from amorphous aggregates to fibrils. How the conformation of a prion dictates the disease phenotype remains unclear. Mice expressing GPI-anchorless or GPI-anchored prion protein exposed to the same infectious prion develop fibrillar or nonfibrillar aggregates, respectively, and show a striking divergence in the disease pathogenesis. To better understand how a prion's physical properties govern the pathogenesis, infectious anchorless prions were passaged in mice expressing anchorless prion protein and the resulting prions were biochemically characterized. Serial passage of anchorless prions led to a significant decrease in the incubation period to terminal disease and altered the biochemical properties, consistent with a transmission barrier effect. After an intraperitoneal exposure, anchorless prions were only weakly neuroinvasive, as prion plaques rarely occurred in the brain yet were abundant in extracerebral sites such as heart and adipose tissue. Anchorless prions consistently showed very high stability in chaotropes or when heated in SDS, and were highly resistant to enzyme digestion. Consistent with the results in mice, anchorless prions from a human patient were also highly stable in chaotropes. These findings reveal that anchorless prions consist of fibrillar and highly stable conformers. The additional finding from our group and others that both anchorless and anchored prion fibrils are poorly neuroinvasive strengthens the hypothesis that a fibrillar prion structure impedes efficient CNS invasion

Elucidating peptide and protein structure and dynamics: UV resonance raman spectroscopy

UV resonance Raman spectroscopy (UVRR) is a powerful method that has the requisite selectivity and sensitivity to incisively monitor biomolecular structure and dynamics in solution. In this Perspective, we highlight applications of UVRR for studying peptide and protein structure and the dynamics of protein and peptide folding. UVRR spectral monitors of protein secondary structure, such as the amide III3 band and the Cα-H band frequencies and intensities, can be used to determine Ramachandran ψ angle distributions for peptide bonds. These incisive, quantitative glimpses into conformation can be combined with kinetic T-jump methodologies to monitor the dynamics of biomolecular conformational transitions. The resulting UVRR structural insight is impressive in that it allows differentiation of, for example, different α-helix-like states that enable differentiating π and 310 states from pure α-helices. These approaches can be used to determine the Gibbs free-energy landscape of individual peptide bonds along the most important protein (un)folding coordinate. Future work will find spectral monitors that probe peptide bond activation barriers that control protein (un)folding mechanisms. In addition, UVRR studies of side chain vibrations will probe the role of side chains in determining protein secondary, tertiary, and quaternary structures. © 2011 American Chemical Society

The first step of hen egg white lysozyme fibrillation, irreversible partial unfolding, is a two-state transition

Amyloid fibril depositions are associated with many neurodegenerative diseases as well as amyloidosis. The detailed molecular mechanism of fibrillation is still far from complete understanding. In our previous study of in vitro fibrillation of hen egg white lysozyme, an irreversible partially unfolded intermediate was characterized. A similarity of unfolding kinetics found for the secondary and tertiary structure of lysozyme using deep UV resonance Raman (DUVRR) and tryptophan fluorescence spectroscopy leads to a hypothesis that the unfolding might be a two-state transition. In this study, chemometric analysis, including abstract factor analysis (AFA), target factor analysis (TFA), evolving factor analysis (EFA), multivariate curve resolution–alternating least squares (ALS), and genetic algorithm, was employed to verify that only two principal components contribute to the DUVRR and fluorescence spectra of soluble fraction of lysozyme during the fibrillation process. However, a definite conclusion on the number of conformers cannot be made based solely on the above spectroscopic data although chemometric analysis suggested the existence of two principal components. Therefore, electrospray ionization mass spectrometry (ESI-MS) was also utilized to address the hypothesis. The protein ion charge state distribution (CSD) envelopes of the incubated lysozyme were well fitted with two principal components. Based on the above analysis, the partial unfolding of lysozyme during in vitro fibrillation was characterized quantitatively and proven to be a two-state transition. The combination of ESI-MS and Raman and fluorescence spectroscopies with advanced statistical analysis was demonstrated to be a powerful methodology for studying protein structural transformations