Amyloid formation is implicated in more than 20 human diseases, yet the mechanism by which fibrils form is not well understood. We use 2D infrared spectroscopy and isotope labeling to monitor the kinetics of fibril formation by human islet amyloid polypeptide (hIAPP or amylin) that is associated with type 2 diabetes. We find that an oligomeric intermediate forms during the lag phase with parallel β-sheet structure in a region that is ultimately a partially disordered loop in the fibril. We confirm the presence of this intermediate, using a set of homologous macrocyclic peptides designed to recognize β-sheets. Mutations and molecular dynamics simulations indicate that the intermediate is on pathway. Disrupting the oligomeric β-sheet to form the partially disordered loop of the fibrils creates a free energy barrier that is the origin of the lag phase during aggregation. These results help rationalize a wide range of previous fragment and mutation studies including mutations in other species that prevent the formation of amyloid plaques.inhibitors | aggregation pathway | vibrational coupling T he misfolding of proteins into β-sheet-rich amyloid fibrils is associated with the pathology of more than 20 human diseases, including Alzheimer's, Parkinson, and Huntington diseases (1). Amyloid plaques are formed by masses of fibrils, but growing evidence suggests that the toxic species may be prefibrillar intermediates (2, 3). As a result, there is much interest in understanding the mechanism by which these proteins form fibrils and identifying intermediates in the aggregation pathway. However, obtaining structural information about intermediate species is difficult due to their transient nature. Solid-state NMR (ssNMR) and X-ray crystallography provide high-resolution structures of fibrils (4, 5) and optical techniques can track structural changes in real time (6, 7), but few techniques have both the structural and the temporal resolution to extract specific structural details about intermediates. Fragments have been trapped in intriguing oligomeric structures that may represent intermediate states (5,8) and transient secondary structures are known to exist from circular dichroism measurements and other experiments (9-11), but for full-length proteins it has been difficult to identify the specific residues that contribute to the secondary structure and thus understand their role in the aggregation mechanism. In this paper, we use 2D infrared (2D IR) spectroscopy and isotope labeling to monitor the structural evolution of the full-length human islet amyloid polypeptide (hIAPP or amylin), a 37-residue peptide implicated in type 2 diabetes. We observe the formation of a structured prefibrillar intermediate in a region that has long been known to influence aggregation, but that does not form well-ordered cross-β structure in the amyloid fibril. Its presence provides unique structural insights into the mechanism of amyloid aggregation and helps unify many seemingly inconsistent prior studies.Many studies on hIAPP have focused ...

The structural eye lens protein γD-crystallin is a major component of cataracts, but its conformation when aggregated is unknown. Using expressed protein ligation, we uniformly 13 C labeled one of the two Greek key domains so that they are individually resolved in two-dimensional (2D) IR spectra for structural and kinetic analysis. Upon acid-induced amyloid fibril formation, the 2D IR spectra reveal that the C-terminal domain forms amyloid β-sheets, whereas the N-terminal domain becomes extremely disordered but lies in close proximity to the β-sheets. Two-dimensional IR kinetics experiments show that fibril nucleation and extension occur exclusively in the C-terminal domain. These results are unexpected because the N-terminal domain is less stable in the monomer form. Isotope dilution experiments reveal that each C-terminal domain contributes two or fewer adjacent β-strands to each β-sheet. From these observations, we propose an initial structural model for γD-crystallin amyloid fibrils. Because only 1 μg of protein is required for a 2D IR spectrum, even poorly expressing proteins can be studied under many conditions using this approach. Thus, we believe that 2D IR and protein ligation will be useful for structural and kinetic studies of many protein systems for which IR spectroscopy can be straightforwardly applied, such as membrane and amyloidogenic proteins. C ataracts are a protein misfolding disease caused by the aggregation of lens crystallin proteins into insoluble deposits that blur vision (1, 2). Because these proteins are not regenerated, damage from UV radiation, oxidative stress, and other chemical modifications accumulates with time (1, 2). As a result, over 50% of the population over 55 develops age-related cataracts (2). Additionally, numerous mutations that destabilize crystallin protein folds are linked to inherited and juvenile-onset cataracts (1). Although the causative factors associated with this disease are known, the structures of the aggregates and the mechanisms by which they form are unknown.Like other protein aggregation diseases such as type II diabetes mellitus and Alzheimer's disease, the molecular structures of proteins in cataracts are difficult to determine. Atomic-level structures have been obtained for some amyloid aggregates of peptides using NMR spectroscopy (3, 4) and X-ray crystallography (5). However, the most widely used techniques for studying aggregate structures and aggregation mechanisms are circular dichroism spectroscopy, fluorescence spectroscopy, and transmission electron microscopy, which provide little detailed structural information. Two-dimensional (2D) IR spectroscopy is emerging as an important tool for studying protein aggregates such as amyloid fibrils (6-8) because it provides bond-by-bond structural resolution on kinetically evolving samples (6, 8-10). Two-dimensional IR spectroscopy probes secondary structure through cross peak couplings and solvent exposure through 2D lineshapes. Its bond-specific structural resolution comes from isotope labeling. Mech...

Patients with type II diabetes exhibit fibrillar deposits of human amylin protein in the pancreas. It has been proposed that amylin oligomers arising along the aggregation or fibril-formation pathways are important in the genesis of the disease. In a step toward understanding these aggregation pathways, in this work we report the conformational preferences of human amylin monomer in solution using molecular simulations and infrared experiments. In particular, we identify a stable conformer that could play a key role in aggregation. We find that amylin adopts three stable conformations: one with an α-helical segment comprising residues 9-17 and a short antiparallel β-sheet comprising residues 24-28 and 31-35; one with an extended antiparallel β-hairpin with the turn region comprising residues 20-23; and one with no particular structure. Using detailed calculations, we determine the relative stability of these various conformations, finding that the β-hairpin conformation is the most stable, followed by the α-helical conformation, and then the unstructured coil. To test our predicted structure, we calculate its infrared spectrum in the amide I stretch regime, which is sensitive to secondary structure through vibrational couplings and linewidths, and compare it to experiment. We find that theoretically predicted spectra are in good agreement with the experimental line shapes presented herein. The implications of the monomer secondary structures on its aggregation pathway and on its interaction with cell membranes are discussed.

Polyglutamine (polyQ) sequences are found in a variety of proteins, and mutational expansion of the polyQ tract is associated with many neurodegenerative diseases. We study the amyloid fibril structure and aggregation kinetics of K 2 Q 24 K 2 W, a model polyQ sequence. Two structures have been proposed for amyloid fibrils formed by polyQ peptides. By forming fibrils composed of both 12 C and 13 C monomers, made possible by protein expression in Escherichia coli, we can restrict vibrational delocalization to measure 2D IR spectra of individual monomers within the fibrils. The spectra are consistent with a β-turn structure in which each monomer forms an antiparallel hairpin and donates two strands to a single β-sheet. Calculated spectra from atomistic molecular-dynamics simulations of the two proposed structures confirm the assignment. No spectroscopically distinct intermediates are observed in rapid-scan 2D IR kinetics measurements, suggesting that aggregation is highly cooperative. Although 2D IR spectroscopy has advantages over linear techniques, the isotope-mixing strategy will also be useful with standard Fourier transform IR spectroscopy.Huntington disease | isotope dilution | antiparallel β-sheets | two-dimensional infrared spectroscopy N umerous neurodegenerative diseases, including Huntington disease, are associated with the mutational expansion of CAG repeats in specific genes. This expansion causes an increase in the length of normally benign polyglutamine (polyQ) tracts in expressed proteins (1, 2). In vivo, polyQ tracts occur embedded within larger proteins, such as the huntingtin protein. Full length proteins are impractical for detailed structural studies, and so isolated polyQ sequences are often used instead in experiments and simulations (3-5). Although isolated polyQ sequences may not aggregate in precisely the same manner as they would in fulllength proteins (3, 6, 7), it has been established that such models yield data that are relevant to some, if not all, polyQ pathologies (8). Additionally, proteolytic polyQ-containing fragments, rather than full-length proteins, may be the primary toxic species in Huntington disease (9) and some types of spinocerebellar ataxia (10). As a consequence, simple polyQ peptides remain an attractive alternative to more complicated proteins for understanding polyQmediated aggregation.A plethora of structures have been suggested for polyQ aggregates, ranging from α-helical coiled coils (11) or β-helices (12) to amyloid fibrils comprising stacked, linear β-sheets (13). Current consensus favors fibrillar structures, but the precise arrangement of the peptides within the fibril remains unclear. Most amyloid proteins form parallel β-sheets within fibrils, although antiparallel sheets do occur (14). Parallel sheets have been observed for glutamine-rich hexapeptides (15), but NMR studies (16), as well as the results presented here, suggest that sequences approaching the pathological length of 40 glutamines adopt an antiparallel structure. Fig. 1 shows two models that hav...

Urea/water is an archetypical “biological” mixture, and is especially well known for its relevance to protein thermodynamics, as urea acts as a protein denaturant at high concentration. This behavior has given rise to an extended debate concerning urea’s influence on water structure. Based on a variety of methods and of definitions of water structure, urea has been variously described as a structure-breaker, a structure-maker, or as remarkably neutral towards water. Because of its sensitivity to microscopic structure and dynamics, vibrational spectroscopy can help resolve these debates. We report experimental and theoretical spectroscopic results for the OD stretch of HOD/H2O/urea mixtures (linear IR, 2DIR, and pump-probe anisotropy decay) and for the CO stretch of urea-D4/D2O mixtures (linear IR only). Theoretical results are obtained using existing approaches for water, and a modification of a frequency map developed for acetamide. All absorption spectra are remarkably insensitive to urea concentration, consistent with the idea that urea only very weakly perturbs water structure. Both this work and experiments by Rezus and Bakker, however, show that water’s rotational dynamics are slowed down by urea. Analysis of the simulations casts doubt on the suggestion that urea immobilizes particular doubly hydrogen bonded water molecules.

Deamidation of asparagine and glutamine is the most common non-enzymatic, post-translational modification. Deamidation can influence the structure, stability, folding, and aggregation of proteins and has been proposed to play a role in amyloid formation. However there are no structural studies of the consequences of deamidation on amyloid fibers, in large part because of the difficulty of studying these materials using conventional methods. Here we examine the effects of deamidation on the kinetics of amyloid formation by amylin, the causative agent of type 2 diabetes. We find that deamidation accelerates amyloid formation and the deamidated material is able to seed amyloid formation by unmodified amylin. Using site-specific isotope labeling and two-dimensional infrared (2D IR) spectroscopy, we show that fibers formed by samples that contain deamidated polypeptide contain reduced amounts of β-sheet. Deamidation leads to disruption of the N-terminal β-sheet between Ala-8 and Ala-13, but β-sheet is still retained near Leu-16. The C-terminal sheet is disrupted near Leu-27. Analysis of potential sites of deamidation together with structural models of amylin fibers reveals that deamidation in the N-terminal β-sheet region may be the cause for the disruption of the fiber structure at both the N- and C-terminal β-sheet. Thus, deamidation is a post-translational modification that creates fibers which have an altered structure, but can still act as a template for amylin aggregation. Deamidation is very difficult to detect with standard methods used to follow amyloid formation, but isotope labeled IR spectroscopy provides a means for monitoring sample degradation and investigating the structural consequences of deamidation.

This article by the Perineural Invasion (PNI) Registry Group aims to clarify clinical and histopathological ambiguities surrounding PNI in non-melanoma skin cancer (NMSC). PNI is reportedly present in approximately 2-6% of cases of NMSC and is associated with greater rates of morbidity and mortality. The distinction between clinical PNI and incidental PNI is somewhat unclear, especially in regard to management and prognosis. One important objective of the PNI Registry is to develop a standardised method of classifying perineural invasion. Hence, in this article we propose a definition for PNI and for its sub-classification. This article also provides a critical analysis of the current literature on the treatment of incidental PNI by evaluating the key cohort studies that have investigated the use of surgery or radiotherapy in the management of incidental PNI. At present, there are no universal clinical guidelines that specify the acceptable treatment of NMSC exhibiting incidental PNI. Consequently, patients often receive surgery with varying wider margins, or radiotherapy despite the limited evidence substantiating such management options. It is evident from the existing literature that current opinion is divided over the benefit of adjuvant radiotherapy. Certain prognostic factors have been proposed, such as the size and depth of tumour invasion, nerve diameter, the presence of multifocal PNI and the type of tumour. The PNI Registry is a web-based registry that has been developed to assist in attaining further data pertaining to incidental PNI in NMSC. It is envisaged that this information will provide the foundation for identifying and defining best practice in managing incidental PNI.

We report that the waiting time delay in 2D IR pulse sequences can be used to suppress signals from structurally disordered regions of amyloid fibrils. At a waiting time delay of 1.0 ps, the random coil vibrational modes of amylin fibrils are no longer detectable, leaving only the sharp excitonic vibrational features of the fibril β-sheets. Isotope labeling with 13C18O reveals that structurally disordered residues decay faster than residues protected from solvent. Since structural disorder is usually accompanied by hydration, we conclude that the shorter lifetimes of random-coil residues is due to solvent exposure. These results indicate that 2D IR pulse sequences can utilize the waiting time to better resolve solvent-protected regions of peptides and that local mode lifetimes should be included in simulations of 2D IR spectra.