One of the characteristics of Alzheimer disease is the presence of neurotoxic deposits in brain tissue, which are largely made up of a short, 39 -42-amino acid-long peptide referred to as amyloid ␤ (A␤).2 〈␤ peptide plays a major role in Alzheimer disease pathogenesis (1). The deposits (plaques) are an active subject of investigation in many research centers in an attempt to design preventative and therapeutic approaches to the disease (2-14). The molecular structures and morphologies of the fibrils comprising the plaques have been studied at various levels with a variety of imaging and spectroscopic techniques including neutron diffraction, atomic force microscopy, cryoelectronmicroscopy,magneticresonancespectroscopy,andfluorescence (8,(11)(12)(13)(15)(16)(17)(18)(19)(20)(21)(22).A typical characteristic of the fibrils is the presence of ribbon-like ␤-sheets with the strands close to perpendicular to the axis of the fibril, whereas the hydrogen bonds between different strands run parallel to the axis. In vitro studies of the full-length fibrils (that comprise 40 -42-residue peptides) demonstrate multiple morphological possibilities for fibril structures that are very much dependent on the details of the growth conditions (23-25). However, the characteristics of molecular structures are largely preserved (11). For example, in the wild-type sequence, only structures that contain parallel ␤-sheets are observed ( Fig. 1) (9, 24 -28). By contrast, antiparallel ␤-sheet structures can found in the D23N (Iowa) mutant (6,13,29,30), which is associated with an early onset of the neurodegeneration process (6). The polymorphs of D23N with the antiparallel ␤-sheet structures are "protofibrils"; i.e. they are relatively short and curved fibril-like intermediates. They are metastable and eventually convert to mature D23N fibrils, which have parallel ␤-sheet structures that are very similar to wild-type A␤ fibrils (13,22,29).Although both parallel and antiparallel structures can display cytotoxicity (13), the level of toxicity varies greatly depending on the morphological form (11,31). These polymorphs are often viewed as twisted or parallel shapes using transmission electron microscopy (11,25,32,33). The antiparallel ␤-sheet structure represents perhaps the most extreme variability in morphology among polymorphs identified up to date. Effective drug development has not been achieved so far, and one of the possible reasons is the complexity of the polymorphs and interconversions between them as well as the presence of conformational diversity within each polymorph.The intrinsic conformational diversity of the A␤ monomer is well known. In solution, A␤ lacks regular ␣-helical or ␤-stranded structure and is recognized as an intrinsically disordered protein (33-36). However, studies of dynamics in the fibril forms at site-specific level are relatively sparse (37). The most common view is that the hydrophobic core of the fibrils is rigid, complemented by other relative flexible regions (33, 38 -40). The flexibility of non-core re...

Amyloid-β (Aβ) peptide is the major component of plaques found in Alzheimer's disease patients. Using solid-state H NMR relaxation performed on selectively deuterated methyl groups, we probed the dynamics in the threefold symmetric and twofold symmetric polymorphs of native Aβ as well as the protofibrils of the D23N mutant. Specifically, we investigated the methyl groups of two leucine residues that belong to the hydrophobic core (L17 and L34) as well as M35 residues belonging to the hydrophobic interface between the cross-β subunits, which has been previously found to be water-accessible. Relaxation measurements performed over 310-140 K and two magnetic field strengths provide insights into conformational variability within and between polymorphs. Core packing variations within a single polymorph are similar to what is observed for globular proteins for the core residues, whereas M35 exhibits a larger degree of variability. M35 site is also shown to undergo a solvent-dependent dynamical transition in which slower amplitude motions of methyl axes are activated at high temperature. The motions, modeled as a diffusion of methyl axis, have activation energy by a factor of 2.7 larger in the twofold compared with the threefold polymorph, whereas D23N protofibrils display a value similar to the threefold polymorph. This suggests enhanced flexibility of the hydrophobic interface in the threefold polymorph. This difference is only observed in the hydrated state and is absent in the dry fibrils, highlighting the role of solvent at the cavity. In contrast, the dynamic behavior of the core is hydration-independent.

Type 2 alveolar epithelial cells (AT2s), facultative progenitor cells of the lung alveolus, play a vital role in the biology of the distal lung. In vitro model systems that incorporate human cells, recapitulate the biology of primary AT2s, and interface with the outside environment could serve as useful tools to elucidate functional characteristics of AT2s in homeostasis and disease. We and others recently adapted human induced pluripotent stem cell–derived AT2s (iAT2s) for air-liquid interface (ALI) culture. Here, we comprehensively characterize the effects of ALI culture on iAT2s and benchmark their transcriptional profile relative to both freshly sorted and cultured primary human fetal and adult AT2s. We find that iAT2s cultured at ALI maintain an AT2 phenotype while upregulating expression of transcripts associated with AT2 maturation. We then leverage this platform to assay the effects of exposure to clinically significant, inhaled toxicants including cigarette smoke and electronic cigarette vapor.

Aromatic residues are important markers of dynamical changes in proteins’ hydrophobic cores. In this work we investigated the dynamics of the F19 side-chain in the core of amyloid fibrils across a wide temperature range of 300 to 140 K. We utilized solid-state 2H NMR relaxation to demonstrate the presence of a solvent-driven dynamical cross-over between different motional regimes, often also referred to as the dynamical transition. In particular, the dynamics are dominated by small-angle fluctuations at low temperatures and by π-flips of the aromatic ring at high temperatures. The cross-over temperature is more than 43 degrees lower for the hydrated state of the fibrils compared to the dry state, indicating that interactions with water facilitate π-flips. Further, cross-over temperatures are shown to be very sensitive to polymorphic states of the fibrils, such as the 2-fold and 3-fold symmetric morphologies of the wild-type protein as well as D23N mutant protofibrils. We speculate that these differences can be attributed, at least partially, to enhanced interactions with water in the 3-fold polymorph, which has been shown to have a water-accessible cavity. Combined with previous studies of methyl group dynamics, the results highlight the presence of multiple dynamics modes in the core of the fibrils, which was originally believed to be quite rigid.

We have investigated the effect of deuteration of non‐exchangeable protons on protein global thermal stability, hydrophobicity, and local flexibility using well‐known thermostable model systems such as the villin headpiece subdomain (HP36) and the third immunoglobulin G‐binding domain of protein G (GB3). Reversed‐phase high‐performance liquid chromatography (RP‐HPLC) measurements as a function of temperature probe global thermal stability in the presence of acetonitrile, while differential scanning calorimetry determines thermal stability in solution. Both indicate small but measurable changes in the order of several degrees. RP‐HPLC also permitted quantification of the effect of deuteration of just three core phenylalanine side chains of HP36. NMR dynamics investigation has focused on methyl axes motions using cross‐correlated relaxation measurements. The analysis of order parameters provided a complex picture indicating that deuteration generally increases motional amplitudes of sub‐nanosecond motion in GB3 but decreases those in HP36. Combined with earlier dynamics measurements at Cα–Cβ sites and backbone sites of GB3, which probed slower time scales, the results point to the need to probe multiple atoms in the protein and variety of time scales to the discern the full complexity of the effects of deuteration on dynamics.