Human islet amyloid polypeptide (hIAPP or Amylin) is a 37 residue hormone that is cosecreted with insulin from the pancreatic islets. The aggregation of hIAPP plays a role in the progression of type 2 diabetes and contributes to the failure of islet cell grafts. Despite considerable effort, little is known about the mode of action of IAPP amyloid inhibitors, and this has limited rational drug design. Insulin is one of the most potent inhibitors of hIAPP fibril formation, but its inhibition mechanism is not understood. In this study, the aggregation of mixtures of hIAPP with insulin, as well as with the separate A and B chains of insulin, were characterized using ion mobility spectrometry-based mass spectrometry and atomic force microscopy. Insulin and the insulin B chain target the hIAPP monomer in its compact isoform and shift the equilibrium away from its extended isoform, an aggregation-prone conformation, and thus inhibit hIAPP from forming β-sheets and subsequently amyloid fibrils. All-atom molecular modeling supports these conclusions.

One of the earliest events in amyloid beta-protein (A beta) self-association is nucleation of A beta monomer folding through formation of a turn at G1y25-Lys28. We report here the effects of structural changes at the center of the turn, G1y25-Ser26, on A beta 42 conformational dynamics and assembly. We used "click peptide" chemistry to quasi-synchronously create A beta 42 from 26-O-acyliso-A beta 42 (iA beta 42) through a pH jump from 3 to 7.4. We also synthesized N-alpha-acetyl-Ser26-iA beta 42 (Ac-iA beta 42), which cannot undergo 0 N acyl chemistry, to study the behavior of this ester form of A beta 42 itself at neutral pH. Data from experiments monitoring increases in beta-sheet formation (thioflavin T, CD), hydrodynamic radius (RH), scattering intensity (quasielastic light scattering spectroscopy), and extent of oligomerization (ion mobility spectroscopy mass spectrometry) were quite consistent. A rank order of Ac-iA beta 42 > iA beta 42 > A beta 42 was observed. Photochemically cross-linked iA beta 42 displayed an oligomer distribution with a prominent dimer band that was not present with A beta 42. These dimers also were observed selectively in iA beta 42 in ion mobility spectrometry experiments. The distinct biophysical behaviors of iA beta 42 and A beta 42 appear to be due to the conversion of iA beta 42 into "pure" A beta 42 monomer, a nascent form of A beta 42 that does not comprise the variety of oligomeric and aggregated states present in pre-existent A beta 42. These results emphasize the importance of the Gly25-Ser26 dipeptide in organizing A beta 42 monomer structure and thus suggest that drugs altering the interactions of this dipeptide with neighboring side-chain atoms or with the peptide backbone could be useful in therapeutic strategies targeting formation of Ap oligomers and higher-order assemblies. (C) 2014 Elsevier Ltd. All rights reserved.

One of the earliest events in amyloid β-protein (Aβ) self-association is nucleation of Aβ monomer folding through formation of a turn at Gly25-Lys28. We report here the effects of structural changes at the center of the turn, Gly25-Ser26, on Aβ42 conformational dynamics and assembly. We used "click peptide" chemistry to quasi-synchronously create Aβ42 from 26-O-acyliso-Aβ42 (iAβ42) through a pH jump from 3 to 7.4. We also synthesized Nα-acetyl-Ser26-iAβ42 (Ac-iAβ42), which cannot undergo O→N acyl chemistry, to study the behavior of this ester form of Aβ42 itself at neutral pH. Data from experiments monitoring increases in β-sheet formation (thioflavin T, CD), hydrodynamic radius (RH), scattering intensity (quasielastic light scattering spectroscopy), and extent of oligomerization (ion mobility spectroscopy-mass spectrometry) were quite consistent. A rank order of Ac-iAβ42>iAβ42>Aβ42 was observed. Photochemically cross-linked iAβ42 displayed an oligomer distribution with a prominent dimer band that was not present with Aβ42. These dimers also were observed selectively in iAβ42 in ion mobility spectrometry experiments. The distinct biophysical behaviors of iAβ42 and Aβ42 appear to be due to the conversion of iAβ42 into "pure" Aβ42 monomer, a nascent form of Aβ42 that does not comprise the variety of oligomeric and aggregated states present in pre-existent Aβ42. These results emphasize the importance of the Gly25-Ser26 dipeptide in organizing Aβ42 monomer structure and thus suggest that drugs altering the interactions of this dipeptide with neighboring side-chain atoms or with the peptide backbone could be useful in therapeutic strategies targeting formation of Aβ oligomers and higher-order assemblies.

Although most cases of Alzheimer's disease (AD) are sporadic, ∼5% of cases are genetic in origin. These cases, known as familial Alzheimer's disease (FAD), are caused by mutations that alter the rate of production or the primary structure of the amyloid β-protein (Aβ). Changes in the primary structure of Aβ alter the peptide's assembly and toxic activity. Recently, a primary working hypothesis for AD has evolved where causation has been attributed to early, soluble peptide oligomer states. Here we posit that both experimental and pathological differences between FAD-related mutants and wild-type Aβ could be reflected in the early oligomer distributions of these peptides. We use ion mobility-based mass spectrometry to probe the structure and early aggregation states of three mutant forms of Aβ40 and Aβ42: Tottori (D7N), Flemish (A21G), and Arctic (E22G). Our results indicate that the FAD-related amino acid substitutions have no noticeable effect on Aβ monomer cross section, indicating there are no major structural changes in the monomers. However, we observe significant changes to the aggregation states populated by the various Aβ mutants, indicating that structural changes present in the monomers are reflected in the oligomers. Moreover, the early oligomer distributions differ for each mutant, suggesting a possible structural basis for the varied pathogenesis of different forms of FAD.

The aggregation and conformation of deoxyguanosine (dG) in an ammonium acetate buffer solution were examined using mass spectrometry, ion mobility, and molecular mechanics/ dynamics calculations. The nano-ESI mass spectrum indicated that 4 and 6 dGs cluster with 1 NH4 ; 11 dGs with 2 NH4 ; 14, 16, and 17 dGs with 3 NH4 ; and 23 dGs with 4 NH4 . The collision cross sections with helium were measured and compared with calculated cross sections of theoretical structures generated by molecular mechanics/dynamics calculations. Three distinct arrival time distribution (ATD) peaks were observed for (4dG NH4) . One peak was assigned to the quadruplex structure of (4dG NH4) , while the other two peaks corresponded to the quadruplex structures of (8dG 2NH4) 2 and (12dG 3NH4) 3 , all with the same m/z. Four ATD peaks were observed for (6dG NH4) and assigned to the globular structure of (6dG NH4) , and the quadruplex structures of (12dG 2NH4) 2 , (18dG 3NH4) 3 , and (24dG 4NH4) 4 . Two ATD peaks were observed for (11dG 2NH4) 2 and assigned to the quadruplex structures of (11dG 2NH4) 2 and (22dG 4NH4) 4 . All of the other clusters in the mass spectrum (14, 16, and 17 dGs with 3 NH4 and 23 dGs with 4 NH4 ) only had one peak in their ATDs and in all cases the theoretical structures in a quadruplex arrangement agreed with the experimental cross sections. These results provide compelling evidence that quadruplexes are present in solution and retain their structure during the spray process, dehydration, and detection. (J Am Soc Mass Spectrom 2005, 16, 989–997) © 2005 American Society for Mass Spectrometry

In recent years, small protein oligomers have been implicated in the aetiology of a number of important amyloid diseases, such as type 2 diabetes, Parkinson's disease and Alzheimer's disease. As a consequence, research efforts are being directed away from traditional targets, such as amyloid plaques, and towards characterization of early oligomer states. Here we present a new analysis method, ion mobility coupled with mass spectrometry, for this challenging problem, which allows determination of in vitro oligomer distributions and the qualitative structure of each of the aggregates. We applied these methods to a number of the amyloid-β protein isoforms of Aβ40 and Aβ42 and showed that their oligomer-size distributions are very different. Our results are consistent with previous observations that Aβ40 and Aβ42 self-assemble via different pathways and provide a candidate in the Aβ42 dodecamer for the primary toxic species in Alzheimer's disease.

The structure of the 21-30 fragment of the amyloid beta-protein (Abeta) was investigated by ion mobility mass spectrometry and replica exchange dynamics simulations. Mutations associated with familial Alzheimer's disease (E22G, E22Q, E22K, and D23N) of Abeta(21-30) were also studied, in order to understand any structural changes that might occur with these substitutions. The structure of the WT peptide shows a bend and a perpendicular turn in the backbone which is maintained by a network of D23 hydrogen bonding. Results for the mutants show that substitutions at E22 do little to alter the overall structure of the fragment. A substitution at D23 resulted in a change of structure for Abeta(21-30). A comparison of these gas-phase studies to previous solution-phase studies reveals that the peptide can fold in the absence of solvent to a structure also seen in solution, highlighting the important role of the D23 hydrogen bonding network in stabilizing the fragment's folded structure.

A beta 40 and A beta 42 are peptides that adopt similar random-coil structures in solution. A beta 42, however, is significantly more neurotoxic than A beta 40 and forms amyloid fibrils much more rapidly than A beta 40. Here, mass spectrometry and ion mobility spectrometry are used to investigate a mixture of A beta 40 and A beta 42. The mass spectrum for the mixed solution shows the presence of a heterooligomer composed of equal parts of A beta 40 and A beta 42. Ion mobility results indicate that this mixed species comprises an oligomer distribution extending to tetramers. A beta 40 atone produces such a distribution, whereas A beta 42 alone produces oligomers as large as dodecamers. This indicates that A beta 40 inhibits A beta 42 oligomerization.

*Corresponding authors. J.-E. Shea i Department of Chemistry and Bioch California, Santa Barbara, CA 93106 addresses: shea@chem.ucsb.edu; bo † C.W., M.M.M., and S.L.B. contri work. Abbreviations used: Aβ, amyloid C-terminal fragment; AD, Alzheime replica exchange molecular dynami mobility mass spectrometry; ATD, a distribution; MD, molecular dynam nano-electrospray ionization; MALD laser desorption/ionization.

The C-terminus of amyloid beta-protein (Abeta) 42 plays an important role in this protein's oligomerization and may therefore be a good therapeutic target for the treatment of Alzheimer's disease. Certain C-terminal fragments (CTFs) of Abeta42 have been shown to disrupt oligomerization and to strongly inhibit Abeta42-induced neurotoxicity. Here we study the structures of selected CTFs [Abeta(x-42); x=29-31, 39] using replica exchange molecular dynamics simulations and ion mobility mass spectrometry. Our simulations in explicit solvent reveal that the CTFs adopt a metastable beta-structure: beta-hairpin for Abeta(x-42) (x=29-31) and extended beta-strand for Abeta(39-42). The beta-hairpin of Abeta(30-42) is converted into a turn-coil conformation when the last two hydrophobic residues are removed, suggesting that I41 and A42 are critical in stabilizing the beta-hairpin in Abeta42-derived CTFs. The importance of solvent in determining the structure of the CTFs is further highlighted in ion mobility mass spectrometry experiments and solvent-free replica exchange molecular dynamics simulations. A comparison between structures with solvent and structures without solvent reveals that hydrophobic interactions are critical for the formation of beta-hairpin. The possible role played by the CTFs in disrupting oligomerization is discussed.

The effect of single amino acid substitutions associated with the Italian (E22K), Arctic (E22G), Dutch (E22Q) and Iowa (D23N) familial forms of Alzheimer's disease and cerebral amyloid angiopathy on the structure of the 21–30 fragment of the Alzheimer amyloid β-protein (Aβ) is investigated by replica-exchange molecular dynamics simulations. The 21–30 segment has been shown in our earlier work to adopt a bend structure in solution that may serve as the folding nucleation site for Aβ. Our simulations reveal that the 24–28 bend motif is retained in all E22 mutants, suggesting that mutations involving residue E22 may not affect the structure of the folding nucleation site of Aβ. Enhanced aggregation in Aβ with familial Alzheimer's disease substitutions may result from the depletion of the E22–K28 salt bridge, which destabilizes the bend structure. Alternately, the E22 mutations may affect longer-range interactions outside the 21–30 segment that can impact the aggregation of Aβ. Substituting at residue D23, on the other hand, leads to the formation of a turn rather than a bend motif, implying that in contrast to E22 mutants, the D23N mutant may affect monomer Aβ folding and subsequent aggregation. Our simulations suggest that the mechanisms by which E22 and D23 mutations affect the folding and aggregation of Aβ are fundamentally different.

Aggregation of alpha-synuclein (alpha-syn), a protein implicated in Parkinson's disease (PD), is believed to progress through formation of a partially folded intermediate. Using nanoelectrospray ionization (nano-ESI) mass spectrometry combined with ion mobility measurements we found evidence for a highly compact partially folded family of structures for alpha-syn and its disease-related A53T mutant with net charges of -6, -7, and -8. For the other early onset PD mutant, A30P, this highly compact population was only evident when the protein had a net charge of -6. When bound to spermine near physiologic pH, all three proteins underwent a charge reduction from the favored solution charge state of -10 to a net charge of -6. This charge reduction is accompanied by a dramatic size reduction of about a factor of 2 (cross section of 2600 A2 (-10 charge state) down to 1430 A2 (-6 charge state)). We conclude that spermine increases the aggregation rate of alpha-syn by inducing a collapsed conformation, which then proceeds to form aggregates.

Chapter 16 Effect of Heat Treatment on the Oxidation of Hot-Pressed Si3N4 as Determined by Infrared Reflection Analysis J. M. Barrett, J. M. Barrett Materials Science and Engineering Dept. University of Florida, Gainesville, Fla. 32611Search for more papers by this authorL. L. Hench, L. L. Hench Materials Science and Engineering Dept. University of Florida, Gainesville, Fla. 32611Search for more papers by this authorS. Bernstein, S. Bernstein Materials Science and Engineering Dept. University of Florida, Gainesville, Fla. 32611Search for more papers by this authorD. E. Clark, D. E. Clark Materials Science and Engineering Dept. University of Florida, Gainesville, Fla. 32611Search for more papers by this authorS. W. Freiman, S. W. Freiman Fracture & Deformation Div. National Bureau of Standards Washington, D.C. 20234Search for more papers by this author J. M. Barrett, J. M. Barrett Materials Science and Engineering Dept. University of Florida, Gainesville, Fla. 32611Search for more papers by this authorL. L. Hench, L. L. Hench Materials Science and Engineering Dept. University of Florida, Gainesville, Fla. 32611Search for more papers by this authorS. Bernstein, S. Bernstein Materials Science and Engineering Dept. University of Florida, Gainesville, Fla. 32611Search for more papers by this authorD. E. Clark, D. E. Clark Materials Science and Engineering Dept. University of Florida, Gainesville, Fla. 32611Search for more papers by this authorS. W. Freiman, S. W. Freiman Fracture & Deformation Div. National Bureau of Standards Washington, D.C. 20234Search for more papers by this author Book Editor(s):William J. Smothers, William J. SmothersSearch for more papers by this author First published: 01 January 1980 https://doi.org/10.1002/9780470291030.ch16Citations: 1Book Series:Ceramic Engineering and Science Proceedings AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Proceedings of the 2nd and 3rd Annual Conference on Composites and Advanced Materials: Ceramic Engineering and Science Proceedings, Volume 1 RelatedInformation

The structural properties of the A beta 42 peptide, a main constituent of the amyloid plaques formed in Alzheimer's disease, were investigated through a combination of ion-mobility mass spectrometry and theoretical modeling. Replica exchange molecular dynamics simulations using a fully atomic description of the peptide and implicit water solvent were performed on the -3 charge state of the peptide, its preferred state under experimental conditions. Equilibrated structures at 300 K were clustered into three distinct families with similar structural features within a family and with significant root mean square deviations between families. An analysis of secondary structure indicates the A beta 42 peptide conformations are dominated by loops and turns but show some helical structure in the C-terminal hydrophobic tail. A second calculation on A beta 42 in a solvent-free environment yields compact structures turned "inside out" from the solution structures (hydrophobic parts on the outside, polar parts on the inside). Ion mobility experiments on the A beta 42-3 charge state electrosprayed from solution yield a bimodal arrival time distribution. This distribution can be quantitatively fit using cross-sections from dehydrated forms of the three families of calculated solution structures and the calculated solvent-free family of structures. Implications of the calculations on the early stages of aggregation of A beta 42 are discussed.

The structural properties of G-quadruplex forming sequences, such as the human telomeric repeat d(T2AG3)n, are of great interest due to their role in cancer and cellular aging. To determine if G-quadruplexes are present in a solvent-free environment, different lengths of the telomeric repeat d(T2AG3)n (where n=1, 2, 4 and 6) and dTG4T were investigated with mass spectrometry, ion mobility and molecular dynamics calculations. Nano-ESI-MS illustrated quadruplex stoichiometries compatible with G-quadruplex structures for each sequence, with dT2AG3 and dTG4T forming 4-strand complexes with two and three NH4+ adducts, d(T2AG3)2 a 2-strand complex, and d(T2AG3)4 and d(T2AG3)6 remaining single-stranded. Experimental cross sections were obtained for all species using ion mobility methods. In all cases, these could be quantitatively matched to model cross sections with specific strand orientations (parallel/antiparallel) and structures. For each species, the solvent-free structures agreed with the solution CD measurements, but the ion mobility/modeling procedure often gave much more detailed structural information.

Folding and self-assembly of the 42-residue amyloid beta-protein (A beta) are linked to Alzheimer's disease (AD). The 21-30 region of A beta, A beta(21-30), is resistant to proteolysis and is believed to nucleate the folding of full-length A beta. The conformational space accessible to the A beta(21-30) peptide is investigated by using replica exchange molecular dynamics simulations in explicit solvent. Conformations belonging to the global free energy minimum (the "native'' state) from simulation are in good agreement with reported NMR structures. These conformations possess a bend motif spanning the central residues V24-K28. This bend is stabilized by a network of hydrogen bonds involving the side chain of residue D23 and the amide hydrogens of adjacent residues G25, S26, N27, and K28, as well as by a salt bridge formed between side chains of K28 and E22. The non-native states of this peptide are compact and retain a native-like bend topology. The persistence of structure in the denatured state may account for the resistance of this peptide to protease degradation and aggregation, even at elevated temperatures.

Oligomeric, neurotoxic amyloid protein assemblies are thought to be causative agents in Alzheimer's and other neurodegenerative diseases. Development of oligomer-specific therapeutic agents requires a mechanistic understanding of the oligomerization process. This is a daunting task because amyloidogenic protein oligomers often are metastable and comprise structurally heterogeneous populations in equilibrium with monomers and fibrils. A single methodological approach cannot elucidate the entire protein assembly process. An integrated multidisciplinary program is required. We discuss here the synergistic application of in hydro, in vacuo, and in silico methods to the study of the amyloid beta-protein, the key pathogenetic agent in Alzheimer's disease.