The presence of β‐sheets in the core of amyloid fibrils raised questions as to whether or not β‐sheet‐containing proteins, such as transthyretin, are predisposed to form such fibrils. However, we ...show here that the molecular structure of amyloid fibrils differs more generally from the β‐sheets in native proteins. This difference is evident from the amide I region of the infrared spectrum and relates to the distribution of the ϕ/ψ dihedral angles within the Ramachandran plot, the average number of strands per sheet, and possibly, the β‐sheet twist. These data imply that amyloid fibril formation from native β‐sheet proteins can involve a substantial structural reorganization.
We report the design and first applications of a tandem mass spectrometer (a quadrupole time-of-flight mass spectrometer) optimized for the transmission and analysis of large macromolecular ...assemblies. Careful control of the pressure gradient in the different pumping stages of the instrument has been found to be essential for the detection of macromolecular particles. Such assemblies are, however, difficult to analyze by tandem-MS approaches, because they give rise to signals above m/z 3000−4000, the limit for commercial quadrupoles. By reducing the frequency of the quadrupole to 300 kHz and using it as a narrow-band mass filter, we show that it is possible to isolate ions from a single peak at m/z 22 000 in a window as narrow as 22 m/z units. Using cesium iodide cluster signals, we show that the mass range in the time-of-flight (TOF) analyzer extends beyond m/z 90 000, in theory to more than m/z 150 000. We also demonstrate that the resolution of the instrument is greater than 3000 at m/z 44 500. Tandem-MS capabilities are illustrated by separating components from heterooligomeric assemblies formed between tetrameric transthyretin, thyroxine, retinol-binding protein, and retinol. Isolation of a single charge state at m/z 5340 in the quadrupole and subsequent collision-induced dissociation (CID) in the gas-filled collision cell leads to the formation of ions from individual subunits and subcomplexes, identified by their mass and charge in the TOF analyzer.
Corporate venture capital and Cambridge McCammon, Margaret G; Pio, Edwina; Barakat, Shima ...
Nature biotechnology,
10/2014, Letnik:
32, Številka:
10
Journal Article
Recenzirano
Odprti dostop
As the pressure on public funding continues to grow, academic scientists are increasingly contemplating the merits of a biotech spin-out venture to advance their research. The challenge today is that ...traditional venture capital has largely moved away from early stage innovations, seeking the safety of more developed, de-risked technologies for their portfolio pipeline. By contrast, the pharmaceutical industry has demonstrated a growing interest in this field and is now a major player in funding biotech startups.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Transthyretin (TTR) amyloidosis is a fatal disease for which new therapeutic approaches are urgently needed. We have designed two palindromic ligands, ...2,2'-(4,4'-(heptane-1,7-diylbis(oxy))bis(3,5-dichloro-4,1-phenylene)) bis(azanediyl)dibenzoic acid (mds84) and 2,2'-(4,4'-(undecane-1,11-diylbis(oxy))bis(3,5-dichloro-4,1|-phenylene)) bis(azanediyl)dibenzoic acid (4ajm15), that are rapidly bound by native wild-type TTR in whole serum and even more avidly by amyloidogenic TTR variants. One to one stoichiometry, demonstrable in solution and by MS, was confirmed by X-ray crystallographic analysis showing simultaneous occupation of both T4 binding sites in each tetrameric TTR molecule by the pair of ligand head groups. Ligand binding by native TTR was irreversible under physiological conditions, and it stabilized the tetrameric assembly and inhibited amyloidogenic aggregation more potently than other known ligands. These superstabilizers are orally bioavailable and exhibit low inhibitory activity against cyclooxygenase (COX). They offer a promising platform for development of drugs to treat and prevent TTR amyloidosis.
The flight of macromolecular complexes in a mass spectrometer Sobott, Frank; McCammon, Margaret G.; Hernández, Helena ...
Philosophical transactions of the Royal Society of London. Series A: Mathematical, physical, and engineering sciences,
02/2005, Letnik:
363, Številka:
1827
Journal Article
Recenzirano
The discovery that conditions can be found such that non-covalent macromolecular complexes can survive the transition from solution to gas phase and remain intact during their flight in a mass ...spectrometer is an intriguing observation. While the nature of the interaction between the components, either ionic, hydrophobic or van der Waals, undoubtedly has an effect on the stability of these gas phase species, the role of small molecules in conferring additional stability is often overlooked. Here we review historical aspects of the development of mass spectrometry for macromolecular complexes with particular focus on the role of small molecules in stabilizing gas-phase complexes. Moreover, we demonstrate how the dissociation of small molecules from subunits within a macromolecular complex can be used to probe the topological arrangement. Overall, therefore, we show that mass spectrometry used in this way is capable of addressing features of the energy landscape not readily accessed by traditional structural biology approaches.
Transthyretin is a tetrameric protein associated with the commonest form of systemic amyloid disease. Using isotopically labeled proteins and mass spectrometry, we compared subunit exchange in ...wild-type transthyretin with that of the variant associated with the most aggressive form of the disease, L55P. Wild-type subunit exchange occurs via both monomers and dimers, whereas exchange via dimers is the dominant mechanism for the L55P variant. Because patients with the L55P mutation are heterozygous, expressing both proteins simultaneously, we also analyzed the subunit exchange reaction between wild-type and L55P tetramers. We found that hybrid tetramers containing two or three L55P subunits dominate in the early stages of the reaction. Surprisingly, we also found that, in the presence of L55P transthyretin, the rate of dissociation of wild-type transthyretin is increased. This implies interactions between the two proteins that accelerate the formation of hybrid tetramers, a result with important implications for transthyretin amyloidosis.
Amyloid fibrils are typically rigid, unbranched structures with diameters of ∼10 nm and lengths up to several micrometres, and are associated with more than 20 diseases including Alzheimer's disease ...and type II diabetes. Insulin is a small, predominantly α-helical protein consisting of 51 residues in two disulfide-linked polypeptide chains that readily assembles into amyloid fibrils under conditions of low pH and elevated temperature. We demonstrate here that both the A-chain and the B-chain of insulin are capable of forming amyloid fibrils in isolation under similar conditions, with fibrillar morphologies that differ from those composed of intact insulin. Both the A-chain and B-chain fibrils were found to be able to cross-seed the fibrillization of the parent protein, although these reactions were substantially less efficient than self-seeding with fibrils composed of full-length insulin. In both cases, the cross-seeded fibrils were morphologically distinct from the seeding material, but shared common characteristics with typical insulin fibrils, including a very similar helical repeat. The broader distribution of heights of the cross-seeded fibrils compared to typical insulin fibrils, however, indicates that their underlying protofilament hierarchy may be subtly different. In addition, and remarkably in view of this seeding behavior, the soluble forms of the A-chain and B-chain peptides were found to be capable of inhibiting insulin fibril formation. Studies using mass spectrometry suggest that this behavior might be attributable to complex formation between insulin and the A-chain and B-chain peptides. The finding that the same chemical form of a polypeptide chain in different physical states can either stimulate or inhibit the conversion of a protein into amyloid fibrils sheds new light on the mechanisms underlying fibril formation, fibril strain propagation and amyloid disease initiation and progression.
We have used tandem mass spectrometry to examine the stoichiometry and binding sites of trp molecules in various assemblies of the protein complex TRAP. The results show that TRAP forms oligomers ...containing 11 and 12 subunits. MS/MS experiments show that up to 11 trp molecules bind to the 12-mer but that during gas-phase dissociation 5 then 6 trp molecules are released reflecting the different gas-phase stabilities of the partially ligated forms. At high trp concentrations, the protein assembles to form a double ring structure. Tandem mass spectrometry reveals that it is composed of 24 subunits with up to 22 molecules of trp. Dissociation of the complex reveals the same dissociation pathway as for the single ring structure, allowing us to propose a model for the assembly of the TRAP 24-mer based on the different environments of trp molecules. More generally, these results demonstrate the power of tandem mass spectrometry for defining the stoichiometry and quaternary structural arrangement of subunits and ligands within a 46-component multiprotein multiligand complex.
We report the secreted expression by Pichia pastoris of two human lysozyme variants F57I and W64R, associated with systemic amyloid disease, and describe their characterization by biophysical ...methods. Both variants have a substantially decreased thermostability compared with wild‐type human lysozyme, a finding that suggests an explanation for their increased propensity to form fibrillar aggregates and generate disease. The secreted yields of the F57I and W64R variants from P. pastoris are 200‐ and 30‐fold lower, respectively, than that of wild‐type human lysozyme. More comprehensive analysis of the secretion levels of 10 lysozyme variants shows that the low yields of these secreted proteins, under controlled conditions, can be directly correlated with a reduction in the thermostability of their native states. Analysis of mRNA levels in this selection of variants suggests that the lower levels of secretion are due to post‐transcriptional processes, and that the reduction in secreted protein is a result of degradation of partially folded or misfolded protein via the yeast quality control system. Importantly, our results show that the human disease‐associated mutations do not have levels of expression that are out of line with destabilizing mutations at other sites. These findings indicate that a complex interplay between reduced native‐state stability, lower secretion levels, and protein aggregation propensity influences the types of mutation that give rise to familial forms of amyloid disease.
In a climate of increased expectation for the translation of research, academic clinical research units are looking at new ways to streamline their operation and maintain effective translational ...support services. Clinical research, although undeniably expensive, is an essential step in the translation of any medical breakthrough, and as a result, many academic clinical research units are actively looking to expand their clinical services despite financial pressures. We examine some of the hybrid academic-business models in 19 clinical research centers within the Clinical and Translational Science Award consortium that are emerging to address the issue of cost recovery of clinical research that is supported by the United States federal government. We identify initiatives that have succeeded or failed, essential supporting and regulatory components, and lessons learned from experience to design an optimal cost recovery model and a timeline for its implementation.
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