| 51. |
- Villebeck, Laila, et al.
(författare)
-
Domain-specific chaperone-induced expansion is required for ß-actin folding : a comparison of ß-actin conformations upon interactions with GroEL and tail-less complex polypeptide 1 ring complex (TRiC)
- 2007
-
Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 46:44, s. 12639-12647
-
Tidskriftsartikel (refereegranskat)abstract
- Actin, an abundant cytosolic protein in eukaryotic cells, is dependent on the interaction with the chaperonin tail-less complex polypeptide 1 ring complex (TRiC) to fold to the native state. The prokaryotic chaperonin GroEL also binds non-native ß-actin, but is unable to guide ß-actin toward the native state. In this study we identify conformational rearrangements in ß-actin, by observing similarities and differences in the action of the two chaperonins. A cooperative collapse of ß-actin from the denatured state to an aggregation-prone intermediate is observed, and insoluble aggregates are formed in the absence of chaperonin. In the presence of GroEL, however, >90% of the aggregation-prone actin intermediate is kept in solution, which shows that the binding of non-native actin to GroEL is effective. The action of GroEL on bound flourescein-labeled ß-actin was characterized, and the structural rearrangement was compared to the case of the ß-actin-TRiC complex, employing the homo fluorescence resonance energy transfer methodology previously used [Villebeck, L., Persson, M., Luan, S.-L., Hammarström, P., Lindgren, M., and Jonsson, B.-H. (2007) Biochemistry 46 (17), 5083-93]. The results suggest that the actin structure is rearranged by a "binding-induced expansion" mechanism in both TRiC and GroEL, but that binding to TRiC, in addition, causes a large and specific separation of two subdomains in the ß-actin molecule, leading to a distinct expansion of its ATP-binding cleft. Moreover, the binding of ATP and GroES has less effect on the GroEL-bound ß-actin molecule than the ATP binding to TRiC, where it leads to a major compaction of the ß-actin molecule. It can be concluded that the specific and directed rearrangement of the ß-actin structure, seen in the natural ß-actin-TRiC system, is vital for guiding ß-actin to the native state. © 2007 American Chemical Society.
|
|
| 52. |
- Wirehn, J., et al.
(författare)
-
Activity, folding, misfolding, and aggregation in vitro of the naturally occurring human tissue factor mutant R200W
- 2005
-
Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 44:18, s. 6755-6763
-
Tidskriftsartikel (refereegranskat)abstract
- Tissue factor (TF), a small transmembrane receptor, binds factor VIIa (FVIIa), and the formed complex initiates blood coagulation by proteolytic activation of substrate factors IX and X. A naturally occurring mutation in the human TF gene was recently reported, where a single-base substitution results in an R200W mutation in the TF extracellular domain [Zawadzki, C., Preudhomme, C., Gavériaux, V., Amouyel, P., and Jude, B. (2002) Thromb. Haemost. 87, 540-541]. This mutation appears to be associated with low monocyte TF expression and may protect against thrombosis but has not been associated with any pathological condition, and individuals who present the heterozygous trait appear healthy. Here, we report the activity, folding, and aggregation behavior of the R200W mutant of the 219-residue soluble extracellular domain of TF (sTFR200W) compared to that of the wild-type protein (sTF wt). No differences in stability or FVIIa cofactor activity but an impaired ability to promote FX activation at physiological conditions between the sTFR200W mutant and sTFwt were evident. Increased binding of 1-anilino-8-naphthalene-sulfonic acid (ANS) to sTFR200W indicated a population of partially folded intermediates during denaturation. sTFR200W showed a dramatically increased propensity for aggregate formation compared to sTFwt at mildly acidic pHs, with an increased rate of aggregation during conditions, promoting the intermediate state. The lowered pH resistance could explain the loss of sTFR200W in vivo because of aggregation of the mutant. The intrinsic structure of the sTF aggregates appears reminiscent of amyloid fibrils, as revealed by thioflavin T fluorescence, atomic force microscopy, and transmission electron microscopy. We conclude that the lowered activity for FX activation and the propensity of the mutant protein to misfold and aggregate will both contribute to decreased coagulation activity in TFR200W carriers, which could protect from thrombotic disease. © 2005 American Chemical Society.
|
|
| 53. |
- Zako, T., et al.
(författare)
-
Structure and cytotoxicity of novel insulin noodle-like filamentous amyloids
- 2009
-
Ingår i: The FEBS Journal. - : Wiley-Blackwell. - 1742-464X .- 1742-4658. ; 276:Suppl. s1, s. 173-173
-
Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Insulin is a small peptide hormone that is known to form proteinassembly called amyloid fibrils under acidic conditions. We havepreviously shown that filamentous (‘noodle’-like) insulin amyloidwhich was morphologically different from fibrous (‘needle’-like)insulin amyloid was formed in the presence of a reducing agent,tris (2-carboxyethyl) phosphine hydrochloride (TCEP). The CDspectra showed that both of insulin fibrils and filaments containa beta-sheet structure. Nevertheless, Thioflavin T (ThT) bindingproperty was very different between them, suggesting a differencein inner structure. In this study, we examined their cell toxicitiesusing two different cell lines with MTT assay, and also examineddifference in their inner structures using novel luminescent conjugatedpolyelectrolyte probes (LCPs)1–4. The cytotoxicity of theinsulin filaments against rat PC12 and human HEK293 cell linewas also extremely low while the fibrils were toxic, suggestingthat the insulin filaments were generally nontoxic. This findingsupports the idea that cell toxicity of amyloids correlates withtheir morphology. The fluorescence measurement in the presenceof Polythiophene acetic acid (PTAA)1–4, one of the conformationsensitive LCPs, showed that PTAA weakly bound to the insulinfilaments and that the insulin fibrils’ spectrum revealed a spectral red shift in comparison to the PTAA-filaments interaction. Thissuggests that the insulin ‘noodle’-like filaments are formed byloose assembly of insulin molecules.References:1. Nilsson et al., Adv Mat 2008; 20: 2639.2. Chem Bio Chem 2006; 7:1096.3. ACS Chem Biol 2007; 4: 553.4. Sigurdson et al., Nature Methods 2007; 4: 1023.
|
|
| 54. |
- Zettersten, Camilla, et al.
(författare)
-
The influence of the thin-layer flow cell design on the mass spectra when coupling electrochemistry to electrospray ionisation mass spectrometry
- 2006
-
Ingår i: Journal of Electroanalytical Chemistry. - : Elsevier BV. - 0022-0728 .- 1873-2569 .- 1572-6657. ; 590:1, s. 90-99
-
Tidskriftsartikel (refereegranskat)abstract
- The influence of the flow cell configuration on the mass spectra obtained when coupling an electrochemical thin-layer flow cell to electrospray mass spectrometry (ESI-MS) has been investigated. It is shown that interferences due to the electrochemical reaction on the counter electrode and/or the absence of 100% conversion efficiency can alter the mass spectra when conventional thin-layer flow cells are used in conjunction with ESI-MS. The effects, which affect the intensities and distribution of the peaks in the mass spectra, can result in the inability to detect products formed at the working electrode. Comparisons of mass spectra, generated after the electrochemical oxidation of a dinuclear Mn complex (where bpmp = 2,6-bis[bis(2-pyridylmethyl) amino]methyl-4-methylphenol) using two different thin-layer flow cells clearly show that the potential dependence and appearance of the mass spectra depend on the flow cell configuration used. The use of a modified thin-layer flow cell, in which the counter electrode had been separated from the working electrode, gave rise to significantly increased intensities for the oxidised MnIII,IV state of the complex. With the conventional unmodified cell, the corresponding complex was only seen for considerably higher oxidation potentials. The different results can be explained by the reduced risk of redox cycling and interferences due to species generated at the counter electrode with the modified cell. As interferences due to the counter electrode reactions likewise may be expected with many coulometric flow cells, the electrochemical cell design clearly needs to be considered when using electrochemistry coupled to ESI-MS to study electrochemical reactions.
|
|
| 55. |
- Åslund, Andreas, et al.
(författare)
-
Novel Pentameric Thiophene Derivatives for in Vitro and in Vivo Optical Imaging of a Plethora of Protein Aggregates in Cerebral Amyloidoses
- 2009
-
Ingår i: ACS CHEMICAL BIOLOGY. - : American Chemical Society (ACS). - 1554-8929 .- 1554-8937. ; 4:8, s. 673-684
-
Tidskriftsartikel (refereegranskat)abstract
- Molecular probes for selective Identification of protein aggregates are important to advance our understanding of the molecular pathogenesis underlying cerebral amyloidoses. Here we report the chemical design of pentameric thiophene derivatives, denoted luminescent conjugated oligothiophenes (LCOs), which could be used for real-time visualization of cerebral protein aggregates in transgenic mouse models of neurodegenerative diseases by multiphoton microscopy. One of the LCOs, p-FTAA, could be utilized for ex vivo spectral assignment of distinct prion deposits from two, mouse-adapted prion strains. p-FTAA also revealed a transient soluble pre-fibrillar non-thioflavinophilic A beta-assemblies during in vitro fibrillation of A beta peptides. In brain tissue samples, A beta deposits and neurofibrillary tangles (NFTs) were readily identified by a strong fluorescence from p-FTAA and the LCO staining showed complete co-localliation with conventional antibodies (6E10 and AT8). In addition, a patchy islet-like staining of individual A beta plaque was unveiled by the anti-oligomer A11 antibody during co-staining with p-FTAA. The major hallmarks of Alzheimers disease, namely, A beta aggregates versus NFTs, could also be distinguished because of distinct emission spectra from p-FTAA. Overall, we demonstrate that LCOs can be utilized as powerful practical research tools for studying protein aggregation diseases and facilitate the study of amyloid origin, evolution and maturation, A beta-tau interactions, and pathogenesis both ex vivo and in vivo.
|
|
| 56. |
- Åslund, Andreas, et al.
(författare)
-
Studies of luminescent conjugated polythiophene derivatives-Enhanced spectral discrimination of protein conformational states
- 2007
-
Ingår i: Bioconjugate chemistry. - : American Chemical Society (ACS). - 1043-1802 .- 1520-4812. ; 18:6, s. 1860-1868
-
Tidskriftsartikel (refereegranskat)abstract
- Improved probes for amyloid fibril formation are advantageous for the early detection and better understanding of this disease-associated process. Here, we report a comparative study of eight luminescent conjugated polythiophene derivates (LCPs) and their discrimination of a protein (insulin) in the native or amyloid-like fibrillar state. For two of the LCPs, the synthesis is reported. Compared to their monomer-based analogues, trimer-based LCPs showed significantly better optical signal specificity for amyloid-like fibrils, seen from increased quantum yield and spectral shift. The trimer-based LCPs alone were highly quenched and showed little interaction with native insulin, as seen from analytical ultracentrifugation and insignificant spectral differences from the trimer-based LCP in buffered and native protein solution. Hence, the trimer-based LCPs showed enhanced discrimination between the amyloid-like fibrillar state and the corresponding native protein.
|
|
