| 1. |
- Dear, Alexander J., et al.
(author)
-
Identification of on- And off-pathway oligomers in amyloid fibril formation
- 2020
-
In: Chemical Science. - : Royal Society of Chemistry (RSC). - 2041-6520 .- 2041-6539. ; 11:24, s. 6236-6247
-
Journal article (peer-reviewed)abstract
- The misfolding and aberrant aggregation of proteins into fibrillar structures is a key factor in some of the most prevalent human diseases, including diabetes and dementia. Low molecular weight oligomers are thought to be a central factor in the pathology of these diseases, as well as critical intermediates in the fibril formation process, and as such have received much recent attention. Moreover, on-pathway oligomeric intermediates are potential targets for therapeutic strategies aimed at interrupting the fibril formation process. However, a consistent framework for distinguishing on-pathway from off-pathway oligomers has hitherto been lacking and, in particular, no consensus definition of on- and off-pathway oligomers is available. In this paper, we argue that a non-binary definition of oligomers' contribution to fibril-forming pathways may be more informative and we suggest a quantitative framework, in which each oligomeric species is assigned a value between 0 and 1 describing its relative contribution to the formation of fibrils. First, we clarify the distinction between oligomers and fibrils, and then we use the formalism of reaction networks to develop a general definition for on-pathway oligomers, that yields meaningful classifications in the context of amyloid formation. By applying these concepts to Monte Carlo simulations of a minimal aggregating system, and by revisiting several previous studies of amyloid oligomers in light of our new framework, we demonstrate how to perform these classifications in practice. For each oligomeric species we obtain the degree to which it is on-pathway, highlighting the most effective pharmaceutical targets for the inhibition of amyloid fibril formation. This journal is
|
|
| 2. |
- Horrocks, Mathew Harry, et al.
(author)
-
Single-molecule imaging of individual amyloid protein aggregates in human biofluids.
- 2016
-
In: ACS chemical neuroscience. - : American Chemical Society (ACS). - 1948-7193. ; 16;7:3, s. 399-406
-
Journal article (peer-reviewed)abstract
- The misfolding and aggregation of proteins into amyloid fibrils characterizes many neurodegenerative disorders such as Parkinson's and Alzheimer's diseases. We report here a method, termed SAVE (single aggregate visualization by enhancement) imaging, for the ultra-sensitive detection of individual amyloid fibrils and oligomers using single-molecule fluorescence microscopy. We demonstrate that this method is able to detect the presence of amyloid aggregates of alpha-synuclein, tau and amyloid-β. In addition, we show that aggregates can also be identified in human cerebrospinal fluid (CSF). Significantly, we see a two-fold increase in the average aggregate concentration in CSF from PD patients compared to age-matched controls. Taken together, we conclude that this method provides an opportunity to characterize the structural nature of amyloid aggregates in a key biofluid, and therefore has the potential to study disease progression in both animal models and humans to enhance our understanding of neurodegenerative disorders.
|
|
| 3. |
- Iesmantavicius, Vytautas, et al.
(author)
-
Helical Propensity in an Intrinsically Disordered Protein Accelerates Ligand Binding
- 2014
-
In: Angewandte Chemie International Edition. - : Wiley. - 1433-7851 .- 1521-3773. ; 53:6, s. 1548-1551
-
Journal article (peer-reviewed)abstract
- Many intrinsically disordered proteins fold upon binding to other macromolecules. The secondary structure present in the well-ordered complex is often formed transiently in the unbound state. The consequence of such transient structure for the binding process is, however, not clear. The activation domain of the activator for thyroid hormone and retinoid receptors (ACTR) is intrinsically disordered and folds upon binding to the nuclear coactivator binding domain (NCBD) of the CREB binding protein. A number of mutants was designed that selectively perturbs the amount of secondary structure in unbound ACTR without interfering with the intermolecular interactions between ACTR and NCBD. Using NMR spectroscopy and fluorescence-monitored stopped-flow kinetic measurements we show that the secondary structure content in helix1 of ACTR indeed influences the binding kinetics. The results thus support the notion of preformed secondary structure as an important determinant for molecular recognition in intrinsically disordered proteins.
|
|
| 4. |
- Karlsson, Elin, et al.
(author)
-
Coupled Binding and Helix Formation Monitored by Synchrotron-Radiation Circular Dichroism
- 2019
-
In: Biophysical Journal. - : CELL PRESS. - 0006-3495 .- 1542-0086. ; 117:4, s. 729-742
-
Journal article (peer-reviewed)abstract
- Intrinsically disordered proteins organize interaction networks in the cell in many regulation and signaling processes. These proteins often gain structure upon binding to their target proteins in multistep reactions involving the formation of both secondary and tertiary structure. To understand the interactions of disordered proteins, we need to understand the mechanisms of these coupled folding and binding reactions. We studied helix formation in the binding of the molten globule-like nuclear coactivator binding domain and the disordered interaction domain from activator of thyroid hormone and retinoid receptors. We demonstrate that helix formation in a rapid binding reaction can be followed by stopped-flow synchrotron-radiation circular dichroism (CD) spectroscopy and describe the design of such a beamline. Fluorescence-monitored binding experiments of activator of thyroid hormone and retinoid receptors and nuclear coactivator binding domain display several kinetic phases, including one concentration-independent phase, which is consistent with an intermediate stabilized at high ionic strength. Time-resolved CD experiments show that almost all helicity is formed upon initial association of the proteins or separated from the encounter complex by only a small energy barrier. Through simulation of mechanistic models, we show that the intermediate observed at high ionic strength likely involves a structural rearrangement with minor overall changes in helicity. Our experiments provide a benchmark for simulations of coupled binding reactions and demonstrate the feasibility of using synchrotron-radiation CD for mechanistic studies of protein-protein interactions.
|
|
| 5. |
- Kjaergaard, Magnus, et al.
(author)
-
Solution structure of recombinant somatomedin B domain from vitronectin produced in Pichia pastoris.
- 2007
-
In: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 16:9
-
Journal article (peer-reviewed)abstract
- The cysteine-rich somatomedin B domain (SMB) of the matrix protein vitronectin is involved in several important biological processes. First, it stabilizes the active conformation of the plasminogen activator inhibitor (PAI-1); second, it provides the recognition motif for cell adhesion via the cognate integrins (alpha(v)beta(3), alpha(v)beta(5), and alpha(IIb)beta(3)); and third, it binds the complex between urokinase-type plasminogen activator (uPA) and its glycolipid-anchored receptor (uPAR). Previous structural studies on SMB have used recombinant protein expressed in Escherichia coli or SMB released from plasma-derived vitronectin by CNBr cleavage. However, different disulfide patterns and three-dimensional structures for SMB were reported. In the present study, we have expressed recombinant human SMB by two different eukaryotic expression systems, Pichia pastoris and Drosophila melanogaster S2-cells, both yielding structurally and functionally homogeneous protein preparations. Importantly, the entire population of our purified, recombinant SMB has a solvent exposure, both as a free domain and in complex with PAI-1, which is indistinguishable from that of plasma-derived SMB as assessed by amide hydrogen ((1)H/(2)H) exchange. This solvent exposure was only reproduced by one of three synthetic SMB products with predefined disulfide connectivities corresponding to those published previously. Furthermore, this connectivity was also the only one to yield a folded and functional domain. The NMR structure was determined for free SMB produced by Pichia and is largely consistent with that solved by X-ray crystallography for SMB in complex with PAI-1.
|
|
