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- Näsström, Thomas, et al.
(författare)
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Synthetic NAC 71-82 Peptides Designed to Produce Fibrils with Different Protofilament Interface Contacts
- 2021
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Ingår i: International Journal of Molecular Sciences. - : MDPI. - 1661-6596 .- 1422-0067. ; 22:17
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Tidskriftsartikel (refereegranskat)abstract
- Alpha-synucleinopathies are featured by fibrillar inclusions in brain cells. Although α-synuclein fibrils display structural diversity, the origin of this diversity is not fully understood. We used molecular dynamics simulations to design synthetic peptides, based on the NAC 71-82 amino acid fragment of α-synuclein, that govern protofilament contacts and generation of twisted fibrillar polymorphs. Four peptides with structures based on either single or double fragments and capped or non-capped ends were selected for further analysis. We determined the fibrillar yield and the structures from these peptides found in the solution after fibrillisation using protein concentration determination assay and circular dichroism spectroscopy. In addition, we characterised secondary structures formed by individual fibrillar complexes using laser-tweezers Raman spectroscopy. Results suggest less mature fibrils, based on the lower relative β-sheet content for double- than single-fragment peptide fibrils. We confirmed this structural difference by TEM analysis which revealed, in addition to short protofibrils, more elongated, twisted and rod-like fibril structures in non-capped and capped double-fragment peptide systems, respectively. Finally, time-correlated single-photon counting demonstrated a difference in the Thioflavin T fluorescence lifetime profiles upon fibril binding. It could be proposed that this difference originated from morphological differences in the fibril samples. Altogether, these results highlight the potential of using peptide models for the generation of fibrils that share morphological features relevant for disease, e.g., twisted and rod-like polymorphs.
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| 2. |
- Karlsson, Björn C. G., et al.
(författare)
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A molecularly imprinted polymer-based detection of Warfarin using time resolved fluorescence spectroscopy
- 2010
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Konferensbidrag (refereegranskat)abstract
- Warfarin is a clinically important drug widely used in the treatment of thrombolic disorders e.g. myocardial infarction and stroke.1 When administered, 99% of the drug present in blood is bound to the transport protein human serum albumin (HSA).2 On account of the fact that HSA demonstrates polymorphism and warfarin has a narrow therapeutic index, careful monitoring of the effect of drug-dosage must be performed.Currently, warfarin’s anticoagulant effect is measured by an indirect method in which the clotting time is measured and correlated to the amount of warfarin present. As current methods for self-monitoring are limited, the development of alternative robust and more sensitive methods is desirable.In this study, we have developed a non-covalent molecularly imprinted polymer3 (MIP) system with selectivity for warfarin.4 The HSA-like binding properties of this MIP were established in previous efforts to develop polymers capable of HSA-like binding of warfarin.5In principle, the fluorophoric nature of warfarin should allow for the fluorescence spectroscopy-based detection of the drug. Recent efforts by us,6-8 using a series of theoretical and spectroscopic studies have highlighted the complex nature of warfarin. In particular, the medium dependent isomerization of this drug illustrates why spectroscopy based methods for the direct detection of the drug has not been forthcoming. Results from these studies have been used to develop a method for the in situ detection of warfarin using time resolved fluorescence spectroscopy.(1) Landefeld, C.; Beyth, R. Anticoagulant-related bleeding - epidemiology, prediction and prevention. Am. J. Med. 1993, 95, 315-328.(2) Yacobi, A.; Udall, J. A.; Levy, G. Comparative pharmacokinetics of coumarin anticoagulants.18 Serum-protein binding as a determinant of warfarin body clearance and anticoagulant effect. Clin. Pharmacol Ther. 1976, 19, 552-558.(3) Alexander, C.; Andersson, H. S.; Andersson, L. I.; Ansell, R. J.; Kirsch, N.; Nicholls, I. A.; O'Mahony, J.; Whitcombe, M. J. Molecular imprinting science and technology: A survey of the literature for the years up to and including 2003. Journal of Molecular Recognition 2006, 19, 106-180.(4) Rosengren, A. M.; Karlsson, B. C. G.; Näslund, I.; Andersson, P. O.; Nicholls, I. A. Time resolved fluorescence spectroscopic detection of the anticoagulant warfarin: A sensor-based method for direct detection in blood plasma. 2010, Submitted.(5) Karlsson, B. C. G.; Rosengren, A. M.; Näslund, I.; Andersson, P. O.; Nicholls, I. A. Synthetic Human Serum Albumin Sudlow I binding site mimics. 2010, Submitted.(6) Karlsson, B. C. G.; Rosengren, A. M.; Andersson, P. O.; Nicholls, I. A. The Spectrophysics of Warfarin: Implications for Protein Binding J. Phys. Chem. B 2007, 111, 10520-10528.(7) Karlsson, B. C. G.; Rosengren, A. M.; Andersson, P. O.; Nicholls, I. A. Molecular Insights on the Two Fluorescence Lifetimes Displayed by Warfarin from Fluorescence Anisotropy and Molecular Dynamics Studies. J. Phys. Chem. B 2009, 113, 7945-7949.(8) Nicholls, I. A.; Karlsson, B. C. G., Rosengren, A. M.. Henschel, H. Warfarin: an Environment-Dependent Switchable Molecular Probe. J. Mol. Recognit. 2010, in press.
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| 3. |
- Karlsson, Björn C. G., et al.
(författare)
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Molecular Insights on the Two Fluorescence Lifetimes Displayed by Warfarin from Fluorescence Anisotropy and Molecular Dynamics Studies
- 2009
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Ingår i: Journal of Physical Chemistry B. - : American Chemical Society (ACS). - 1520-6106 .- 1520-5207. ; 113:22, s. 7945-7949
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Tidskriftsartikel (refereegranskat)abstract
- A series of steady-state fluorescence anisotropy experiments has been performed to demonstrate the presence of a deprotonated open side chain form of warfarin in organic environments. We explain the observed emission-wavelength-dependent anisotropy of warfarin in ethanol, 2-propanol, and acetonitrile due to the coexistence of neutral isomers and deprotonated open side chain forms displaying different fluorescence decay kinetics. To investigate solvent-solute interactions in more detail, a series of molecular dynamics simulations was performed to study warfarin solvation and to predict the time scale of rotational diffusion displayed by this compound. Predictions obtained provide an explanation for the nonzero values in anisotropy observed for neutral isomers of warfarin associated with the short fluorescence lifetime (tau < 0.1 ns) and for an approximately zero anisotropy observed for the deprotonated open side chain form, which is associated with the longer fluorescence lifetime (tau = 0.5-1.6 ns). Finally, we address the potential use of fluorescence anisotropy for an increased understanding of the structural diversity of warfarin in protein binding pockets.
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| 4. |
- Karlsson, Björn C. G., et al.
(författare)
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Synthetic Human Serum Albumin Sudlow I Binding Site Mimics
- 2010
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Ingår i: Journal of Medicinal Chemistry. - : American Chemical Society (ACS). - 0022-2623 .- 1520-4804. ; 53:22, s. 7932-7937
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Tidskriftsartikel (refereegranskat)abstract
- Here, we report the design, synthesis, and characterization of molecularly imprinted polymer (MIP) derived mimics of the human serum albumin (HSA) Sudlow I site-the binding site for the anticoagulant warfarin. MIP design was based upon a combination of experimental (H-1 NMR) and computational (molecular dynamics) methods, Two MIPs and corresponding nonimprinted reference polymers were synthesized and characterized (scanning electron microscopy; nitrogen sorption; and Fourier transform infrared spectroscopy). MIP-ligand recognition was examined using radioligand binding studies, where the largest number of selective sites was found in a warfarin-imprinted methacrylic acid ethylene dimethacrylate copolymer (MAA-MIP). The warfarin selectivity of this MIP was confirmed using radioligand displacement and zonal chromatographic studies. A direct comparison of MIP-warfarin binding characteristics with those of the HSA Sudlow I binding site was made, and similarities in site population (per gram polymer or protein) and affinities were observed. The warfarin selectivity of the MIP suggests its potential for use as a recognition element in a MIP-based warfarin sensor and even as a model to aid in understanding and steering blood-plasma protein-regulated transport processes or even for the development of warfarin sensors.
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