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Nanomechanical properties of human prion protein amyloid as probed by force spectroscopy.

ABSTRACT: Amyloids are associated with a number of protein misfolding disorders, including prion diseases. In this study, we used single-molecule force spectroscopy to characterize the nanomechanical properties and molecular structure of amyloid fibrils formed by human prion protein PrP90-231. Force-extension curves obtained by specific attachment of a gold-covered atomic force microscope tip to engineered Cys residues could be described by the worm-like chain model for entropic elasticity of a polymer chain, with the size of the N-terminal segment that could be stretched entropically depending on the tip attachment site. The data presented here provide direct information about the forces required to extract an individual monomer from the core of the PrP90-231 amyloid, and indicate that the beta-sheet core of this amyloid starts at residue approximately 164-169. The latter finding has important implications for the ongoing debate regarding the structure of PrP amyloid.

SUBMITTER: Ganchev DN

PROVIDER: S-EPMC2527243 | biostudies-literature | 2008 Sep

REPOSITORIES: biostudies-literature

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Nanomechanical properties of human prion protein amyloid as probed by force spectroscopy.

Ganchev Dragomir N DN Cobb Nathan J NJ Surewicz Krystyna K Surewicz Witold K WK

Biophysical journal 20080606 6

Amyloids are associated with a number of protein misfolding disorders, including prion diseases. In this study, we used single-molecule force spectroscopy to characterize the nanomechanical properties and molecular structure of amyloid fibrils formed by human prion protein PrP90-231. Force-extension curves obtained by specific attachment of a gold-covered atomic force microscope tip to engineered Cys residues could be described by the worm-like chain model for entropic elasticity of a polymer ch ...[more]

PMID: 18539633

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Project description:Amyloid aggregates of a C-truncated Y145Stop mutant of human prion protein, huPrP23-144, associated with a heritable amyloid angiopathy, have previously been shown to contain a compact, relatively rigid, and beta-sheet-rich approximately 30-residue amyloid core near the C-terminus under physiologically relevant conditions. In contrast, the remaining huPrP23-144 residues display considerable conformational dynamics, as evidenced by the absence of corresponding signals in cross-polarization (CP)-based solid-state NMR (SSNMR) spectra under ambient conditions and their emergence in analogous spectra recorded at low temperature on frozen fibril samples. Here, we present the direct observation of residues comprising the flexible N-terminal domain of huPrP23-144 amyloid by using 2D J-coupling-based magic-angle spinning (MAS) SSNMR techniques. Chemical shifts for these residues indicate that the N-terminal domain is effectively an ensemble of protein chains with random-coil-like conformations. Interestingly, a detailed analysis of signal intensities in CP-based 3D SSNMR spectra suggests that non-negligible molecular motions may also be occurring on the NMR time scale within the relatively rigid core of huPrP23-144 amyloid. To further investigate this hypothesis, quantitative measurements of backbone dipolar order parameters and transverse spin relaxation rates were performed for the core residues. The observed order parameters indicate that, on the submicrosecond time scale, these residues are effectively rigid and experience only highly restricted and relatively uniform motions similar to those characteristic for well-structured regions of microcrystalline proteins. On the other hand, significant variations in magnitude of transverse spin relaxation rates were noted for residues present at different locations within the core region and correlated with observed differences in spectral intensities. While interpreted only qualitatively at the present time, the extent of the observed variations in transverse relaxation rates is consistent with the presence of relatively slow, microsecond-millisecond time scale chemical exchange type phenomena within the huPrP23-144 amyloid core.

Project description:BackgroundIncreasing applications of titanium dioxide (TiO2) fine particles (FPs) and nanoparticles (NPs) require coupled knowledge improvement concerning their biokinetic effects. Neutrophils are quickly recruited to titanium implantation areas. Neutrophils mechanical properties display a crucial role on cell physiology and immune responsive functions. Then, micro and nanomechanical characterization assessed by force spectroscopy (FS) technique has been largely applied in this field.ResultsScanning electron microscopy (SEM) images highlighted neutrophils morphological changes along TiO2 FPs and NPs aggregates exposure time (1, 5, and 30 min) compared to controls. FS approaches showed an increasing on attraction forces to TiO2 FPs and NPs treated neutrophils. This group depicted stronger stiffness features than controls just at 1 min of exposure. Treated neutrophils showed a tendency to increase adhesive properties after 1 and 5 min of exposure. These cells maintained comparatively higher elasticity behavior for a longer time possibly due to intense phagocytosis and cell stiffness opposing to the tip indentation. Neutrophils activation caused by FPs and NPs uptake could be related to increasing dissipated energy results.ConclusionsMechanical modifications resulted from TiO2 FPs and NPs aggregates interaction with neutrophils showed increasing stiffness and also cell morphology alteration. Cells treatment by this metal FPs and NPs caused an increase in attractive forces. This event was mainly observed on the initial exposure times probably regarding to the interaction of neutrophils membrane and phagocytosis. Similar results were found to adhesion forces and dissipated energy outcomes. Treated cells presented comparatively higher elasticity behavior for a longer time. SEM images clearly suggested cell morphology alteration along time course probably related to activation, cytoskeleton rearrangement and phagocytosis. This scenario with increase in stiffness strongly suggests a direct relationship over neutrophil rolling, arrest, and transmigration. Scrutinizing these interactions represents an essential step to clarify the mechanisms involved on treatments containing micro and nanomaterials and their fates on the organisms.

Dataset Information

Nanomechanical properties of human prion protein amyloid as probed by force spectroscopy.

Publications

Nanomechanical properties of human prion protein amyloid as probed by force spectroscopy.

Similar Datasets

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