Lability Landscape and Protease Resistance of Human Insulin Amyloid: A New Insight into Its Molecular Properties (original) (raw)
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Quantitative Characterization of Metastability and Heterogeneity of Amyloid Aggregates
Biophysical Journal, 2018
Amyloids are heterogeneous assemblies of extremely stable fibrillar aggregates of proteins. Although biological activities of the amyloids are dependent on its conformation, quantitative evaluation of heterogeneity of amyloids has been difficult. Here we use disaggregation of the amyloids of tetramethylrhodamine-labeled Ab (TMR-Ab) to characterize its stability and heterogeneity. Disaggregation of TMR-Ab amyloids, monitored by fluorescence recovery of TMR, was negligible in native buffer even at low nanomolar concentrations but the kinetics increased exponentially with addition of denaturants such as urea or GdnCl. However, dissolution of TMR-Ab amyloids is different from what is expected in the case of thermodynamic solubility. For example, the fraction of soluble amyloids is found to be independent of total concentration of the peptide at all concentrations of the denaturants. Additionally, soluble fraction is dependent on growth conditions such as temperature, pH, and aging of the amyloids. Furthermore, amyloids undissolved in a certain concentration of the denaturant do not show any further dissolution after dilution in the same solvent; instead, these require higher concentrations of the denaturant. Taken together, our results indicate that amyloids are a heterogeneous ensemble of metastable states. Furthermore, dissolution of each structurally homogeneous member requires a unique threshold concentration of denaturant. Fraction of soluble amyloids as a function of concentration of denaturants is found to be sigmoidal. The sigmoidal curve becomes progressively steeper with progressive seeding of the amyloids, although the midpoint remains unchanged. Therefore, heterogeneity of the amyloids is a major determinant of the steepness of the sigmoidal curve. The sigmoidal curve can be fit assuming a normal distribution for the population of the amyloids of various kinetic stabilities. We propose that the mean and the standard deviation of the normal distribution provide quantitative estimates of mean kinetic stability and heterogeneity, respectively, of the amyloids in a certain preparation.
Characterization of the nanoscale properties of individual amyloid fibrils
Proceedings of The National Academy of Sciences, 2006
We report the detailed mechanical characterization of individual amyloid fibrils by atomic force microscopy and spectroscopy. These self-assembling materials, formed here from the protein insulin, were shown to have a strength of 0.6 +/- 0.4 GPa, comparable to that of steel (0.6-1.8 GPa), and a mechanical stiffness, as measured by Young's modulus, of 3.3 +/- 0.4 GPa, comparable to that of silk (1-10 GPa). The values of these parameters reveal that the fibrils possess properties that make these structures highly attractive for future technological applications. In addition, analysis of the solution-state growth kinetics indicated a breakage rate constant of 1.7 +/- 1.3 x 10(-8) s(-1), which reveals that a fibril 10 mum in length breaks spontaneously on average every 47 min, suggesting that internal fracturing is likely to be of fundamental importance in the proliferation of amyloid fibrils and therefore for understanding the progression of their associated pathogenic disorders.
Biophysical Journal, 2010
To understand and tackle amyloid-related diseases, it is crucial to investigate the factors that modulate amyloid formation of proteins. Our previous studies proved that the N47A mutant of the a-spectrin SH3 (Spc-SH3) domain forms amyloid fibrils quickly under mildly acidic conditions. Here, we analyze how experimental conditions influence the kinetics of assembly and the final morphology of the fibrils. Early formation of curly fibrils occurs after a considerable conformational change of the protein and the concomitant formation of small oligomers. These processes are strongly accelerated by an increase in salt concentration and temperature, and to a lesser extent by a reduction in pH. The rate-limiting step in these events has a high activation enthalpy, which is significantly reduced by an increase in NaCl concentration. At low-to-moderate NaCl concentrations, the curly fibrils convert to straight and twisted amyloid fibrils after long incubation times, but only in the presence of soluble species in the mixture, which suggests that the curly fibrils and the twisted amyloid fibrils are diverging assembly pathways. The results suggest that the influence of environmental variables on protein solvation is crucial in determining the nucleation kinetics, the pathway of assembly, and the final fibril morphology.
Communication: Watching Amyloid Fibrils Grow by Time-lapse Atomic Force Microscopy
Late-onset diabetes is typically associated with amyloid deposits of ®brillar amylin in the pancreatic islets. Aqueous synthetic human amylin spontaneously forms polymorphic ®brils in vitro, and this system was used to examine the dynamics of ®bril assembly. By time-lapse atomic force microscopy (AFM), the growth of individual amylin ®brils on a mica surface was observed over several hours. Prominent was the assembly of a proto®bril with an elongation rate in these experiments of 1.1(AE0.5) nm/minute. The assembly of higher order polymorphic ®brils was also observed. Growth of the proto®brils was bidirectional, i.e. it occurred by elongation at both ends. This ability of AFM to continuously monitor growth, directionality, and changes in morphology for individual ®brils, provides a signi®cant advantage over spectroscopy-based bulk methods which average the growth of many ®brils and typically require 100 to 1000-fold more protein. The time-lapse AFM procedure used for human amylin here is thus likely to be applicable to ®bril formation from other amyloid proteins and peptides.
Stable, Metastable, and Kinetically Trapped Amyloid Aggregate Phases
Biomacromolecules, 2015
Self-assembly of proteins into amyloid fibrils plays a key role in a multitude of human disorders that range from Alzheimer's disease to type II diabetes. Compact oligomeric species, observed early during amyloid formation, are reported as the molecular entities responsible for the toxic effects of amyloid self-assembly. However, the relation between early-stage oligomeric aggregates and late-stage rigid fibrils, which are the hallmark structure of amyloid plaques, has remained unclear. We show that these different structures occupy well-defined regions in a peculiar phase diagram. Lysozyme amyloid oligomers and their curvilinear fibrils only form after they cross a salt and protein concentration-dependent threshold. We also determine a boundary for the onset of amyloid oligomer precipitation. The oligomeric aggregates are structurally distinct from rigid fibrils and are metastable against nucleation and growth of rigid fibrils. These experimentally determined boundaries match well with colloidal model predictions that account for salt-modulated charge repulsion. The model also incorporates the metastable and kinetic character of oligomer phases. Similarities and differences of amyloid oligomer assembly to metastable liquid−liquid phase separation of proteins and to surfactant aggregation are discussed.
Kinetics of different processes in human insulin amyloid formation
Journal of molecular …, 2007
Human insulin has long been known to form amyloid fibrils under given conditions. The molecular basis of insulin aggregation is relevant for modeling the amyloidogenesis process, which is involved in many pathologies, as well as for improving delivery systems, used for diabetes treatments. Insulin aggregation displays a wide variety of morphologies, from small oligomeric filaments to huge floccules, and therefore different specific processes are likely to be intertwined in the overall aggregation. In the present work, we studied the aggregation kinetics of human insulin at low pH and different temperatures and concentrations. The structure and the morphogenesis of aggregates on a wide range of length scales (from monomeric proteins to elongated fibrils and larger aggregates networks) have been monitored by using different experimental techniques: time-lapse atomic force microscopy (AFM), quasi-elastic light-scattering (QLS), small and large angle static light-scattering, thioflavin T fluorescence, and optical microscopy. Our experiments, along with the analysis of scattered intensity distribution, show that fibrillar aggregates grow following a thermally activated heterogeneous coagulation mechanism, which includes both tipto-tip elongation and lateral thickening. Also, the association of fibrils into bundles and larger clusters (up to tens of microns) occurs simultaneously and is responsible for an effective lag-time.
Watching amyloid fibrils grow by time-lapse atomic force microscopy 1 1Edited by W. Baumeister
Journal of Molecular Biology, 1999
Late-onset diabetes is typically associated with amyloid deposits of ®brillar amylin in the pancreatic islets. Aqueous synthetic human amylin spontaneously forms polymorphic ®brils in vitro, and this system was used to examine the dynamics of ®bril assembly. By time-lapse atomic force microscopy (AFM), the growth of individual amylin ®brils on a mica surface was observed over several hours. Prominent was the assembly of a proto®bril with an elongation rate in these experiments of 1.1(AE0.5) nm/minute. The assembly of higher order polymorphic ®brils was also observed. Growth of the proto®brils was bidirectional, i.e. it occurred by elongation at both ends. This ability of AFM to continuously monitor growth, directionality, and changes in morphology for individual ®brils, provides a signi®cant advantage over spectroscopy-based bulk methods which average the growth of many ®brils and typically require 100 to 1000-fold more protein. The time-lapse AFM procedure used for human amylin here is thus likely to be applicable to ®bril formation from other amyloid proteins and peptides.
Watching amyloid fibrils grow by time-lapse atomic force microscopy1
Journal of Molecular Biology, 1999
Late-onset diabetes is typically associated with amyloid deposits of ®brillar amylin in the pancreatic islets. Aqueous synthetic human amylin spontaneously forms polymorphic ®brils in vitro, and this system was used to examine the dynamics of ®bril assembly. By time-lapse atomic force microscopy (AFM), the growth of individual amylin ®brils on a mica surface was observed over several hours. Prominent was the assembly of a proto®bril with an elongation rate in these experiments of 1.1(AE0.5) nm/minute. The assembly of higher order polymorphic ®brils was also observed. Growth of the proto®brils was bidirectional, i.e. it occurred by elongation at both ends. This ability of AFM to continuously monitor growth, directionality, and changes in morphology for individual ®brils, provides a signi®cant advantage over spectroscopy-based bulk methods which average the growth of many ®brils and typically require 100 to 1000-fold more protein. The time-lapse AFM procedure used for human amylin here is thus likely to be applicable to ®bril formation from other amyloid proteins and peptides.
Proceedings of the National Academy of Sciences, 1996
We have studied the fibrillogenesis of synthetic amyloid 13-protein-(1-40) fragment (Aj3) in 0.1 M HCI. At low pH, Af3 formed fibrils at a rate amenable to detailed monitoring by quasi-elastic light-scattering spectroscopy. Examination of the fibrils with circular dichroism spectroscopy and electron microscopy showed them to be highly similar to those found in amyloid plaques. We determined the hydrodynamic radii ofA,8 aggregates during the entire process of fibril nucleation and growth. Above an Aj3 concentration of :0.1 mM, the initial rate of elongation and the final size of fibrils were independent of Aj3 concentration. Below an A,j concen