23 research outputs found
Amyloids: From molecular structure to mechanical properties
AbstractMany proteins of diverse sequence, structure and function self-assemble into morphologically similar fibrillar aggregates known as amyloids. Amyloids are remarkable polymers in several respects. First of all, amyloids can be formed from proteins with very different amino acid sequences; the common denominator is that the individual proteins constituting the amyloid fold predominantly into a β-sheet structure. Secondly, the formation of the fibril occurs through non-covalent interactions between primarily the β-sheets, causing the monomers to stack into fibrils. The fibrils are remarkably robust, considering that the monomers are bound non-covalently. Finally, a common characteristic of fibrils is their unbranched, straight, fiber-like structure arising from the intertwining of the multiple β-sheet filaments. These remarkably ordered and stable nanofibrils can be useful as building blocks for protein-based functional materials, but they are also implicated in severe neurodegenerative diseases. The overall aim of this article is to highlight recent efforts aimed at obtaining insights into amyloid proteins on different length scales. Starting from molecular information on amyloids, single fibril properties and mechanical properties of networks of fibrils are described. Specifically, we focus on the self-assembly of amyloid protein fibrils composed of peptides and denatured model proteins, as well as the influence of inhibitors of fibril formation. Additionally, we will demonstrate how the application of recently developed vibrational spectroscopic techniques has emerged as a powerful approach to gain spatially resolved information on the structure–function relation of amyloids. While spectroscopy provides information on local molecular conformations and protein secondary structure, information on the single fibril level has been developed by diverse microscopic techniques. The approaches to reveal basic mechanical properties of single fibrils like bending rigidity, shear modulus, ultimate tensile strength and fracture behavior are illustrated. Lastly, mechanics of networks of amyloid fibrils, typically forming viscoelastic gels are outlined, with a focus on (micro-) rheological properties. The resulting fundamental insights are essential for the rational design of novel edible and biodegradable protein-based polymers, but also to devise therapeutic strategies to combat amyloid assembly and accumulation during pathogenic disorders
Morphology and Persistence Length of Amyloid Fibrils Are Correlated to Peptide Molecular Structure
Nanoscale Heterogeneity of the Molecular Structure of Individual hIAPP Amyloid Fibrils Revealed with Tip-Enhanced Raman Spectroscopy
Type 2 diabetes mellitus is characterized by the pathological deposition of fibrillized protein, known as amyloids. It is thought that oligomers and/or amyloid fibrils formed from human islet amyloid polypeptide (hIAPP or amylin) cause cell death by membrane damage. The molecular structure of hIAPP amyloid fibrils is dominated by β-sheet structure, as probed with conventional infrared and Raman vibrational spectroscopy. However, with these techniques it is not possible to distinguish between the core and the surface structure of the fibrils. Since the fibril surface crucially affects amyloid toxicity, it is essential to know its structure. Here the surface molecular structure and amino acid residue composition of hIAPP fibrils are specifically probed with nanoscale resolution using tip-enhanced Raman spectroscopy (TERS). The fibril surface mainly contains unordered or α-helical structures, in contrast to the β-sheet-rich core. This experimentally validates recent models of hIAPP amyloids based on NMR measurements. Spatial mapping of the surface structure reveals a highly heterogeneous surface structure. Finally, TERS can probe fibrils formed on a lipid interface, which is more representative of amyloids in vivo
Nanoscale Heterogeneity of the Molecular Structure of Individual hIAPP Amyloid Fibrils Revealed with Tip-Enhanced Raman Spectroscopy
Type 2 diabetes mellitus is characterized by the pathological deposition of fibrillized protein, known as amyloids. It is thought that oligomers and/ or amyloid fibrils formed from human islet amyloid polypeptide (hIAPP or amylin) cause cell death by membrane damage. The molecular structure of hIAPP amyloid fibrils is dominated by beta-sheet structure, as probed with conventional infrared and Raman vibrational spectroscopy. However, with these techniques it is not possible to distinguish between the core and the surface structure of the fibrils. Since the fibril surface crucially affects amyloid toxicity, it is essential to know its structure. Here the surface molecular structure and amino acid residue composition of hIAPP fibrils are specifically probed with nanoscale resolution using tip-enhanced Raman spectroscopy (TERS). The fibril surface mainly contains unordered or alpha-helical structures, in contrast to the beta-sheet-rich core. This experimentally validates recent models of hIAPP amyloids based on NMR measurements. Spatial mapping of the surface structure reveals a highly heterogeneous surface structure. Finally, TERS can probe fibrils formed on a lipid interface, which is more representative of amyloids in vivo
Amyloids: From molecular structure to mechanical properties
Many proteins of diverse sequence, structure and function self-assemble into morphologically similar fibrillar aggregates known as amyloids. Amyloids are remarkable polymers in several respects. First of all, amyloids can be formed from proteins with very different amino acid sequences; the common denominator is that the individual proteins constituting the amyloid fold predominantly into a β-sheet structure. Secondly, the formation of the fibril occurs through non-covalent interactions between primarily the β-sheets, causing the monomers to stack into fibrils. The fibrils are remarkably robust, considering that the monomers are bound non-covalently. Finally, a common characteristic of fibrils is their unbranched, straight, fiber-like structure arising from the intertwining of the multiple β-sheet filaments. These remarkably ordered and stable nanofibrils can be useful as building blocks for protein-based functional materials, but they are also implicated in severe neurodegenerative diseases. The overall aim of this article is to highlight recent efforts aimed at obtaining insights into amyloid proteins on different length scales. Starting from molecular information on amyloids, single fibril properties and mechanical properties of networks of fibrils are described. Specifically, we focus on the self-assembly of amyloid protein fibrils composed of peptides and denatured model proteins, as well as the influence of inhibitors of fibril formation. Additionally, we will demonstrate how the application of recently developed vibrational spectroscopic techniques has emerged as a powerful approach to gain spatially resolved information on the structure-function relation of amyloids. While spectroscopy provides information on local molecular conformations and protein secondary structure, information on the single fibril level has been developed by diverse microscopic techniques. The approaches to reveal basic mechanical properties of single fibrils like bending rigidity, shear modulus, ultimate tensile strength and fracture behavior are illustrated. Lastly, mechanics of networks of amyloid fibrils, typically forming viscoelastic gels are outlined, with a focus on (micro-) rheological properties. The resulting fundamental insights are essential for the rational design of novel edible and biodegradable protein-based polymers, but also to devise therapeutic strategies to combat amyloid assembly and accumulation during pathogenic disorders
Nanoscale Heterogeneity of the Molecular Structure of Individual hIAPP Amyloid Fibrils Revealed with Tip-Enhanced Raman Spectroscopy
Type 2 diabetes mellitus is characterized by the pathological deposition of fibrillized protein, known as amyloids. It is thought that oligomers and/ or amyloid fibrils formed from human islet amyloid polypeptide (hIAPP or amylin) cause cell death by membrane damage. The molecular structure of hIAPP amyloid fibrils is dominated by beta-sheet structure, as probed with conventional infrared and Raman vibrational spectroscopy. However, with these techniques it is not possible to distinguish between the core and the surface structure of the fibrils. Since the fibril surface crucially affects amyloid toxicity, it is essential to know its structure. Here the surface molecular structure and amino acid residue composition of hIAPP fibrils are specifically probed with nanoscale resolution using tip-enhanced Raman spectroscopy (TERS). The fibril surface mainly contains unordered or alpha-helical structures, in contrast to the beta-sheet-rich core. This experimentally validates recent models of hIAPP amyloids based on NMR measurements. Spatial mapping of the surface structure reveals a highly heterogeneous surface structure. Finally, TERS can probe fibrils formed on a lipid interface, which is more representative of amyloids in vivo
The Polyphenol EGCG Inhibits Amyloid Formation Less Efficiently at Phospholipid Interfaces than in Bulk Solution
Age-related diseases, like Alzheimer’s disease
and type
2 diabetes mellitus, are characterized by protein misfolding and the
subsequent pathological deposition of fibrillized protein, also called
amyloid. Several classes of amyloid-inhibitors have recently been
tested, traditionally under bulk conditions. However, it has become
apparent that amyloid fibrils and oligomers assemble and exert their
cytotoxic effect at cellular membranes, rather than in bulk solution.
Knowledge is therefore required of inhibitor activity specifically
at the phospholipid membrane interface. Here we show, using surface-specific
sum-frequency generation (SFG) spectroscopy and atomic force microscopy
(AFM), that the commonly used (−)-epigallocatechin gallate
(EGCG) is a much less efficient amyloid inhibitor at a phospholipid
interface than in bulk solution. Moreover, EGCG is not able to disaggregate
existing amyloid fibrils at a phospholipid interface, in contrast
to its behavior in bulk. Our results show that interfaces significantly
affect the efficiency of inhibition by EGCG inhibitors and should
therefore be considered during the design and testing of amyloid inhibitors
Shear stress-dependent downregulation of the adhesion-G protein-coupled receptor CD97 on circulating leukocytes upon contact with its ligand CD55
Adhesion G protein-coupled receptors (aGPCRs) are two-subunit molecules, consisting of an adhesive extracellular α subunit that couples noncovalently to a seven-transmembrane β subunit. The cooperation between the two subunits and the effect of endogenous ligands on the functioning of aGPCRs is poorly understood. In this study, we investigated the interaction between the pan-leukocyte aGPCR CD97 and its ligand CD55. We found that leukocytes from CD55-deficient mice express significantly increased levels of cell surface CD97 that normalized after transfer into wild-type mice because of contact with CD55 on both leukocytes and stromal cells. Downregulation of both CD97 subunits occurred within minutes after first contact with CD55 in vivo, which correlated with an increase in plasma levels of soluble CD97. In vitro, downregulation of CD97 on CD55-deficient leukocytes cocultured with wild-type blood cells was strictly dependent on shear stress. In vivo, CD55-mediated downregulation of CD97 required an intact circulation and was not observed on cells that lack contact with the blood stream, such as microglia. Notably, de novo ligation of CD97 did not activate signaling molecules constitutively engaged by CD97 in cancer cells, such as ERK and protein kinase B/Akt. We conclude that CD55 downregulates CD97 surface expression on circulating leukocytes by a process that requires physical forces, but based on current evidence does not induce receptor signaling. This regulation can restrict CD97-CD55-mediated cell adhesion to tissue site
Multimodal Spectroscopic Study of Amyloid Fibril Polymorphism
Amyloid
fibrils are a large class of self-assembled protein aggregates
that are formed from unstructured peptides and unfolded proteins.
The fibrils are characterized by a universal β-sheet core stabilized
by hydrogen bonds, but the molecular structure of the peptide subunits
exposed on the fibril surface is variable. Here we show that multimodal
spectroscopy using a range of bulk- and surface-sensitive techniques
provides a powerful way to dissect variations in the molecular structure
of polymorphic amyloid fibrils. As a model system, we use fibrils
formed by the milk protein β-lactoglobulin, whose morphology
can be tuned by varying the protein concentration during formation.
We investigate the differences in the molecular structure and composition
between long, straight fibrils versus short, wormlike fibrils. We
show using mass spectrometry that the peptide composition of the two
fibril types is similar. The overall molecular structure of the fibrils
probed with various bulk-sensitive spectroscopic techniques shows
a dominant contribution of the β-sheet core but no difference
in structure between straight and wormlike fibrils. However, when
probing specifically the surface of the fibrils with nanometer resolution
using tip-enhanced Raman spectroscopy (TERS), we find that both fibril
types exhibit a heterogeneous surface structure with mainly unordered
or α-helical structures and that the surface of long, straight
fibrils contains markedly more β-sheet structure than the surface
of short, wormlike fibrils. This finding is consistent with previous
surface-specific vibrational sum-frequency generation (VSFG) spectroscopic
results (VandenAkker et al. J. Am. Chem. Soc., 2011, 133, 18030−18033, DOI: 10.1021/ja206513r). In conclusion, only advanced vibrational spectroscopic techniques
sensitive to surface structure such as TERS and VSFG are able to reveal
the difference in structure that underlies the distinct morphology
and rigidity of different amyloid fibril polymorphs that have been
observed for a large range of food and disease-related proteins