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In JoVE (1)
Other Publications (22)
- The Journal of Biological Chemistry
- European Biophysics Journal : EBJ
- European Journal of Biochemistry / FEBS
- The Journal of Biological Chemistry
- Biochemistry
- Biophysical Journal
- Biophysical Journal
- Biophysical Journal
- The Journal of Biological Chemistry
- The Journal of Biological Chemistry
- The Journal of Biological Chemistry
- The Journal of Biological Chemistry
- Trends in Biochemical Sciences
- Journal of Molecular Biology
- IUBMB Life
- Journal of Molecular Biology
- Biochemical and Biophysical Research Communications
- Journal of Lipid Research
- Chemistry & Biology
- The Journal of Biological Chemistry
- Methods (San Diego, Calif.)
- Methods in Molecular Biology (Clifton, N.J.)
Articles by Danny M. Hatters in JoVE
JoVE General
ReAsH/FlAsH Labeling and Image Analysis of Tetracysteine Sensor Proteins in Cells
The biarsenical dyes FlAsH and ReAsH bind specifically to tetracysteine motifs in proteins and can selectively label proteins in live cells. Recently this labeling strategy has been used to develop sensors for different protein conformations or oligomeric states. We describe the labeling approach and methods to quantitatively analyze binding.
Other articles by Danny M. Hatters on PubMed
Macromolecular Crowding Accelerates Amyloid Formation by Human Apolipoprotein C-II
Human apolipoprotein C-II (apoC-II) slowly forms amyloid fibers in lipid-free solutions at physiological pH and salt concentrations (Hatters, D. M., MacPhee, C. E., Lawrence, L. J., Sawyer, W. H., and Howlett, G. J. (2000) Biochemistry 39, 8276--8283). Measurements of the time dependence of solution turbidity, thioflavin T reactivity, and the amount of sedimentable aggregate reveal that the rate and extent of amyloid formation are significantly increased by the addition of an inert polymer, dextran T10, at concentrations exceeding 20 g/liter. High dextran concentrations do not alter the secondary structure of the protein, fiber morphology, or the thioflavin T and Congo Red binding capacity of apoC-II amyloid. Analytical ultracentrifugation studies show that monomeric apoC-II does not associate significantly with dextran. The observed dependence of the overall rate of amyloid formation on dextran concentration may be accounted for quantitatively by a simple model for nonspecific volume exclusion. The model predicts that an increase in the fractional volume occupancy of macromolecules in a physiological fluid can nonspecifically accelerate the formation of amyloid fibers by any amyloidogenic protein.
The Structural Basis for Amyloid Formation by Plasma Apolipoproteins: a Review
The formation of amyloid and other protein deposits in vivo is synonymous with many pathological conditions such as Alzheimer's disease, Creutzfeldt-Jakob disease and Parkinson's disease. Interestingly, many plasma apolipoproteins are also associated with amyloid deposits, including apolipoprotein (apo) A-I, apoA-II and apoE. Apolipoproteins share a number of structural and conformational properties, namely a large proportion of class A amphipathic alpha-helices and limited conformational stability in the absence of lipid. Other proteins that form amyloid such as alpha-synuclein and serum amyloid A also contain amphipathic alpha-helical domains similar to those found in apolipoproteins. In this review we develop a hypothesis to account for the widespread occurrence of apolipoproteins in amyloid deposits. We describe the conformational stability of human apoC-II and the stabilization of alpha-helical structure in the presence of phospholipid. We propose that lipid-free apoC-II forms partially folded intermediates prone to amyloid formation. Parameters that affect apolipoprotein lipid binding in vivo, such as protein and lipid oxidation or protein truncations and mutations, could promote apolipoprotein-related pathologies including those associated within amyloid deposits of atherosclerotic plaques.
Suppression of Apolipoprotein C-II Amyloid Formation by the Extracellular Chaperone, Clusterin
The effect of the extracellular chaperone, clusterin, on amyloid fibril formation by lipid-free human apolipoprotein C-II (apoC-II) was investigated. Sub-stoichiometric levels of clusterin, derived from either plasma or semen, potently inhibit amyloid formation by apoC-II. Inhibition is dependent on apoC-II concentration, with more effective inhibition by clusterin observed at lower concentrations of apoC-II. The average sedimentation coefficient of apoC-II fibrils formed from apoC-II (0.3 mg.mL-1) is reduced by coincubation with clusterin (10 microg x mL(-1)). In contrast, addition of clusterin (0.1 mg x mL(-1)) to preformed apoC-II amyloid fibrils (0.3 mg x mL(-1)) does not affect the size distribution after 2 days. This sedimentation velocity data suggests that clusterin inhibits fibril growth but does not promote fibril dissociation. Electron micrographs indicate similar morphologies for amyloid fibrils formed in the presence or absence of clusterin. The substoichiometric nature of the inhibition suggests that clusterin interacts with transient amyloid nuclei leading to dissociation of the monomeric subunits. We propose a general role for clusterin in suppressing the growth of extracellular amyloid.
Apolipoprotein E4 Forms a Molten Globule. A Potential Basis for Its Association with Disease
The amino-terminal domain of apolipoprotein (apo) E4 is less susceptible to chemical and thermal denaturation than the apoE3 and apoE2 domains. We compared the urea denaturation curves of the 22-kDa amino-terminal domains of the apoE isoforms at pH 7.4 and 4.0. At pH 7.4, apoE3 and apoE4 reflected an apparent two-state denaturation. The midpoints of denaturation were 5.2 and 4.3 m urea, respectively. At pH 4.0, a pH value known to stabilize folding intermediates, apoE4 and apoE3 displayed the same order of denaturation but with distinct plateaus, suggesting the presence of a stable folding intermediate. In contrast, apoE2 proved the most stable and lacked the distinct plateau observed with the other two isoforms and could be fitted to a two-state unfolding model. Analysis of the curves with a three-state unfolding model (native, intermediate, and unfolded) showed that the apoE4 folding intermediate reached its maximal concentration ( approximately 90% of the mixture) at 3.75 m, whereas the apoE3 intermediate was maximal at 4.75 m ( approximately 80%). These results are consistent with apoE4 being more susceptible to unfolding than apoE3 and apoE2 and more prone to form a stable folding intermediate. The structure of the apoE4 folding intermediate at pH 4.0 in 3.75 m urea was characterized using pepsin proteolysis, Fourier transform infrared spectroscopy, and dynamic light scattering. From these studies, we conclude that the apoE4 folding intermediate is a single molecule with the characteristics of a molten globule. We propose a model of the apoE4 molten globule in which the four-helix bundle of the amino-terminal domain is partially opened, generating a slightly elongated structure and exposing the hydrophobic core. Since molten globules have been implicated in both normal and abnormal physiological function, the differential abilities of the apoE isoforms to form a molten globule may contribute to the isoform-specific effects of apoE in disease.
Cross-linking and Amyloid Formation by N- and C-terminal Cysteine Derivatives of Human Apolipoprotein C-II
We have investigated the effect of disulfide cross-linking on amyloid formation by human apolipoprotein (apo) C-II. Three derivatives of apoC-II were generated by inserting a cysteine residue on either the N-terminus (C(N)-apoC-II), C-terminus (C(C)-apoC-II), or both termini (C(N)C(C)-apoC-II). Under reducing conditions, all derivatives formed amyloid with a fibrous ribbon morphology similar to that of wild-type apoC-II. Under oxidizing conditions, C(N)- and C(N)C(C)-apoC-II formed a highly tangled network of fibrils, suggesting that the addition of an N-terminal cysteine to apoC-II promotes interfibril disulfide cross-links. Fibrils formed by C(C)-apoC-II under oxidizing conditions were closely packed but less tangled than fibrils formed by the C(N) and C(N)C(C) derivatives. The frequency of closed ring structures was more than doubled for C(C)-apoC-II compared to wild-type apoC-II. The kinetics of fibril formation by all cysteine derivatives was markedly enhanced under oxidizing conditions, suggesting that disulfide cross-linking promotes amyloid formation. Substoichiometric levels of preformed C(N)- and C(C)-apoC-II dimers accelerate amyloid formation by wild-type apoC-II. These data suggest that the N- and C-termini of apoC-II are close together in the amyloid fibril such that covalent cross-linking of either the N or C end of apoC-II promotes nucleation and the "seeding" of fibril growth.
Sedimentation Velocity Analysis of Flexible Macromolecules: Self-association and Tangling of Amyloid Fibrils
A novel bead modeling technique has been developed for the analysis of the sedimentation velocity behavior of flexible fibrils. The method involves the generation of a family of bead models representing a sample of the conformations available to the molecule and the calculation of the sedimentation coefficients of these models by established techniques. This approach has been used to investigate the size distribution of amyloid fibrils formed by human apolipoprotein C-II (apoC-II). ApoC-II fibrils have a simple and homogeneous ribbon morphology with no evidence of amorphous aggregation. Freshly prepared apoC-II forms fibrils with systematically larger sedimentation coefficients upon increasing protein concentration (modes of 100, 300, and 800 for apoC-II concentrations of 0.3, 0.7, and 1.0 mg/mL, respectively). The sedimentation coefficient distributions are not affected by rotor speed, and are not significantly changed by dilution once the fibrils are formed. The kinetics of aggregation (1 mg/mL apoC-II) as assessed using thioflavin T and preparative pelleting assays reveal that monomeric apoC-II is depleted after approximately 12 h incubation at room temperature. In contrast, the sedimentation coefficient distribution of fibrils continues to grow larger over a period of 48 h to an average value of 800 S. Calculations using the bead modeling procedure suggest maximum sedimentation coefficients for individual apoC-II fibrils to be around 100 S. The larger experimentally observed sedimentation coefficients for apoC-II fibrils indicate an extensive and time-dependent tangling or association of the fibrils to form specific networks.
Phospholipid Complexation and Association with Apolipoprotein C-II: Insights from Mass Spectrometry
The interactions between phospholipid molecules in suspensions have been studied by using mass spectrometry. Electrospray mass spectra of homogeneous preparations formed from three different phospholipid molecules demonstrate that under certain conditions interactions between 90 and 100 lipid molecules can be preserved. In the presence of apolipoprotein C-II, a phospholipid binding protein, a series of lipid molecules and the protein were observed in complexes. The specificity of binding was demonstrated by proteolysis; the resulting mass spectra reveal lipid-bound peptides that encompass the proposed lipid-binding domain. The mass spectra of heterogeneous suspensions and their complexes with apolipoprotein C-II demonstrate that the protein binds simultaneously to two different phospholipids. Moreover, when apolipoprotein C-II is added to lipid suspensions formed with local concentrations of the same lipid molecule, the protein is capable of remodeling the distribution to form one that is closer to a statistical arrangement. These observations demonstrate a capacity for apolipoprotein C-II to change the topology of the phospholipid surface. More generally, these results highlight the fact that mass spectrometry can be used to probe lipid interactions in both homogeneous and heterogeneous suspensions and demonstrate reorganization of the distribution of lipids upon surface binding of apolipoprotein C-II.
The Circularization of Amyloid Fibrils Formed by Apolipoprotein C-II
Amyloid fibrils have historically been characterized by diagnostic dye-binding assays, their fibrillar morphology, and a "cross-beta" x-ray diffraction pattern. Whereas the latter demonstrates that amyloid fibrils have a common beta-sheet core structure, they display a substantial degree of morphological variation. One striking example is the remarkable ability of human apolipoprotein C-II amyloid fibrils to circularize and form closed rings. Here we explore in detail the structure of apoC-II amyloid fibrils using electron microscopy, atomic force microscopy, and x-ray diffraction studies. Our results suggest a model for apoC-II fibrils as ribbons approximately 2.1-nm thick and 13-nm wide with a helical repeat distance of 53 nm +/- 12 nm. We propose that the ribbons are highly flexible with a persistence length of 36 nm. We use these observed biophysical properties to model the apoC-II amyloid fibrils either as wormlike chains or using a random-walk approach, and confirm that the probability of ring formation is critically dependent on the fibril flexibility. More generally, the ability of apoC-II fibrils to form rings also highlights the degree to which the common cross-beta superstructure can, as a function of the protein constituent, give rise to great variation in the physical properties of amyloid fibrils.
Fibrillar Amyloid Protein Present in Atheroma Activates CD36 Signal Transduction
The self-association of proteins to form amyloid fibrils has been implicated in the pathogenesis of a number of diseases including Alzheimer's, Parkinson's, and Creutzfeldt-Jakob diseases. We recently reported that the myeloid scavenger receptor CD36 initiates a signaling cascade upon binding to fibrillar beta-amyloid that stimulates recruitment of microglia in the brain and production of inflammatory mediators. This receptor plays a key role in the pathogenesis of atherosclerosis, prompting us to evaluate whether fibrillar proteins were present in atherosclerotic lesions that could initiate signaling via CD36. We show that apolipoprotein C-II, a component of very low and high density lipoproteins, readily forms amyloid fibrils that initiate macrophage inflammatory responses including reactive oxygen production and tumor necrosis factor alpha expression. Using macrophages derived from wild type and Cd36(-/-) mice to distinguish CD36-specific events, we show that fibrillar apolipoprotein C-II activates a signaling cascade downstream of this receptor that includes Lyn and p44/42 MAPKs. Interruption of this signaling pathway through targeted deletion of Cd36 or blocking of p44/42 MAPK activation inhibits macrophage tumor necrosis factor alpha gene expression. Finally, we demonstrate that apolipoprotein C-II in human atheroma co-localizes to regions positive for markers of amyloid and macrophage accumulation. Together, these data characterize a CD36-dependent signaling cascade initiated by fibrillar amyloid species that may promote atherogenesis.
Engineering Conformational Destabilization into Mouse Apolipoprotein E. A Model for a Unique Property of Human Apolipoprotein E4
Apolipoprotein (apo) E4 is a major risk factor for Alzheimer and cardiovascular diseases. ApoE4 differs from the two other common isoforms (apoE2 and apoE3) by its lower resistance to denaturation and greater propensity to form partially folded intermediates. As a first step to determine the importance of stability differences in vivo, we reengineered a partially humanized variant of the amino-terminal domain of mouse apoE (T61R mouse apoE) to acquire a destabilized conformation like that of apoE4. For this process, we determined the crystal structure of wild-type mouse apoE, which, like apoE4, forms a four-helix bundle, and identified two structural differences in the turn between helices 2 and 3 and in the middle of helix 3 as potentially destabilizing sites. Introducing mutations G83T and N113G at these sites destabilized the mouse apoE conformation. The mutant mouse apoE more rapidly remodeled phospholipid than T61R mouse apoE, which supports the hypothesis that a destabilized conformation promotes apoE4 lipid binding.
Modulation of Apolipoprotein E Structure by Domain Interaction: Differences in Lipid-bound and Lipid-free Forms
Interaction of the amino- and carboxyl-terminal domains in apolipoprotein (apo) E, referred to as domain interaction, is predicted to be more pronounced in apoE4 than in apoE3 and to underlie the association of apoE4 with Alzheimer and cardiovascular diseases. However, direct physical proof for the domain interaction concept is lacking. To address this issue, fluorescence resonance energy transfer and electron paramagnetic resonance spectroscopy were used to probe the spatial proximity of the two domains of apoE. Both methods demonstrated that the two domains are closer in both lipid-free and phospholipid-bound apoE4 than in apoE3 as a result of domain interaction. In addition, as shown by electron paramagnetic resonance, the domains of apoE4 move apart to resemble more closely the distance in apoE3 when the isoforms are bound to triglyceride-rich emulsion particles. These results demonstrate that domain interaction is a structural property of apoE4 and that apoE adopts different conformations when complexed to different lipids.
Model of Biologically Active Apolipoprotein E Bound to Dipalmitoylphosphatidylcholine
Apolipoprotein (apo)E plays a critical role in cholesterol transport, through high affinity binding to the low density lipoprotein receptor. This interaction requires apoE to be associated with a lipoprotein particle. To determine the structure of biologically active apoE on a lipoprotein particle, we crystallized dipalmitoylphosphatidylcholine particles containing two apoE molecules and determined the molecular envelope of apoE at 10 Angstroms resolution. On the basis of the molecular envelope and supporting biochemical evidence, we propose a model in which each apoE molecule is folded into a helical hairpin with the binding region for the low density lipoprotein receptor at its apex.
Apolipoprotein E Structure: Insights into Function
Human apolipoprotein E (apoE) is a member of the family of soluble apolipoproteins. Through its interaction with members of the low-density lipoprotein receptor family, apoE has a key role in lipid transport both in the plasma and in the central nervous system. Its three common structural isoforms differentially affect the risk of developing atherosclerosis and neurodegenerative disorders, including Alzheimer's disease. Because the function of apoE is dictated by its structure, understanding the structural properties of apoE and its isoforms is required both to determine its role in disease and for the development of therapeutic strategies.
Amino-terminal Domain Stability Mediates Apolipoprotein E Aggregation into Neurotoxic Fibrils
The three isoforms of apolipoprotein (apo) E are strongly associated with different risks for Alzheimer's disease: apoE4>apoE3>apoE2. Here, we show at physiological salt concentrations and pH that native tetramers of apoE form soluble aggregates in vitro that bind the amyloid dyes thioflavin T and Congo red. However, unlike classic amyloid fibrils, the aggregates adopt an irregular protofilament-like morphology and are seemingly highly alpha-helical. The aggregates formed at substantially different rates (apoE4>apoE3>apoE2) and were significantly more toxic to cultured neuronal cells than the tetramer. Since the three isoforms have large differences in conformational stability that can influence aggregation and amyloid pathways, we tested the effects of mutations that increased or decreased stability. Decreasing the conformational stability of the amino-terminal domain of apoE increased aggregation rates and vice versa. Our findings provide a new perspective for an isoform-specific pathogenic role for apoE aggregation in which differences in the conformational stability of the amino-terminal domain mediate neurodegeneration.
Protein Misfolding Inside Cells: the Case of Huntingtin and Huntington's Disease
Huntington's disease is one of the several neurodegenerative diseases caused by dominant mutations that expand the number of glutamine codons within an existing poly-glutamine (polyQ) repeat sequence of a gene. An expanded polyQ sequence in the huntingtin gene is known to cause the huntingtin protein to aggregate and form intracellular inclusions as disease progresses. However, the role that polyQ-induced aggregation plays in disease is yet to be fully determined. This review focuses on key questions remaining for how the expanded polyQ sequences affect the aggregation properties of the huntingtin protein and the corresponding effects on cellular machinery. The scope includes the technical challenges that remain for rigorously assessing the effects of aggregation on the cellular machinery.
Insight on the Molecular Envelope of Lipid-bound Apolipoprotein E from Electron Paramagnetic Resonance Spectroscopy
Although a high-resolution X-ray structure for the N-terminal domain of apolipoprotein E (apoE) in the lipid-free state has been solved, our knowledge of the structure of full-length apoE in a lipid-bound state is limited to an X-ray model fitting a molecular envelope at 10-A resolution. To add molecular detail to the molecular envelope, we used cysteine mutagenesis to incorporate spin labels for analysis with electron paramagnetic resonance (EPR) spectroscopy. Twelve cysteine residues were introduced singly and in pairs at unique locations throughout apoE4 and labeled with an EPR spin probe. The labeled apoE4 was combined with dipalmitoylphosphatidylcholine, the particles were purified, and spectra were determined for 24 combinations (single and double) of the cysteine mutants. Data on the conformation, mobility, distance, and surface exposure of regions revealed by the cysteine probes were modeled into the molecular envelope of apoE bound to dipalmitoylphosphatidylcholine that had been determined by X-ray analysis. This EPR model of apoE in a native lipid-bound state validates the structural model derived from X-ray analysis and provides additional insight into apoE structure-function relationships.
Conformational Detection of Prion Protein with Biarsenical Labeling and FlAsH Fluorescence
Prion diseases are associated with the misfolding of the host-encoded cellular prion protein (PrP(C)) into a disease associated form (PrP(Sc)). Recombinant PrP can be refolded into either an alpha-helical rich conformation (alpha-PrP) resembling PrP(C) or a beta-sheet rich, protease resistant form similar to PrP(Sc). Here, we generated tetracysteine tagged recombinant PrP, folded this into alpha- or beta-PrP and determined the levels of FlAsH fluorescence. Insertion of the tetracysteine tag at three different sites within the 91-111 epitope readily distinguished beta-PrP from alpha-PrP upon FlAsH labeling. Labelling of tetracysteine tagged PrP in the alpha-helical form showed minimal fluorescence, whereas labeling of tagged PrP in the beta-sheet form showed high fluorescence indicating that this region is exposed upon conversion. This highlights a region of PrP that can be implicated in the development of diagnostics and is a novel, protease free mechanism for distinguishing PrP(Sc) from PrP(C). This technique may also be applied to any protein that undergoes conformational change and/or misfolding such as those involved in other neurodegenerative disorders including Alzheimer's, Huntington's and Parkinson's diseases.
VLDL Lipolysis Products Increase VLDL Fluidity and Convert Apolipoprotein E4 into a More Expanded Conformation
Our previous work indicated that apolipoprotein (apo) E4 assumes a more expanded conformation in the postprandial period. The postprandial state is characterized by increased VLDL lipolysis. In this article, we tested the hypothesis that VLDL lipolysis products increase VLDL particle fluidity, which mediates expansion of apoE4 on the VLDL particle. Plasma from healthy subjects was collected before and after a moderately high-fat meal and incubated with nitroxyl-spin labeled apoE. ApoE conformation was examined by electron paramagnetic resonance spectroscopy using targeted spin probes on cysteines introduced in the N-terminal (S76C) and C-terminal (A241C) domains. Further, we synthesized a novel nitroxyl spin-labeled cholesterol analog, which gave insight into lipoprotein particle fluidity. Our data revealed that the order of lipoprotein fluidity was HDL approximately LDL
Conformation Sensors That Distinguish Monomeric Proteins from Oligomers in Live Cells
Proteins prone to misfolding form large macroscopic deposits in many neurodegenerative diseases. Yet the in situ aggregation kinetics remains poorly understood because of an inability to demarcate precursor oligomers from monomers. We developed a strategy for mapping the localization of soluble oligomers and monomers directly in live cells. Sensors for mutant huntingtin, which forms aggregates in Huntington's disease, were made by introducing a tetracysteine motif into huntingtin that becomes occluded from binding biarsenical fluorophores in oligomers, but not monomers. Up to 70% of the diffusely distributed huntingtin molecules appeared as submicroscopic oligomers in individual neuroblastoma cells expressing mutant huntingtin. We anticipate the sensors to enable insight into cellular mechanisms mediated by oligomers and monomers and for the approach to be adaptable more generally in the study of protein self-association.
Tracking Mutant Huntingtin Aggregation Kinetics in Cells Reveals Three Major Populations That Include an Invariant Oligomer Pool
Huntington disease is caused by expanded polyglutamine sequences in huntingtin, which procures its aggregation into intracellular inclusion bodies (IBs). Aggregate intermediates, such as soluble oligomers, are predicted to be toxic to cells, yet because of a lack of quantitative methods, the kinetics of aggregation in cells remains poorly understood. We used sedimentation velocity analysis to define and compare the heterogeneity and flux of purified huntingtin with huntingtin expressed in mammalian cells under non-denaturing conditions. Non-pathogenic huntingtin remained as hydrodynamically elongated monomers in vitro and in cells. Purified polyglutamine-expanded pathogenic huntingtin formed elongated monomers (2.4 S) that evolved into a heterogeneous aggregate population of increasing size over time (100-6,000 S). However, in cells, mutant huntingtin formed three major populations: monomers (2.3 S), oligomers (mode s(20,w) = 140 S) and IBs (mode s(20,w) = 320,000 S). Strikingly, the oligomers did not change in size heterogeneity or in their proportion of total huntingtin over 3 days despite continued monomer conversion to IBs, suggesting that oligomers are rate-limiting intermediates to IB formation. We also determined how a chaperone known to modulate huntingtin toxicity, Hsc70, influences in-cell huntingtin partitioning. Hsc70 decreased the pool of 140 S oligomers but increased the overall flux of monomers to IBs, suggesting that Hsc70 reduces toxicity by facilitating transfer of oligomers into IBs. Together, our data suggest that huntingtin aggregation is streamlined in cells and is consistent with the 140 S oligomers, which remain invariant over time, as a constant source of toxicity to cells irrespective of total load of insoluble aggregates.
Sedimentation Velocity Analysis of Amyloid Oligomers and Fibrils Using Fluorescence Detection
The assembly of proteins into large fibrillar aggregates, known as amyloid fibrils, is associated with a number of common and debilitating diseases. In some cases, proteins deposit extracellularly, while in others the aggregation is intracellular. A common feature of these diseases is the presence of aggregates of different sizes, including mature fibrils, small oligomeric precursors, and other less well understood structural forms such as amorphous aggregates. These various species possess distinct biochemical, biophysical, and pathological properties. Here, we detail a number of techniques that can be employed to examine amyloid fibrils and oligomers using a fluorescence-detection system (FDS) coupled with the analytical ultracentrifuge. Sedimentation velocity analysis using fluorescence detection is a particularly useful method for resolving the complex heterogeneity present in amyloid systems and can be used to characterize aggregation in exceptional detail. Furthermore, the fluorescence detection module provides a number of particularly attractive features for the analysis of aggregating proteins. It expands the practical range of concentrations of aggregating proteins under study, which is useful for greater insight into the aggregation process. It also enables the assessment of aggregation behavior in complex biological solutions, such as cell lysates, and the assessment of processes that regulate in-cell or extracellular aggregation kinetics. Four methods of fluorescent detection that are compatible with the current generation of FDS instrumentation are described: (1) Detection of soluble amyloid fibrils using a covalently bound fluorophore. (2) Detection of amyloid fibrils using an extrinsic dye that emits fluorescence when bound to fibrils. (3) Detection of fluorescently-labeled lipids and their interaction with oligomeric amyloid intermediates. (4) Detection of green fluorescence protein (GFP) constructs and their interactions within mammalian cell lysates.
Diagnostics for Amyloid Fibril Formation: Where to Begin?
Twenty-five proteins are known to form amyloid fibrils in vivo in association with disease (Westermark et al., Amyloid 12:1-4, 2005). However, the fundamental ability of a protein to form amyloid-like fibrils is far more widespread than in just the proteins associated with disease, and indeed this property can provide insight into the basic thermodynamics of folding and misfolding pathways. But how does one determine whether a protein has formed amyloid-like fibrils? In this chapter, we cover the basic steps toward defining the amyloid-like properties of a protein and how to measure the kinetics of fibrillization. We describe several basic tests for aggregation and the binding to two classic amyloid-reactive dyes, Congo Red, and thioflavin T, which are key indicators to the presence of fibrils.
