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Analysis Of Fibril Elongation By Surface Plasmon Resonance
by Michelle J. Cannon and David G. Myszka
Figure 1. Fibril structure. Left: Ab peptides aggregate to form protofilaments.(4) The protofilaments assemble into amyloid fibrils. Right: A scanning electron microscopy image of Ab fibrils.
Figure 2. Fibril elongation on Sensor Chip C1 immobilized with 6000 RU or 2500 RU Ab (sensorgrams a and b, respectively).
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Introduction
Peptide aggregates are associated with several diseases, including Alzheimer's and Huntington's Disease. Surface plasmon resonance (SPR) -amyloid peptide (Ab) (1-40) was monitored by injecting the peptide over fibril and reference surfaces. Ab monomers were found to interact only with surfaces containing immobilized fibrils. Atomic Force Microscopy (AFM) has been used to image sensor chip surfaces before and after fibril extension to demonstrate growth of immobilized fibers.(1, 2) SPR kinetic data revealed that the association and dissociation of peptide to the fibrils were multi-phasic. A model that described the elongation events includes a dock, lock, and block mechanism.(3) Additional experiments, including the variation of both contact time and peptide concentration, were performed to validate the reaction model. These results illustrate how SPR technology can be used to analyze the initial events in polymerization reactions.
Background
Ab is known to aggregate with other monomer peptide units to form protofilaments and fibrils (Figure 1). Amyloid fibrils are primary constituents of plaques in the brain of patients with Alzheimer's disease. Very little is known about fibril structure at the molecular level and how Ab peptide units are added to a fibril tip. Analysis of this process is critical to understanding the mechanism of fibril assembly. Traditionally, the methods used to evaluate the kinetics of fibril growth have been fluorescence spectroscopy and ELISA. These approaches give information about the elongation process over a period of days. SPR monitors binding reactions in real time. Therefore, both long-term fibril growth and short-term addition of monomer units can be analyzed.
Figure 3. Stability of a fibril-coated Sensor Chip C1. Ab was injected for 6 seconds and dissociation was monitored for 30 minutes.
Figure 4: The structure of Ab fibrils on Sensor Chip C1 before (A) and after (B) elongation. The bar indicates a length of 1 mm.
Figure 5. A model for Ab fibril elongation. The kinetic rate constants k1 and k-1 describe the reversible interaction of Ab monomer interacting with a b-amyloid fibril as the monomer docks and binds to the growing fibril. Rate constants k2, k-2, k3, and k-3 indicate first order conformational change reaction kinetics and relate to locking the monomer into the growing fibril.(1)
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The assay
Ab fibrils were immobilized at 2500 and 6000 RU on Sensor Chip C1 by using standard amine-coupling chemistry. Control surfaces of an underivatized carboxymethyl dextran layer and an immobilized Ab peptide surface were also used for the analysis. The soluble peptide was passed over all surfaces to monitor polymerization of the fibrils in real time.
Fibril elongation
10 mM soluble peptide was injected for 30 seconds over fibril and control surfaces at 100 ml/min. In Figure 2, the responses shown by sensorgrams (a) and (b) represent fibril elongation on surfaces of 6000 RU and 2500 RU, respectively. Sensorgrams (c) and (d) show the responses of Ab injected over Ab peptide and reference surfaces, respectively. Fibril elongation is dependent upon and proportional to the fibril concentration on the sensor chip surface, and Ab does not interact with the reference or peptide surfaces.
Monitoring background fibril decay
The decay of a freshly prepared fibril surface was monitored for 30 minutes (Figure 3, sensorgram (a)). The response shown by sensorgram (b) represents Ab (10 mM) that was injected over a fibril surface for 6 seconds and the dissociation phase was monitored for 30 minutes. The solid line (sensorgram (c)) represents a reference baseline.
Surface analysis
Hasegawa et al. analyzed the structure of Ab fibrils on Sensor Chip C1 before and after elongation by AFM.(1) Figure 4 shows the structure of sonicated fibrils just after immobilization (A) and after the addition of a two-fold increase in monomer mass to the surface (B). Binding of the monomer to the immobilized fibril surface significantly increased the length of the fibrils. This work validates the binding events observed using surface plasmon resonance technology by showing that the peptide forms well ordered, extended fibrils and is not merely adsorbing nonspecifically onto the surface of the sensor chip.
Reaction model
Fibril elongation model
bA (shown as A in Figure 5) reversibly binds to a unique site on the end of the fibril tip (B) in a "docking" step." Once bound, the peptide conformation rearranges, as shown in the "locking" step. This leads to a more stable complex, thereby regenerating the recognition site on the end of the fibril. Further addition of Ab to the fibril during polymerization causes a "blocking" of the release of previously bound peptides.
Model testing
Varying A association phase time
10 mM of Ab was injected over a fibril surface for 0.20, 0.33, 1.0, 3.0, 9.0, and 27 minutes (Figure 6A). Responses were overlaid by zeroing the response on the y-axis prior to the injection and zeroing the time on the x-axis at the start of the dissociation phase. The amount of peptide captured onto the surface increased dramatically with longer contact times. Fibril growth was proportional to the Ab fibril contact time.
Figure 6. Model testing by varying the association phase time. (A) Before data normalization; (B) After data normalization; (C) Concentration and contact time of Ab are fixed to achieve an immobilization level of 20 RU after association.
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Data normalization
The data generated by varying Ab contact time were normalized by setting the responses at the start of the dissociation phase to 1 (Figure 6B). As the contact times increased, a lower decay rate of the fibril was observed.
Varying contact time and Ab concentration
The concentration and contact time of Ab were varied to keep the amount of peptide bound at the end of the association phase constant at 20 RU (Figure 6C). The extended contact time allowed a larger amount of peptide to be stably incorporated into the growing fiber.
The results observed for varying association time, analyte concentration, and normalizing at the beginning of the dissociation phase to determine the amount bound, are consistent with those expected for docking and locking.
Summary
Surface plasmon resonance analysis provides an opportunity to measure the initial events in fibril elongation. Soluble Ab peptides were shown to bind rapidly to fibril surfaces but not to immobilized monomeric Ab peptides, which self-aggregate only very slowly. Elongation was dependent upon the fibril concentration immobilized on the sensor chip as well as the concentration of Ab monomer and amount of time that the monomer was in contact with the fibers. Fibril surfaces also show slow linear background decay. All of these observations are consistent with a polymerization model where monomer binds to the end of a growing fibril tip. The structures of the immobilized and extended fibrils were confirmed by AFM experiments. The work shows how the Biacore assay can be used to study mechanistic aspects of Ab fibril formation. It also suggests how Biacore can be used as a tool to analyze other polymerization and aggregation systems in detail.
About the authors
Michelle J. Cannon, PhD and David G. Myszka, PhD work at The Center for Biomolecular Interaction Analysis at the University of Utah in Salt Lake City.
They can be reached at www.cores.utah.edu/interaction.
More information about surface plasmon resonance technology is available from: Biacore
References
1. Hasegawa, K., Yamada, M. and Naiki, H. Biochemistry 41:13489-98 (2003).
2. Myszka, D. G., Wood, S.J. and Biere, A.L. Methods Enzymol 309:386-402 (1999).
3. Kheterpal, I., Williams, A., Murphy, C., Bledsoe, B. and Wetzel, R. Biochemistry 40:11757-67 (2001).
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