Ishan Deshmukh and Dr. Kazushige Yokoyama Printing · Ishan Deshmukh and Dr. Kazushige Yokoyama...
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Interfacial Interaction of Amyloid Beta Peptide 1-40 with ThT Ishan Deshmukh and Dr. Kazushige Yokoyama BACKGROUND One of the main causes of many neurodegenerative diseases, such as Alzheimer’s disease or Parkinson’s disease, is the deformation of a specific protein, called amyloidogenic protein. While extensive studies of these proteins have been conducted, an enormous amount of effort is needed to interpret the study outcome due to the complexity of the proteins involved. The amyloid beta peptide (Aβ) is highly regarded as a critical monomer that reversibly conforms an insoluble fibril that leads to Alzheimer’s disease. The mechanism of fibril formation is considered to be a polymerization of a nucleus unit consisting of Aβ oligomers under a reversible process. While extensive studies have been conducted on fibril properties and character, only limited information is available on these initial oligomers. The proteins, when immobilized at an interface, can possess different properties than their counterparts dispersed in solution. The sheet structure was confirmed on fluorinated nanoparticles, implying that self assembly over nanoscale interfacial environment plays a key role in fibrillogenesis. A folding or unfolding of monomer peptide is highly associated with the critical stage of intermediate oligomer formation, leading to fibrillogenesis. While direct probe of plausible interfacial conformation may be challenging, monitoring a change in the dynamics of fluorophore attached to a folding peptide is possible. RESULTS DISCUSSION CONCLUSIONS The Aβ1-40 adsorbed gold colloid provided the ThT fluorescence assay with monitoring free ThT as well as the Aβ1-40 interacted with ThT. Through exploration of fluorescence probing over various sizes of gold colloidal surfaces in the pH range of 1 to 12, the plausible section of ThT-Aβ1-40 binding is concluded to be a sequence of 22Glu and 23Asp through electro-static interaction (–N+⋅⋅⋅ [ -22Glu– - 23Asp – -]), avoiding the direct interaction between ThT and Ab1-40,as it took a conformational change by folding. The folded conformation was estimated to shorten an interaction distance between attachment sites and ThT, resulting in a stabilization of excited states of ThT. Figure 4: The fluorescence decay time of band II of ThT with Aβ 1-40 coated over 10 nm gold colloid (i) in blue and ThT with Aβ 1-40 coated over 100 nm gold colloid (ii) in red at pH 6.5. The decay time (i) profile was almost identical with that of ThT alone. Figure 3: The fluorescence spectra of ThT alone (dotted line) and ThT with Aβ 1-40 (solid line). Figure 5a: A surface plot of pH dependent average fluorescence decay time, <t>, for gold colloidal size ranging between 10 nm and 100 nm size. The value of <t> was monitored at a band I. Figure 5b: . A surface plot of pH dependent average fluorescence decay time, <t>, for gold colloidal size ranging between 10 nm and 100 nm size. The value of <t> was monitored at a band II. Figure 5c: A surface plot of pH dependent average fluorescence decay time, ∆<τ>; differences between band I and band II. METHODS Figure 1. The stable conformation of Aβ may depend on the interfacial environment. Images a and b illustrate the folded and unfolded conformations that may be experienced by Aβ attached to different nanocolloidal interfacial environments. The Tht was attached to gold nanocolloids of various sizes ranging from 10 to 100 nm in diameter which provided the peptide with a nanoscale interfacial environment to conform on. Additionally, changes to the chemical environment were made by altering the pH from 2 to 12 and its influence on the solution was studied. Figure 2. Sketch showing sequence of Aβ 1-40 monomer (left) and molecular structure of ThT (right) Figure 6. Representative sketch indicating the position of ThT dye attached to an unfolded (at acidic condition) Aβ 1-40 adsorbed over gold colloidal surface. a) ThT is adsorbed at the hydrophobic section of Aβ 1-40 . b) ThT adsorbed at the hydrophilic segment exposed to the outward for aggregate formation. c) ThT adsorbed at the pivotal section of Aβ 1-40 . In the lower box, the situation of Aβ 1-40 adsorbed gold colloid are illustrated for each case. Figure 7. A schematic sketch indicating a particular segment(s) used for ThT attachment in the case of folded (a) and unfolded (b) Aβ 1-40 . Here, yellow circle indicates ThT dye and D– and E– are 22 Glu– and 23 Asp–, respectively. Each box shows a blow up of the ThT attachment site. An identification of the ThT attachment site confirms that both 1) Aβ 1-40 adsorbs on the gold colloidal surface through any parts of hydrophobic segments (23-40) and that 2) hydrophilic segments (1-16) are used for networking with the other Aβ 1-40 monomer adsorbed on gold colloid to form colloidal aggregates. The formation of networking with the other Aβ 1-40 is a very significant implication of the formation of oligomers at an interface between adjacent gold colloids. This allows these oligomers to act like a networking chain for the colloidal aggregates. However, an extraction of a particular oligomeric form was not conclusive through this study. Currently, a morphology of amyloidogenic peptide coated gold colloidal aggregates are being studied by TEM (Transmission Electron Microscopy). This networking conformation can provide a great hint of how networking segment is spatially (geometrically) oriented. Two approaches were used to study the dynamics of the peptide fluorescence, spectroscopy, and subnanosecond fluorescence decay time measurements. Fluorescence spectroscopy was used to understand the possible oligomeric structures formed by the Aβ 1-40 . Time correlated single photon counting (TCSPC) was used to measure the energy relaxation time of the fluorescein in order to indirectly probe the time scale of the conformational changes exhibited by the Aβ 1-40 . 353
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Interfacial Interaction of Amyloid Beta Peptide 1-40 with ThT
Ishan Deshmukh and Dr. Kazushige Yokoyama
BACKGROUND
One of the main causes of many neurodegenerative diseases, such as Alzheimer’sdisease or Parkinson’s disease, is the deformation of a specific protein, calledamyloidogenic protein. While extensive studies of these proteins have beenconducted, an enormous amount of effort is needed to interpret the study outcomedue to the complexity of the proteins involved. The amyloid beta peptide (Aβ) ishighly regarded as a critical monomer that reversibly conforms an insoluble fibril thatleads to Alzheimer’s disease. The mechanism of fibril formation is considered to be apolymerization of a nucleus unit consisting of Aβ oligomers under a reversibleprocess. While extensive studies have been conducted on fibril properties andcharacter, only limited information is available on these initial oligomers. Theproteins, when immobilized at an interface, can possess different properties thantheir counterparts dispersed in solution. The sheet structure was confirmed onfluorinated nanoparticles, implying that self assembly over nanoscale interfacialenvironment plays a key role in fibrillogenesis. A folding or unfolding of monomerpeptide is highly associated with the critical stage of intermediate oligomerformation, leading to fibrillogenesis. While direct probe of plausible interfacialconformation may be challenging, monitoring a change in the dynamics offluorophore attached to a folding peptide is possible.
RESULTS DISCUSSION
CONCLUSIONSThe Aβ1-40 adsorbed gold colloid provided the ThT fluorescence assay withmonitoring free ThT as well as the Aβ1-40 interacted with ThT. Through explorationof fluorescence probing over various sizes of gold colloidal surfaces in the pH range of1 to 12, the plausible section of ThT-Aβ1-40 binding is concluded to be a sequence of22Glu and 23Asp through electro-static interaction (–N+⋅⋅⋅ [ -22Glu– - 23Asp – -]),avoiding the direct interaction between ThT and Ab1-40,as it took a conformationalchange by folding. The folded conformation was estimated to shorten an interactiondistance between attachment sites and ThT, resulting in a stabilization of excitedstates of ThT.
Figure 4: The fluorescence decay time of band II ofThT with Aβ1-40 coated over 10 nm gold colloid (i) inblue and ThT with Aβ1-40 coated over 100 nm goldcolloid (ii) in red at pH 6.5. The decay time (i) profilewas almost identical with that of ThT alone.
Figure 3: The fluorescence spectra of ThT alone(dotted line) and ThT with Aβ1-40 (solid line).
Figure 5a: A surface plot of pH dependent average fluorescence decay time, <t>, forgold colloidal size ranging between 10 nm and 100 nm size. The value of <t> wasmonitored at a band I.
Figure 5b: . A surface plot of pH dependent average fluorescence decay time, <t>, forgold colloidal size ranging between 10 nm and 100 nm size. The value of <t> wasmonitored at a band II.
Figure 5c: A surface plot of pH dependent average fluorescence decay time, ∆<τ>; differences between band I and band II.
METHODS
Figure 1. The stable conformation
of Aβ may depend on the interfacial
environment. Images a and b
illustrate the folded and unfolded
conformations that may be
experienced by Aβ attached to
different nanocolloidal interfacial
environments.
The Tht was attached to gold nanocolloids of various sizes ranging from 10 to 100 nmin diameter which provided the peptide with a nanoscale interfacial environment toconform on. Additionally, changes to the chemical environment were made by alteringthe pH from 2 to 12 and its influence on the solution was studied.
Figure 2. Sketch showing sequence of Aβ1-40 monomer (left) and molecular structure of ThT (right)
Figure 6. Representative sketch indicating the position of ThT dye attached to an unfolded (at acidiccondition) Aβ1-40 adsorbed over gold colloidal surface. a) ThT is adsorbed at the hydrophobic sectionof Aβ1-40. b) ThT adsorbed at the hydrophilic segment exposed to the outward for aggregateformation. c) ThT adsorbed at the pivotal section of Aβ1-40. In the lower box, the situation of Aβ1-40
adsorbed gold colloid are illustrated for each case.
Figure 7. A schematic sketch indicating a particular segment(s) used for ThT attachment in the caseof folded (a) and unfolded (b) Aβ1-40. Here, yellow circle indicates ThT dye and D– and E– are 22Glu–and 23Asp–, respectively. Each box shows a blow up of the ThT attachment site.
An identification of the ThT attachment site confirms that both 1) Aβ1-40 adsorbs onthe gold colloidal surface through any parts of hydrophobic segments (23-40) andthat 2) hydrophilic segments (1-16) are used for networking with the other Aβ1-40
monomer adsorbed on gold colloid to form colloidal aggregates. The formation ofnetworking with the other Aβ1-40 is a very significant implication of the formation ofoligomers at an interface between adjacent gold colloids. This allows these oligomersto act like a networking chain for the colloidal aggregates. However, an extraction ofa particular oligomeric form was not conclusive through this study. Currently, amorphology of amyloidogenic peptide coated gold colloidal aggregates are beingstudied by TEM (Transmission Electron Microscopy). This networking conformationcan provide a great hint of how networking segment is spatially (geometrically)oriented.
Two approaches were used to studythe dynamics of the peptidefluorescence, spectroscopy, andsubnanosecond fluorescence decaytime measurements. Fluorescencespectroscopy was used tounderstand the possible oligomericstructures formed by the Aβ1-40.Time correlated single photoncounting (TCSPC) was used tomeasure the energy relaxation timeof the fluorescein in order toindirectly probe the time scale ofthe conformational changesexhibited by the Aβ1-40.
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