Atomic force microscopy of self-assembled biomolecular structures and their interaction with metallic nanoparticles
We applied AFM to study biomolecular wires, both out of interest in thei r biological functions and in the framework of nanotechnology based fabr ication. We have focused on two different kinds of protein wires: Insuli n fibrils and microtubules. Microtubules are an important constituent of the cyto...
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Format: | Dissertation |
| Sprache: | eng |
| Online-Zugang: | Volltext bestellen |
| Tags: |
Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
|
| container_end_page | |
|---|---|
| container_issue | |
| container_start_page | |
| container_title | |
| container_volume | |
| creator | Gysemans, Maarten |
| description | We applied AFM to study biomolecular wires, both out of interest in thei r biological functions and in the framework of nanotechnology based fabr ication. We have focused on two different kinds of protein wires: Insuli n fibrils and microtubules. Microtubules are an important constituent of the cytoskeleton and fulfill multiple vital functions in the cell. Insu lin fibrils on the other hand are amyloid fibrils without a clear biolog ical role, but with intriguing polymerization properties that make them an interesting model system for the formation of amyloid fibrils. Both p rotein wires are formed by a self-assembly process and have robust struc tural properties, which make them interesting candidates to act as a tem plate for the formation of metal wires. A first part of this thesis deals with the characterization of the compl ex substructure of insulin fibrils. The multipathway polymerization proc ess of insulin results in fibrils that can be divided into two groups, e ach characterized by a different structural motif: Helical fibrils compo sed of protofilaments, and chains of rod-like segments. We observed both types of structures with AFM in great detail. The helical fibrils exist with a large variety of periodicities and heights, indicating that they can be formed by different numbers of protofilaments. These protofilame nts cannot directly be distinguished in the fibrils and their number has to be inferred from the height of the fibril. By studying defective hel ical fibrils that have frayed into their individual protofilaments, we w ere able to distinguish the individual protofilaments, confirming the fi bril substructure. This led to the observation that the protofilaments t hat constitute a helical fibril exhibit a periodicity themselves, which is not observed in protofilaments that are not involved in the formation of a helical fibril. The assembly of the fibrils was observed to occur in a hierarchical way: Two protofilaments twist around each other to for m a pair, and two pairs then twist around each other to form a 4-filamen t fibril. Another important assembly pathway leads to insulin fibrils consisting o f rod-like segments that are associated in a head to tail fashion. We we re able to elucidate this polymerization pathway with high-resolution AF M. Strikingly, all of the rod-like segments are uniform in size within o ne fibril, but differ greatly in size between different fibrils. This in dicates the importance of lateral interactions in |
| format | Dissertation |
| fullrecord | <record><control><sourceid>kuleuven_FZOIL</sourceid><recordid>TN_cdi_kuleuven_dspace_123456789_233942</recordid><sourceformat>XML</sourceformat><sourcesystem>PC</sourcesystem><sourcerecordid>123456789_233942</sourcerecordid><originalsourceid>FETCH-kuleuven_dspace_123456789_2339423</originalsourceid><addsrcrecordid>eNqNjDkOwkAMANNQIOAP7ihQCrJcKREC8QD6yGwcscJZR2svx--h4AFUM8VoxoXuTfrgoZPkCb6WRL0Mb5AOlLgrUZX6K1ML1yC9MPnMmEAtZW85kQLGFuxGIUGIRgm9BYnwDHaDngyZv_uIUQZMFjyTTotRh6w0-3FSzE_Hy-Fc3jNTflBsWh3QU7Os3Gq92e7qpnKuXlVuUiz-Kxt7mfv_-wHIxFb4</addsrcrecordid><sourcetype>Institutional Repository</sourcetype><iscdi>true</iscdi><recordtype>dissertation</recordtype></control><display><type>dissertation</type><title>Atomic force microscopy of self-assembled biomolecular structures and their interaction with metallic nanoparticles</title><source>Lirias (KU Leuven Association)</source><creator>Gysemans, Maarten</creator><creatorcontrib>Gysemans, Maarten ; Van Haesendonck, Christian ; Snauwaert, Johan</creatorcontrib><description>We applied AFM to study biomolecular wires, both out of interest in thei r biological functions and in the framework of nanotechnology based fabr ication. We have focused on two different kinds of protein wires: Insuli n fibrils and microtubules. Microtubules are an important constituent of the cytoskeleton and fulfill multiple vital functions in the cell. Insu lin fibrils on the other hand are amyloid fibrils without a clear biolog ical role, but with intriguing polymerization properties that make them an interesting model system for the formation of amyloid fibrils. Both p rotein wires are formed by a self-assembly process and have robust struc tural properties, which make them interesting candidates to act as a tem plate for the formation of metal wires. A first part of this thesis deals with the characterization of the compl ex substructure of insulin fibrils. The multipathway polymerization proc ess of insulin results in fibrils that can be divided into two groups, e ach characterized by a different structural motif: Helical fibrils compo sed of protofilaments, and chains of rod-like segments. We observed both types of structures with AFM in great detail. The helical fibrils exist with a large variety of periodicities and heights, indicating that they can be formed by different numbers of protofilaments. These protofilame nts cannot directly be distinguished in the fibrils and their number has to be inferred from the height of the fibril. By studying defective hel ical fibrils that have frayed into their individual protofilaments, we w ere able to distinguish the individual protofilaments, confirming the fi bril substructure. This led to the observation that the protofilaments t hat constitute a helical fibril exhibit a periodicity themselves, which is not observed in protofilaments that are not involved in the formation of a helical fibril. The assembly of the fibrils was observed to occur in a hierarchical way: Two protofilaments twist around each other to for m a pair, and two pairs then twist around each other to form a 4-filamen t fibril. Another important assembly pathway leads to insulin fibrils consisting o f rod-like segments that are associated in a head to tail fashion. We we re able to elucidate this polymerization pathway with high-resolution AF M. Strikingly, all of the rod-like segments are uniform in size within o ne fibril, but differ greatly in size between different fibrils. This in dicates the importance of lateral interactions in the nucleation of cons ecutive segments: A once formed segment is involved in the formation of more segments that have similar shapes and dimensions. Some of the segme nts reveal a substructure that consists of a narrow lateral protrusion t owards one side along the length of the segment. Remarkably, this protru sion was observed on segments in different fibrils, independent of their size, implying that the substructure of the segments is a characteristi c of this insulin polymerization pathway. The fibrils that are polymerized from B-chain insulin are quite differen t from the complex polymorphism of fibrils polymerized from native insul in. Our measurements revealed two types of fibrils: Small, simple filame nts with a height around 2.2 nm and thick, helical fibrils with heights between 5.5 and 8.0 nm. These two fibril types do not seem to interact w ith each other and we could not observe any substructure in the larger f ibrils. This indicates that, in contrast to the case of native insulin f ibrils, the thick helical fibrils are not composed of the thin filaments . Instead, both fibril types may result from different polymerization pa thways, whose occurrence depends on insulin concentration and the presen ce of a denaturating agent. Microtubules and their interaction with MAPs were the subject of a fruit ful collaboration with the Laboratory of Experimental Genetics and Trans genesis, the Laboratory for Molecular Cell Biology and the Laboratory of Biomolecular Dynamics. In the framework of this collaboration, we succe eded in direct visualization with AFM of microtubules conjugated with no rmal and mutated forms of the protein tau in different phosphorylation s tates. These different tau forms were made available and their binding t o microtubules was tested. Subsequently, the microtubule-tau complexes w ere deposited on substrates that are suitable for AFM, and then imaged t o observe directly how the tau is attached to the microtubule walls. The effect of hyperphosphorylation of tau was evident, as the massive detac hment of tau from the microtubules resulted in unfolding of the microtub ules and damage to their structure. Our AFM images also elucidate the me chanism behind the good binding of the mutant tau-P301L to microtubules. This binding was rather unexpected because the P301L-mutation is known to be involved in neurodegenerative diseases. We observed that the mutat ed tau appears to bind to the microtubule walls in the form of large, so lid tau aggregates. Our direct visualization with AFM of individual micr otubule-tau complexes thus proved to be complementary to more standard b iochemical characterization techniques and provided new insights in the interactions of microtubules with the protein tau. Another important part of this thesis was related to the metallization o f protein wires. Our approach to metallization essentially consisted of two steps: First, metal particles with sizes of a few nanometers were at tached to the walls of the protein template. In a second step these smal l particles were enlarged by silver enhancement. We explored several way s to attach the initial metal particles to the protein template, includi ng direct attachment of preformed gold colloids and electroless plating by reduction of silver or palladium ions. Gold colloids with sizes of 10 nm were found to readily attach to insulin fibrils and microtubules. Go ld particles do, however, not bind to the substrates. We observed that i nsulin fibrils or microtubules which are densely covered with gold collo ids adhere badly to the substrate. Uncovered stretches of the biomolecul e are required to anchor the metal-biomolecule complex to the substrate, limiting the density of gold colloids on the template that can be achie ved. The best results were obtained by incubating insulin fibrils with A g+ ions, followed by their immobilization on a substrate and subsequent application of a NaBH4 reduction bath. After optimalization of the param eters of the reaction, this procedure resulted in fibrils that are dense ly covered with silver particles with sizes of a few nanometers. Our met allization technique proved very efficient in comparison with other appr oaches reported in literature. The stabilization provided by the substra te during the reduction process ensures a high yield of densely covered, individual fibrils. High resolution TEM images, provided by the EMAT gr oup, University of Antwerp, revealed that the silver particles are organ ized in a helical way. This indicates that the metal particles bind to t he insulin fibrils at specific binding sites and adopt the helical struc ture of the template. The arrangement of the silver particles was verifi ed with circular dichroism, an optical technique that is sensitive to ch iral structures. Upon irradiation with UV light, the metallized insulin fibrils displayed a circular dichroism band at wavelengths just below 40 0~nm. This wavelength region is characteristic for the light absorption by small silver particles in optical experiments. The presence of the ci rcular dichroism band nicely confirms the helical arrangement of the sil ver particles. Next, the insulin fibrils that were densely covered with small silver pa rticles were enlarged further by application of silver enhancer. This re sulted in fibrils that are coated with a silver layer that is about 50 n m thick, which is continuous as far as can be judged from AFM images. Ho wever, our electrical conductance measurements only reveal a very poor c onductivity of the metallized fibrils. Two typical results were obtained with the conductance measurements: Next to fibrils that exhibited a lin ear behavior with resistance values of several GOhm at low voltages, als o fibrils with asymmetric I-V characteristics and a zero current plateau around zero bias were observed. These results indicate that the silver enhancement is not sufficient to enlarge the particles to form continuous wires. Charging effects of the silver particles may explain the nonlinear shape of the I-V curves. Also , contamination and oxidation effects at the intergrain boundaries may c omplicate the conductivity of the chains of silver particles. The metall ized fibrils proved to be very sensitive to changes caused by currents t hat can flow at high voltages and to voltage peaks during the connection of the sample to large contact pads by ultrasonic wire-bonding. Thermal ly induced displacement of the silver particles could be observed with A FM after the conductance measurements. Increasing the silver enhancement time might solve these problems, but will lead to considerably thicker and more irregular wires. Reports in literature suggest that a better so lution may be provided by the application of gold enhancement to the sil ver particles, which covers them with a gold layer that is insensitive t o oxidation effects. Another interesting possibility to probe the electrical properties of th e metallized fibrils is to the study the response of the wires to voltag es applied at different frequencies (10 kHz < f < 10 MHz). A pplying an oscillating voltage to the wires induces displacement current s, which are much less hampered by a finite distance between the Ag nano particles or by oxidation of the interfaces between adjacent particles. First results indicate that the capacitance of the wires can be determin ed as a function of frequency. A typical value for the capacitance C is 3.8 pF. Using the freque</description><language>eng</language><creationdate>2009</creationdate><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>311,315,776,27837</link.rule.ids><linktorsrc>$$Uhttps://lirias.kuleuven.be/handle/123456789/233942$$EView_record_in_KU_Leuven_Association$$FView_record_in_$$GKU_Leuven_Association$$Hfree_for_read</linktorsrc></links><search><creatorcontrib>Gysemans, Maarten</creatorcontrib><title>Atomic force microscopy of self-assembled biomolecular structures and their interaction with metallic nanoparticles</title><description>We applied AFM to study biomolecular wires, both out of interest in thei r biological functions and in the framework of nanotechnology based fabr ication. We have focused on two different kinds of protein wires: Insuli n fibrils and microtubules. Microtubules are an important constituent of the cytoskeleton and fulfill multiple vital functions in the cell. Insu lin fibrils on the other hand are amyloid fibrils without a clear biolog ical role, but with intriguing polymerization properties that make them an interesting model system for the formation of amyloid fibrils. Both p rotein wires are formed by a self-assembly process and have robust struc tural properties, which make them interesting candidates to act as a tem plate for the formation of metal wires. A first part of this thesis deals with the characterization of the compl ex substructure of insulin fibrils. The multipathway polymerization proc ess of insulin results in fibrils that can be divided into two groups, e ach characterized by a different structural motif: Helical fibrils compo sed of protofilaments, and chains of rod-like segments. We observed both types of structures with AFM in great detail. The helical fibrils exist with a large variety of periodicities and heights, indicating that they can be formed by different numbers of protofilaments. These protofilame nts cannot directly be distinguished in the fibrils and their number has to be inferred from the height of the fibril. By studying defective hel ical fibrils that have frayed into their individual protofilaments, we w ere able to distinguish the individual protofilaments, confirming the fi bril substructure. This led to the observation that the protofilaments t hat constitute a helical fibril exhibit a periodicity themselves, which is not observed in protofilaments that are not involved in the formation of a helical fibril. The assembly of the fibrils was observed to occur in a hierarchical way: Two protofilaments twist around each other to for m a pair, and two pairs then twist around each other to form a 4-filamen t fibril. Another important assembly pathway leads to insulin fibrils consisting o f rod-like segments that are associated in a head to tail fashion. We we re able to elucidate this polymerization pathway with high-resolution AF M. Strikingly, all of the rod-like segments are uniform in size within o ne fibril, but differ greatly in size between different fibrils. This in dicates the importance of lateral interactions in the nucleation of cons ecutive segments: A once formed segment is involved in the formation of more segments that have similar shapes and dimensions. Some of the segme nts reveal a substructure that consists of a narrow lateral protrusion t owards one side along the length of the segment. Remarkably, this protru sion was observed on segments in different fibrils, independent of their size, implying that the substructure of the segments is a characteristi c of this insulin polymerization pathway. The fibrils that are polymerized from B-chain insulin are quite differen t from the complex polymorphism of fibrils polymerized from native insul in. Our measurements revealed two types of fibrils: Small, simple filame nts with a height around 2.2 nm and thick, helical fibrils with heights between 5.5 and 8.0 nm. These two fibril types do not seem to interact w ith each other and we could not observe any substructure in the larger f ibrils. This indicates that, in contrast to the case of native insulin f ibrils, the thick helical fibrils are not composed of the thin filaments . Instead, both fibril types may result from different polymerization pa thways, whose occurrence depends on insulin concentration and the presen ce of a denaturating agent. Microtubules and their interaction with MAPs were the subject of a fruit ful collaboration with the Laboratory of Experimental Genetics and Trans genesis, the Laboratory for Molecular Cell Biology and the Laboratory of Biomolecular Dynamics. In the framework of this collaboration, we succe eded in direct visualization with AFM of microtubules conjugated with no rmal and mutated forms of the protein tau in different phosphorylation s tates. These different tau forms were made available and their binding t o microtubules was tested. Subsequently, the microtubule-tau complexes w ere deposited on substrates that are suitable for AFM, and then imaged t o observe directly how the tau is attached to the microtubule walls. The effect of hyperphosphorylation of tau was evident, as the massive detac hment of tau from the microtubules resulted in unfolding of the microtub ules and damage to their structure. Our AFM images also elucidate the me chanism behind the good binding of the mutant tau-P301L to microtubules. This binding was rather unexpected because the P301L-mutation is known to be involved in neurodegenerative diseases. We observed that the mutat ed tau appears to bind to the microtubule walls in the form of large, so lid tau aggregates. Our direct visualization with AFM of individual micr otubule-tau complexes thus proved to be complementary to more standard b iochemical characterization techniques and provided new insights in the interactions of microtubules with the protein tau. Another important part of this thesis was related to the metallization o f protein wires. Our approach to metallization essentially consisted of two steps: First, metal particles with sizes of a few nanometers were at tached to the walls of the protein template. In a second step these smal l particles were enlarged by silver enhancement. We explored several way s to attach the initial metal particles to the protein template, includi ng direct attachment of preformed gold colloids and electroless plating by reduction of silver or palladium ions. Gold colloids with sizes of 10 nm were found to readily attach to insulin fibrils and microtubules. Go ld particles do, however, not bind to the substrates. We observed that i nsulin fibrils or microtubules which are densely covered with gold collo ids adhere badly to the substrate. Uncovered stretches of the biomolecul e are required to anchor the metal-biomolecule complex to the substrate, limiting the density of gold colloids on the template that can be achie ved. The best results were obtained by incubating insulin fibrils with A g+ ions, followed by their immobilization on a substrate and subsequent application of a NaBH4 reduction bath. After optimalization of the param eters of the reaction, this procedure resulted in fibrils that are dense ly covered with silver particles with sizes of a few nanometers. Our met allization technique proved very efficient in comparison with other appr oaches reported in literature. The stabilization provided by the substra te during the reduction process ensures a high yield of densely covered, individual fibrils. High resolution TEM images, provided by the EMAT gr oup, University of Antwerp, revealed that the silver particles are organ ized in a helical way. This indicates that the metal particles bind to t he insulin fibrils at specific binding sites and adopt the helical struc ture of the template. The arrangement of the silver particles was verifi ed with circular dichroism, an optical technique that is sensitive to ch iral structures. Upon irradiation with UV light, the metallized insulin fibrils displayed a circular dichroism band at wavelengths just below 40 0~nm. This wavelength region is characteristic for the light absorption by small silver particles in optical experiments. The presence of the ci rcular dichroism band nicely confirms the helical arrangement of the sil ver particles. Next, the insulin fibrils that were densely covered with small silver pa rticles were enlarged further by application of silver enhancer. This re sulted in fibrils that are coated with a silver layer that is about 50 n m thick, which is continuous as far as can be judged from AFM images. Ho wever, our electrical conductance measurements only reveal a very poor c onductivity of the metallized fibrils. Two typical results were obtained with the conductance measurements: Next to fibrils that exhibited a lin ear behavior with resistance values of several GOhm at low voltages, als o fibrils with asymmetric I-V characteristics and a zero current plateau around zero bias were observed. These results indicate that the silver enhancement is not sufficient to enlarge the particles to form continuous wires. Charging effects of the silver particles may explain the nonlinear shape of the I-V curves. Also , contamination and oxidation effects at the intergrain boundaries may c omplicate the conductivity of the chains of silver particles. The metall ized fibrils proved to be very sensitive to changes caused by currents t hat can flow at high voltages and to voltage peaks during the connection of the sample to large contact pads by ultrasonic wire-bonding. Thermal ly induced displacement of the silver particles could be observed with A FM after the conductance measurements. Increasing the silver enhancement time might solve these problems, but will lead to considerably thicker and more irregular wires. Reports in literature suggest that a better so lution may be provided by the application of gold enhancement to the sil ver particles, which covers them with a gold layer that is insensitive t o oxidation effects. Another interesting possibility to probe the electrical properties of th e metallized fibrils is to the study the response of the wires to voltag es applied at different frequencies (10 kHz < f < 10 MHz). A pplying an oscillating voltage to the wires induces displacement current s, which are much less hampered by a finite distance between the Ag nano particles or by oxidation of the interfaces between adjacent particles. First results indicate that the capacitance of the wires can be determin ed as a function of frequency. A typical value for the capacitance C is 3.8 pF. Using the freque</description><fulltext>true</fulltext><rsrctype>dissertation</rsrctype><creationdate>2009</creationdate><recordtype>dissertation</recordtype><sourceid>FZOIL</sourceid><recordid>eNqNjDkOwkAMANNQIOAP7ihQCrJcKREC8QD6yGwcscJZR2svx--h4AFUM8VoxoXuTfrgoZPkCb6WRL0Mb5AOlLgrUZX6K1ML1yC9MPnMmEAtZW85kQLGFuxGIUGIRgm9BYnwDHaDngyZv_uIUQZMFjyTTotRh6w0-3FSzE_Hy-Fc3jNTflBsWh3QU7Os3Gq92e7qpnKuXlVuUiz-Kxt7mfv_-wHIxFb4</recordid><startdate>20090529</startdate><enddate>20090529</enddate><creator>Gysemans, Maarten</creator><scope>FZOIL</scope></search><sort><creationdate>20090529</creationdate><title>Atomic force microscopy of self-assembled biomolecular structures and their interaction with metallic nanoparticles</title><author>Gysemans, Maarten</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-kuleuven_dspace_123456789_2339423</frbrgroupid><rsrctype>dissertations</rsrctype><prefilter>dissertations</prefilter><language>eng</language><creationdate>2009</creationdate><toplevel>online_resources</toplevel><creatorcontrib>Gysemans, Maarten</creatorcontrib><collection>Lirias (KU Leuven Association)</collection></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext_linktorsrc</fulltext></delivery><addata><au>Gysemans, Maarten</au><format>dissertation</format><genre>dissertation</genre><ristype>THES</ristype><Advisor>Van Haesendonck, Christian</Advisor><Advisor>Snauwaert, Johan</Advisor><btitle>Atomic force microscopy of self-assembled biomolecular structures and their interaction with metallic nanoparticles</btitle><date>2009-05-29</date><risdate>2009</risdate><abstract>We applied AFM to study biomolecular wires, both out of interest in thei r biological functions and in the framework of nanotechnology based fabr ication. We have focused on two different kinds of protein wires: Insuli n fibrils and microtubules. Microtubules are an important constituent of the cytoskeleton and fulfill multiple vital functions in the cell. Insu lin fibrils on the other hand are amyloid fibrils without a clear biolog ical role, but with intriguing polymerization properties that make them an interesting model system for the formation of amyloid fibrils. Both p rotein wires are formed by a self-assembly process and have robust struc tural properties, which make them interesting candidates to act as a tem plate for the formation of metal wires. A first part of this thesis deals with the characterization of the compl ex substructure of insulin fibrils. The multipathway polymerization proc ess of insulin results in fibrils that can be divided into two groups, e ach characterized by a different structural motif: Helical fibrils compo sed of protofilaments, and chains of rod-like segments. We observed both types of structures with AFM in great detail. The helical fibrils exist with a large variety of periodicities and heights, indicating that they can be formed by different numbers of protofilaments. These protofilame nts cannot directly be distinguished in the fibrils and their number has to be inferred from the height of the fibril. By studying defective hel ical fibrils that have frayed into their individual protofilaments, we w ere able to distinguish the individual protofilaments, confirming the fi bril substructure. This led to the observation that the protofilaments t hat constitute a helical fibril exhibit a periodicity themselves, which is not observed in protofilaments that are not involved in the formation of a helical fibril. The assembly of the fibrils was observed to occur in a hierarchical way: Two protofilaments twist around each other to for m a pair, and two pairs then twist around each other to form a 4-filamen t fibril. Another important assembly pathway leads to insulin fibrils consisting o f rod-like segments that are associated in a head to tail fashion. We we re able to elucidate this polymerization pathway with high-resolution AF M. Strikingly, all of the rod-like segments are uniform in size within o ne fibril, but differ greatly in size between different fibrils. This in dicates the importance of lateral interactions in the nucleation of cons ecutive segments: A once formed segment is involved in the formation of more segments that have similar shapes and dimensions. Some of the segme nts reveal a substructure that consists of a narrow lateral protrusion t owards one side along the length of the segment. Remarkably, this protru sion was observed on segments in different fibrils, independent of their size, implying that the substructure of the segments is a characteristi c of this insulin polymerization pathway. The fibrils that are polymerized from B-chain insulin are quite differen t from the complex polymorphism of fibrils polymerized from native insul in. Our measurements revealed two types of fibrils: Small, simple filame nts with a height around 2.2 nm and thick, helical fibrils with heights between 5.5 and 8.0 nm. These two fibril types do not seem to interact w ith each other and we could not observe any substructure in the larger f ibrils. This indicates that, in contrast to the case of native insulin f ibrils, the thick helical fibrils are not composed of the thin filaments . Instead, both fibril types may result from different polymerization pa thways, whose occurrence depends on insulin concentration and the presen ce of a denaturating agent. Microtubules and their interaction with MAPs were the subject of a fruit ful collaboration with the Laboratory of Experimental Genetics and Trans genesis, the Laboratory for Molecular Cell Biology and the Laboratory of Biomolecular Dynamics. In the framework of this collaboration, we succe eded in direct visualization with AFM of microtubules conjugated with no rmal and mutated forms of the protein tau in different phosphorylation s tates. These different tau forms were made available and their binding t o microtubules was tested. Subsequently, the microtubule-tau complexes w ere deposited on substrates that are suitable for AFM, and then imaged t o observe directly how the tau is attached to the microtubule walls. The effect of hyperphosphorylation of tau was evident, as the massive detac hment of tau from the microtubules resulted in unfolding of the microtub ules and damage to their structure. Our AFM images also elucidate the me chanism behind the good binding of the mutant tau-P301L to microtubules. This binding was rather unexpected because the P301L-mutation is known to be involved in neurodegenerative diseases. We observed that the mutat ed tau appears to bind to the microtubule walls in the form of large, so lid tau aggregates. Our direct visualization with AFM of individual micr otubule-tau complexes thus proved to be complementary to more standard b iochemical characterization techniques and provided new insights in the interactions of microtubules with the protein tau. Another important part of this thesis was related to the metallization o f protein wires. Our approach to metallization essentially consisted of two steps: First, metal particles with sizes of a few nanometers were at tached to the walls of the protein template. In a second step these smal l particles were enlarged by silver enhancement. We explored several way s to attach the initial metal particles to the protein template, includi ng direct attachment of preformed gold colloids and electroless plating by reduction of silver or palladium ions. Gold colloids with sizes of 10 nm were found to readily attach to insulin fibrils and microtubules. Go ld particles do, however, not bind to the substrates. We observed that i nsulin fibrils or microtubules which are densely covered with gold collo ids adhere badly to the substrate. Uncovered stretches of the biomolecul e are required to anchor the metal-biomolecule complex to the substrate, limiting the density of gold colloids on the template that can be achie ved. The best results were obtained by incubating insulin fibrils with A g+ ions, followed by their immobilization on a substrate and subsequent application of a NaBH4 reduction bath. After optimalization of the param eters of the reaction, this procedure resulted in fibrils that are dense ly covered with silver particles with sizes of a few nanometers. Our met allization technique proved very efficient in comparison with other appr oaches reported in literature. The stabilization provided by the substra te during the reduction process ensures a high yield of densely covered, individual fibrils. High resolution TEM images, provided by the EMAT gr oup, University of Antwerp, revealed that the silver particles are organ ized in a helical way. This indicates that the metal particles bind to t he insulin fibrils at specific binding sites and adopt the helical struc ture of the template. The arrangement of the silver particles was verifi ed with circular dichroism, an optical technique that is sensitive to ch iral structures. Upon irradiation with UV light, the metallized insulin fibrils displayed a circular dichroism band at wavelengths just below 40 0~nm. This wavelength region is characteristic for the light absorption by small silver particles in optical experiments. The presence of the ci rcular dichroism band nicely confirms the helical arrangement of the sil ver particles. Next, the insulin fibrils that were densely covered with small silver pa rticles were enlarged further by application of silver enhancer. This re sulted in fibrils that are coated with a silver layer that is about 50 n m thick, which is continuous as far as can be judged from AFM images. Ho wever, our electrical conductance measurements only reveal a very poor c onductivity of the metallized fibrils. Two typical results were obtained with the conductance measurements: Next to fibrils that exhibited a lin ear behavior with resistance values of several GOhm at low voltages, als o fibrils with asymmetric I-V characteristics and a zero current plateau around zero bias were observed. These results indicate that the silver enhancement is not sufficient to enlarge the particles to form continuous wires. Charging effects of the silver particles may explain the nonlinear shape of the I-V curves. Also , contamination and oxidation effects at the intergrain boundaries may c omplicate the conductivity of the chains of silver particles. The metall ized fibrils proved to be very sensitive to changes caused by currents t hat can flow at high voltages and to voltage peaks during the connection of the sample to large contact pads by ultrasonic wire-bonding. Thermal ly induced displacement of the silver particles could be observed with A FM after the conductance measurements. Increasing the silver enhancement time might solve these problems, but will lead to considerably thicker and more irregular wires. Reports in literature suggest that a better so lution may be provided by the application of gold enhancement to the sil ver particles, which covers them with a gold layer that is insensitive t o oxidation effects. Another interesting possibility to probe the electrical properties of th e metallized fibrils is to the study the response of the wires to voltag es applied at different frequencies (10 kHz < f < 10 MHz). A pplying an oscillating voltage to the wires induces displacement current s, which are much less hampered by a finite distance between the Ag nano particles or by oxidation of the interfaces between adjacent particles. First results indicate that the capacitance of the wires can be determin ed as a function of frequency. A typical value for the capacitance C is 3.8 pF. Using the freque</abstract><oa>free_for_read</oa></addata></record> |
| fulltext | fulltext_linktorsrc |
| identifier | |
| ispartof | |
| issn | |
| language | eng |
| recordid | cdi_kuleuven_dspace_123456789_233942 |
| source | Lirias (KU Leuven Association) |
| title | Atomic force microscopy of self-assembled biomolecular structures and their interaction with metallic nanoparticles |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-02-10T09%3A24%3A18IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-kuleuven_FZOIL&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft.genre=dissertation&rft.btitle=Atomic%20force%20microscopy%20of%20self-assembled%20biomolecular%20structures%20and%20their%20interaction%20with%20metallic%20nanoparticles&rft.au=Gysemans,%20Maarten&rft.date=2009-05-29&rft_id=info:doi/&rft_dat=%3Ckuleuven_FZOIL%3E123456789_233942%3C/kuleuven_FZOIL%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_id=info:pmid/&rfr_iscdi=true |