Publikation: From Structure to Disorder : Exploring Protein Conformational Dynamics by Electron Paramagnetic Resonance Spectroscopy
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The investigation of protein structures is crucial for gaining insights into their biological function and, by that, into the underlying processes of life. Additionally, comprehending the relationship between protein structure and diseases is vital for developing effective treatments. However, it is essential to recognize that proteins are not only characterized by their rigid structure, but also by their flexible and with that dynamic nature. Proteins can undergo conformational changes, interact with other molecules and adapt their function in response to those structural modifications. In this context, EPR spectroscopy emerges as an ideal tool for investigating the conformational dynamics of proteins. Dynamic processes and related conformational changes can be followed in solution by continuous wave and rapid scan EPR. Pulsed EPR techniques, like DEER or RIDME offer precise information about the three dimensional arrangement and the flexibility within and between protein domains. By employing site-directed spin labeling, EPR spectroscopy can be effectively applied to study large and complex biological systems, including the intracellular environment. In several examples this work demonstrates, that EPR spectroscopy plays a pivotal role in unraveling the conformational behavior of proteins, from very rigid appearing protein domains, to intrinsically disordered proteins, and shedding light on their various functional mechanisms. The muscle fibril is a construct consisting of multiple proteins of different structures and dynamic properties that allow the contraction and relaxation of muscles. Additionally, the elasticity of the sarcomers, the smallest functional unit of striated muscle tissue, can be finely regulated, for example in an event of stress. Involved in this regulation is titin, a large protein, stretched along half of the sarcomere and its binding partner CARP. In a stress situation, like an injury or hypertrophy, CARP is up-regulated and can crosslink titin to the actin filaments, which increases the stiffness of the sarcomers. To investigate this titin/CARP interaction and evaluate the role of protein flexibility in that interaction, SDSL, followed by DEER measurements were applied. Hence the three-dimensional arrangement of the UN2A domain of titin was studied. UN2A is the domain that is supposed to be involved in CARP binding and therefore in the regulation of this cross-linking mechanism. In this work, a calculated model of the three-helix bundle structure of UN2A was validated experimentally. Additional information about the flexibility of the three helices was gained by the same DEER distance distributions. The entire UN2A domain exhibited a highly defined structure with no intrinsic flexibility observed between the three helices. Furthermore, it was demonstrated, that the UN2A domain keeps its conformation and rigidity upon interaction with CARP. In summary, distance distributions obtained by DEER spectroscopy were applied to study the three-dimensional folding of the UNA2 domain and its rigidity, alone and when interacting with CARP. A complete contrary behavior is shown by intrinsically disordered proteins (IDPs). IDPs are disordered in solution and only adopt a specific conformation and fulfill their function upon interaction with other biomolecules or in specific environments. In this work, two IDPs were studied, alpha-synculein and amyloid beta, both being involved in neurodegenerative diseases. Alpha-synuclein has the ability to bind negatively charged lipids and with that, switch to an alpha-helical conformation. However, within the resolution of the spectroscopic methods, alpha-synuclein persists disordered in the cell, despite the presence of available membranes. Therefore, the membrane binding of alpha-synuclein inside cells was studied using SDSL and rapid scan EPR spectroscopy. The spin label is able to detect changes in the flexibility, which can be observed in changes of the spectral shape. Upon membrane binding, the flexibility of the spin label in the membrane binding region of alpha-synuclein is restricted by the membrane. Therefore, the EPR spectrum can be used to detect and quantify membrane binding. By using rapid scan EPR spectroscopy, this membrane binding was detected in a time-resolved manner, with a time resolution of minutes that allowed to measure lipid binding kinetics of alpha-synuclein in the cell. However, the limit to detect the intrinsic flexibility of the protein backbone using a spin label is the linker flexibility of the label, which adds a certain offset to all measurements. Therefore, the spin labeled amino acid PROF was developed and incorporated in the intrinsically disordered peptide amyloid beta (Aβ). It contains a more rigid linker, than commonly used spin labels like MTSSL, but does not perturb the native disordered conformation of Aβ. Because Aβ binds Cu2+ with a nanomolar affinity and Cu2+ was also found in the Aβ-containing plaques in the brain of Alzheimer’s disease patients, the influence of Cu2+ on the conformation and flexibility of Aβ is of interest. This was exploited to further improve the resolution of the distance determination. As a second spin label the Cu2+-ion bound by Aβ was used. Cu2+ is complexed by four amino acids in the N-terminal region of Aβ, and therefore no additional flexibility should be introduced by this paramagnetic center. Additionally, by incorporation of only one spin labeled amino acid, perturbation of the native disordered state of Aβ was minimized. Using RIDME, it was feasible to obtain the distance distribution between the PROF amino acid and the bound Cu2+ of monomeric Aβ in solution. This system was applied to study the distribution of Cu2+ in monomeric Aβ peptides. Here, it was shown that with sub-equimolar concentrations of Cu2+, the Cu2+-ions distribute between the Aβ peptides in a time range of seconds to minutes. In the future, this system might be exploited to study the changes in conformation and flexibility during the whole process of fibrillization, starting from monomers to oligomers to whole fibrils. An even more elaborated approach to map the flexibility of specific domains within a protein, is multilateration. Using a set of distance distributions, for instance obtained by DEER spectroscopy, the location and the degree of flexibility of a protein domain can be visualized and quantified with multilateration. Using distance information between several known positions in the protein and one unknown location, the coordinates of the unknown location can be calculated, working basically like the Global Position System GPS. This multilateration approach was applied on the protein kinase Akt1. Akt1 is a two-domain protein, consisting of a membrane-binding PH domain and a catalytically active kinase domain, both connected via a flexible linker. The conformation of those two domains towards each other is most probable involved in regulating the activity of kinase. Several kinase inhibitors, developed to be applied for instance in cancer therapy, influence this conformation. Therefore, this two domain conformational equilibrium is relevant to investigate. Due to the flexible linker that connects the two domains, a large degree of flexibility is expected to appear between the domains. Therefore, EPR spectroscopy with subsequent multilateration was applied to study Akt1 and its conformational equilibrium. In this work, the influence of binding two different classes of kinase inhibitors and its native binding partner PIP3 was studied. Additionally, the influence of the E17K mutation, the most found mutation in Akt1 related cancer, was evaluated. The kinase domain was used as fixed reference point and the location of the PH domain was calculated in respect to it. It was shown that in the apo kinase the two domains display a certain degree flexibility between them, which could not be concluded by an existing X-ray structure before. The allosteric inhibitor, however, locked the two domains closely together, inducing a rigid conformation of the kinase, thereby blocking the access to the active site in the kinase domain. The ATP-competitive inhibitors, binding directly to the active site in the kinase domain, had a contrary effect, releasing the two domains from each other, therefore exhibiting a very high degree of flexibility in between them. A similar open and flexible conformation was detected when binding to PIP3 containing lipids, confirming that separation of the two domains is important for PIP3-mediated membrane binding and subsequent activation of the kinase by phosphorylation. Surprisingly, the E17K mutation showed no effect on this whole conformational equilibrium of Akt1. This demonstrates that rather a changed affinity for the phospholipids explains the increased activity in the mutant, than a shift towards the open conformation, induced by an inversion in charge of the mutated amino acid. In conclusion, all these studies show that site-directed spin labeling in combination with a whole selection of different EPR spectroscopy methods is an effective method to study the protein conformational dynamic in relevant environments. Monitoring the flexibility of a spin label in the membrane binding region of alphasynuclein with rapid scan EPR was exploited to follow membrane binding of alphasynuclein inside cells. By incorporation of a spin labeled amino acid in Aβ in combination with Cu2+ as a second paramagnetic center an experimental setup to evaluate the flexibility of Aβ through EPR distance information was established. Using DEER distance distributions the rigid UN2A domain of titin was studied. DEER with subsequent multilateration was applied to map the conformational dynamic of the two-domain kinase Akt1. All these examples illustrate that EPR spectroscopy can offer distinctive insights into protein conformational dynamics, thereby complementing other techniques such as X-ray crystallography and NMR spectroscopy in the investigaton of protein structure and dynamic. This synergy effectively aids in obtaining a comprehensive and realistic understanding of the complex processes in which proteins are involved.
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STEHLE, Juliane, 2024. From Structure to Disorder : Exploring Protein Conformational Dynamics by Electron Paramagnetic Resonance Spectroscopy [Dissertation]. Konstanz: Universität KonstanzBibTex
@phdthesis{Stehle2024Struc-70417, year={2024}, title={From Structure to Disorder : Exploring Protein Conformational Dynamics by Electron Paramagnetic Resonance Spectroscopy}, author={Stehle, Juliane}, address={Konstanz}, school={Universität Konstanz} }
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Additionally, comprehending the relationship between protein structure and diseases is vital for developing effective treatments. However, it is essential to recognize that proteins are not only characterized by their rigid structure, but also by their flexible and with that dynamic nature. Proteins can undergo conformational changes, interact with other molecules and adapt their function in response to those structural modifications. In this context, EPR spectroscopy emerges as an ideal tool for investigating the conformational dynamics of proteins. Dynamic processes and related conformational changes can be followed in solution by continuous wave and rapid scan EPR. Pulsed EPR techniques, like DEER or RIDME offer precise information about the three dimensional arrangement and the flexibility within and between protein domains. By employing site-directed spin labeling, EPR spectroscopy can be effectively applied to study large and complex biological systems, including the intracellular environment. In several examples this work demonstrates, that EPR spectroscopy plays a pivotal role in unraveling the conformational behavior of proteins, from very rigid appearing protein domains, to intrinsically disordered proteins, and shedding light on their various functional mechanisms. The muscle fibril is a construct consisting of multiple proteins of different structures and dynamic properties that allow the contraction and relaxation of muscles. Additionally, the elasticity of the sarcomers, the smallest functional unit of striated muscle tissue, can be finely regulated, for example in an event of stress. Involved in this regulation is titin, a large protein, stretched along half of the sarcomere and its binding partner CARP. In a stress situation, like an injury or hypertrophy, CARP is up-regulated and can crosslink titin to the actin filaments, which increases the stiffness of the sarcomers. To investigate this titin/CARP interaction and evaluate the role of protein flexibility in that interaction, SDSL, followed by DEER measurements were applied. Hence the three-dimensional arrangement of the UN2A domain of titin was studied. UN2A is the domain that is supposed to be involved in CARP binding and therefore in the regulation of this cross-linking mechanism. In this work, a calculated model of the three-helix bundle structure of UN2A was validated experimentally. Additional information about the flexibility of the three helices was gained by the same DEER distance distributions. The entire UN2A domain exhibited a highly defined structure with no intrinsic flexibility observed between the three helices. Furthermore, it was demonstrated, that the UN2A domain keeps its conformation and rigidity upon interaction with CARP. In summary, distance distributions obtained by DEER spectroscopy were applied to study the three-dimensional folding of the UNA2 domain and its rigidity, alone and when interacting with CARP. A complete contrary behavior is shown by intrinsically disordered proteins (IDPs). IDPs are disordered in solution and only adopt a specific conformation and fulfill their function upon interaction with other biomolecules or in specific environments. In this work, two IDPs were studied, alpha-synculein and amyloid beta, both being involved in neurodegenerative diseases. Alpha-synuclein has the ability to bind negatively charged lipids and with that, switch to an alpha-helical conformation. However, within the resolution of the spectroscopic methods, alpha-synuclein persists disordered in the cell, despite the presence of available membranes. Therefore, the membrane binding of alpha-synuclein inside cells was studied using SDSL and rapid scan EPR spectroscopy. The spin label is able to detect changes in the flexibility, which can be observed in changes of the spectral shape. Upon membrane binding, the flexibility of the spin label in the membrane binding region of alpha-synuclein is restricted by the membrane. Therefore, the EPR spectrum can be used to detect and quantify membrane binding. By using rapid scan EPR spectroscopy, this membrane binding was detected in a time-resolved manner, with a time resolution of minutes that allowed to measure lipid binding kinetics of alpha-synuclein in the cell. However, the limit to detect the intrinsic flexibility of the protein backbone using a spin label is the linker flexibility of the label, which adds a certain offset to all measurements. Therefore, the spin labeled amino acid PROF was developed and incorporated in the intrinsically disordered peptide amyloid beta (Aβ). It contains a more rigid linker, than commonly used spin labels like MTSSL, but does not perturb the native disordered conformation of Aβ. Because Aβ binds Cu<sup>2+</sup> with a nanomolar affinity and Cu<sup>2+</sup> was also found in the Aβ-containing plaques in the brain of Alzheimer’s disease patients, the influence of Cu2+ on the conformation and flexibility of Aβ is of interest. This was exploited to further improve the resolution of the distance determination. As a second spin label the Cu<sup>2+</sup>-ion bound by Aβ was used. Cu<sup>2+</sup> is complexed by four amino acids in the N-terminal region of Aβ, and therefore no additional flexibility should be introduced by this paramagnetic center. Additionally, by incorporation of only one spin labeled amino acid, perturbation of the native disordered state of Aβ was minimized. Using RIDME, it was feasible to obtain the distance distribution between the PROF amino acid and the bound Cu<sup>2+</sup> of monomeric Aβ in solution. This system was applied to study the distribution of Cu<sup>2+</sup> in monomeric Aβ peptides. Here, it was shown that with sub-equimolar concentrations of Cu<sup>2+</sup>, the Cu<sup>2+</sup>-ions distribute between the Aβ peptides in a time range of seconds to minutes. In the future, this system might be exploited to study the changes in conformation and flexibility during the whole process of fibrillization, starting from monomers to oligomers to whole fibrils. An even more elaborated approach to map the flexibility of specific domains within a protein, is multilateration. Using a set of distance distributions, for instance obtained by DEER spectroscopy, the location and the degree of flexibility of a protein domain can be visualized and quantified with multilateration. Using distance information between several known positions in the protein and one unknown location, the coordinates of the unknown location can be calculated, working basically like the Global Position System GPS. This multilateration approach was applied on the protein kinase Akt1. Akt1 is a two-domain protein, consisting of a membrane-binding PH domain and a catalytically active kinase domain, both connected via a flexible linker. The conformation of those two domains towards each other is most probable involved in regulating the activity of kinase. Several kinase inhibitors, developed to be applied for instance in cancer therapy, influence this conformation. Therefore, this two domain conformational equilibrium is relevant to investigate. Due to the flexible linker that connects the two domains, a large degree of flexibility is expected to appear between the domains. Therefore, EPR spectroscopy with subsequent multilateration was applied to study Akt1 and its conformational equilibrium. In this work, the influence of binding two different classes of kinase inhibitors and its native binding partner PIP<sub>3</sub> was studied. Additionally, the influence of the E17K mutation, the most found mutation in Akt1 related cancer, was evaluated. The kinase domain was used as fixed reference point and the location of the PH domain was calculated in respect to it. It was shown that in the apo kinase the two domains display a certain degree flexibility between them, which could not be concluded by an existing X-ray structure before. The allosteric inhibitor, however, locked the two domains closely together, inducing a rigid conformation of the kinase, thereby blocking the access to the active site in the kinase domain. The ATP-competitive inhibitors, binding directly to the active site in the kinase domain, had a contrary effect, releasing the two domains from each other, therefore exhibiting a very high degree of flexibility in between them. A similar open and flexible conformation was detected when binding to PIP<sub>3</sub> containing lipids, confirming that separation of the two domains is important for PIP<sub>3</sub>-mediated membrane binding and subsequent activation of the kinase by phosphorylation. Surprisingly, the E17K mutation showed no effect on this whole conformational equilibrium of Akt1. This demonstrates that rather a changed affinity for the phospholipids explains the increased activity in the mutant, than a shift towards the open conformation, induced by an inversion in charge of the mutated amino acid. In conclusion, all these studies show that site-directed spin labeling in combination with a whole selection of different EPR spectroscopy methods is an effective method to study the protein conformational dynamic in relevant environments. Monitoring the flexibility of a spin label in the membrane binding region of alphasynuclein with rapid scan EPR was exploited to follow membrane binding of alphasynuclein inside cells. By incorporation of a spin labeled amino acid in Aβ in combination with Cu<sup>2+</sup> as a second paramagnetic center an experimental setup to evaluate the flexibility of Aβ through EPR distance information was established. Using DEER distance distributions the rigid UN2A domain of titin was studied. DEER with subsequent multilateration was applied to map the conformational dynamic of the two-domain kinase Akt1. All these examples illustrate that EPR spectroscopy can offer distinctive insights into protein conformational dynamics, thereby complementing other techniques such as X-ray crystallography and NMR spectroscopy in the investigaton of protein structure and dynamic. This synergy effectively aids in obtaining a comprehensive and realistic understanding of the complex processes in which proteins are involved.</dcterms:abstract> <dc:creator>Stehle, Juliane</dc:creator> <dcterms:isPartOf rdf:resource="https://kops.uni-konstanz.de/server/rdf/resource/123456789/29"/> <dspace:isPartOfCollection rdf:resource="https://kops.uni-konstanz.de/server/rdf/resource/123456789/29"/> <dcterms:hasPart rdf:resource="https://kops.uni-konstanz.de/bitstream/123456789/70417/4/Stehle_2-10080xkno1bgu7.pdf"/> <dcterms:available rdf:datatype="http://www.w3.org/2001/XMLSchema#dateTime">2024-07-18T05:06:28Z</dcterms:available> <dc:contributor>Stehle, Juliane</dc:contributor> <dcterms:issued>2024</dcterms:issued> <dc:date rdf:datatype="http://www.w3.org/2001/XMLSchema#dateTime">2024-07-18T05:06:28Z</dc:date> <dc:rights>terms-of-use</dc:rights> <bibo:uri rdf:resource="https://kops.uni-konstanz.de/handle/123456789/70417"/> </rdf:Description> </rdf:RDF>