On the molecular organization of a succinyl-CoA-producing cell-free system: A cryo-EM and computational approach
Projektleiter:
Projektbearbeiter:
Panagiotis Kastritis,
Skalidis Ioannis
Finanzierung:
Haushalt;
Forschergruppen:
Summary:
Background: In recent years, the importance of metabolites in cellular signalling and metabolic regulation has gained considerable attention. Metabolites such as acetyl-coenzyme A, α-ketoglutarate and palmitic acid play a key role in the regulation of cellular communication, inflammation and even malignancy. These metabolites are regulated by large enzyme complexes known as "metabolons" that control the availability of these important molecules. To perform their functions, metabolons rely on multiple proteins that form dynamic communities, with flexibility provided by unstructured regions that control the availability of reaction intermediates.
Preliminary work: The enzymes responsible for the production of acetyl-coenzyme A, α-ketoglutarate and palmitic acid are part of large macromolecular associations, namely the pyruvate dehydrogenase complex, the oxoglutarate dehydrogenase complex and fatty acid synthase. These complexes consist of structured and unstructured domains that are crucial for regulating the production and availability of metabolites. To comprehensively understand their structural organisation, two prerequisites must be met: (a) the development of a system that preserves the native context of these complexes through biochemical techniques, and (b) the application of advanced structural methods to visualise and interpret their structures.
Objective: In this work, a cell-free system from the thermophilic fungus Chaetomium thermophilum with the ability to produce succinyl-CoA will be used to investigate the structural details of megadalton-sized protein communities. An AI-driven pipeline will be used to perform a de novo structural characterisation of these communities, providing insight into their organisation and how structural flexibility affects their function. In addition, the study will further elucidate the organisation of the oxoglutarate dehydrogenase complex and, in particular, investigate the structure and function of the E2o core. Kinetic characterisation of enzymatic reactions, mass spectrometry-based proteomics and cross-linking mass spectrometry will provide insights into the composition and interactions of the complex.
Methods: The research uses cryogenic electron microscopy, integrative computational structural biology techniques, mass spectrometry-based proteomics and artificial intelligence-based protein modelling to analyse the structural and functional aspects of the protein complexes under study.
Implications: This research provides new insights into the structural and functional aspects of metabolons and their dynamic protein communities, with a focus on the oxoglutarate dehydrogenase complex. Understanding the structural role of flexibility within these complexes is crucial for deciphering their biological significance and potential applications in various fields.
Alignment with the Sustainable Development Goals (SDGs): This study is aligned with several SDGs. It directly contributes to SDG 3 (health and well-being) and SDG 9 (industry, innovation and infrastructure) by advancing our understanding of cellular processes with potential applications in biotechnology and health. It also indirectly supports SDG 4 (Quality of Education) by promoting high quality teaching and research. The collaborative nature of this research also underlines its alignment with SDG 17 (Partnerships for the Goals), as it promotes collaboration between different stakeholders to address pressing scientific and societal challenges. Furthermore, it aligns with SDG 11 (Sustainable Cities and Communities) by exploring sustainable sources of metabolite production and improving our understanding of the underlying biological processes that can lead to more sustainable practices.
Background: In recent years, the importance of metabolites in cellular signalling and metabolic regulation has gained considerable attention. Metabolites such as acetyl-coenzyme A, α-ketoglutarate and palmitic acid play a key role in the regulation of cellular communication, inflammation and even malignancy. These metabolites are regulated by large enzyme complexes known as "metabolons" that control the availability of these important molecules. To perform their functions, metabolons rely on multiple proteins that form dynamic communities, with flexibility provided by unstructured regions that control the availability of reaction intermediates.
Preliminary work: The enzymes responsible for the production of acetyl-coenzyme A, α-ketoglutarate and palmitic acid are part of large macromolecular associations, namely the pyruvate dehydrogenase complex, the oxoglutarate dehydrogenase complex and fatty acid synthase. These complexes consist of structured and unstructured domains that are crucial for regulating the production and availability of metabolites. To comprehensively understand their structural organisation, two prerequisites must be met: (a) the development of a system that preserves the native context of these complexes through biochemical techniques, and (b) the application of advanced structural methods to visualise and interpret their structures.
Objective: In this work, a cell-free system from the thermophilic fungus Chaetomium thermophilum with the ability to produce succinyl-CoA will be used to investigate the structural details of megadalton-sized protein communities. An AI-driven pipeline will be used to perform a de novo structural characterisation of these communities, providing insight into their organisation and how structural flexibility affects their function. In addition, the study will further elucidate the organisation of the oxoglutarate dehydrogenase complex and, in particular, investigate the structure and function of the E2o core. Kinetic characterisation of enzymatic reactions, mass spectrometry-based proteomics and cross-linking mass spectrometry will provide insights into the composition and interactions of the complex.
Methods: The research uses cryogenic electron microscopy, integrative computational structural biology techniques, mass spectrometry-based proteomics and artificial intelligence-based protein modelling to analyse the structural and functional aspects of the protein complexes under study.
Implications: This research provides new insights into the structural and functional aspects of metabolons and their dynamic protein communities, with a focus on the oxoglutarate dehydrogenase complex. Understanding the structural role of flexibility within these complexes is crucial for deciphering their biological significance and potential applications in various fields.
Alignment with the Sustainable Development Goals (SDGs): This study is aligned with several SDGs. It directly contributes to SDG 3 (health and well-being) and SDG 9 (industry, innovation and infrastructure) by advancing our understanding of cellular processes with potential applications in biotechnology and health. It also indirectly supports SDG 4 (Quality of Education) by promoting high quality teaching and research. The collaborative nature of this research also underlines its alignment with SDG 17 (Partnerships for the Goals), as it promotes collaboration between different stakeholders to address pressing scientific and societal challenges. Furthermore, it aligns with SDG 11 (Sustainable Cities and Communities) by exploring sustainable sources of metabolite production and improving our understanding of the underlying biological processes that can lead to more sustainable practices.
Anmerkungen
Summa cum laude (1.0)
Geräte im Projekt
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Kontakt
Jun.-Prof. Dr. Panagiotis Kastritis
Martin-Luther-Universität Halle-Wittenberg
Naturwissenschaftliche Fakultät I
Institut für Biochemie und Biotechnologie
Kurt-Mothes-Straße 3
06120
Halle (Saale)
Tel.:+49 345 5524983
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