Determination of Dissociation Constants for the Interaction of Myosin-5a with its Cargo Protein Using Microscale Thermophoresis (MST)
Myosin-5a (Myo5a) is an actin-dependent molecular motor that recognizes a diverse range of cargo proteins through its tail domain, playing a crucial role in the transport and localization of various organelles within the cell. We have identified a new interaction between Myo5a and its cargo protein...
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Bio-protocol LLC
2025-02-01
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| author | Rui Zhou Jiabin Pan Xiang-Dong Li |
| author_facet | Rui Zhou Jiabin Pan Xiang-Dong Li |
| author_sort | Rui Zhou |
| collection | DOAJ |
| description | Myosin-5a (Myo5a) is an actin-dependent molecular motor that recognizes a diverse range of cargo proteins through its tail domain, playing a crucial role in the transport and localization of various organelles within the cell. We have identified a new interaction between Myo5a and its cargo protein melanophilin (Mlph), i.e., the interaction between the middle tail domain of Myo5a (Myo5a-MTD) and the actin-binding domain of Mlph (Mlph-ABD), by GST pulldown assay. We then intend to obtain the dissociation constant between Myo5a-MTD and Mlph-ABD using isothermal titration calorimetry (ITC) or microscale thermophoresis (MST), both of which are two commonly used methods for determining quantitative data on protein interactions. The advantages of MST over ITC include less protein usage, shorter operation time, and higher sensitivity. In this protocol, we present a method for using MST to determine the dissociation constants of Myo5a-MTD and Mlph-ABD, which were purified through overexpression in bacteria using affinity chromatography. The dissociation constant values obtained directly reflect the binding strength between these two proteins and provide a foundation for the isolation and purification of the complex in the future. |
| format | Article |
| id | doaj-art-234d2ed3f9e8495397fc7e03dded1212 |
| institution | Kabale University |
| issn | 2331-8325 |
| language | English |
| publishDate | 2025-02-01 |
| publisher | Bio-protocol LLC |
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| spelling | doaj-art-234d2ed3f9e8495397fc7e03dded12122025-02-07T08:16:46ZengBio-protocol LLCBio-Protocol2331-83252025-02-0115310.21769/BioProtoc.5176Determination of Dissociation Constants for the Interaction of Myosin-5a with its Cargo Protein Using Microscale Thermophoresis (MST)Rui Zhou0Jiabin Pan1Xiang-Dong Li2Group of Cell Motility and Muscle Contraction, State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing, ChinaUniversity of Chinese Academy of Sciences, Beijing, ChinaGroup of Cell Motility and Muscle Contraction, State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing, ChinaUniversity of Chinese Academy of Sciences, Beijing, ChinaGroup of Cell Motility and Muscle Contraction, State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing, ChinaUniversity of Chinese Academy of Sciences, Beijing, ChinaMyosin-5a (Myo5a) is an actin-dependent molecular motor that recognizes a diverse range of cargo proteins through its tail domain, playing a crucial role in the transport and localization of various organelles within the cell. We have identified a new interaction between Myo5a and its cargo protein melanophilin (Mlph), i.e., the interaction between the middle tail domain of Myo5a (Myo5a-MTD) and the actin-binding domain of Mlph (Mlph-ABD), by GST pulldown assay. We then intend to obtain the dissociation constant between Myo5a-MTD and Mlph-ABD using isothermal titration calorimetry (ITC) or microscale thermophoresis (MST), both of which are two commonly used methods for determining quantitative data on protein interactions. The advantages of MST over ITC include less protein usage, shorter operation time, and higher sensitivity. In this protocol, we present a method for using MST to determine the dissociation constants of Myo5a-MTD and Mlph-ABD, which were purified through overexpression in bacteria using affinity chromatography. The dissociation constant values obtained directly reflect the binding strength between these two proteins and provide a foundation for the isolation and purification of the complex in the future.https://bio-protocol.org/en/bpdetail?id=5176&type=0 |
| spellingShingle | Rui Zhou Jiabin Pan Xiang-Dong Li Determination of Dissociation Constants for the Interaction of Myosin-5a with its Cargo Protein Using Microscale Thermophoresis (MST) Bio-Protocol |
| title | Determination of Dissociation Constants for the Interaction of Myosin-5a with its Cargo Protein Using Microscale Thermophoresis (MST) |
| title_full | Determination of Dissociation Constants for the Interaction of Myosin-5a with its Cargo Protein Using Microscale Thermophoresis (MST) |
| title_fullStr | Determination of Dissociation Constants for the Interaction of Myosin-5a with its Cargo Protein Using Microscale Thermophoresis (MST) |
| title_full_unstemmed | Determination of Dissociation Constants for the Interaction of Myosin-5a with its Cargo Protein Using Microscale Thermophoresis (MST) |
| title_short | Determination of Dissociation Constants for the Interaction of Myosin-5a with its Cargo Protein Using Microscale Thermophoresis (MST) |
| title_sort | determination of dissociation constants for the interaction of myosin 5a with its cargo protein using microscale thermophoresis mst |
| url | https://bio-protocol.org/en/bpdetail?id=5176&type=0 |
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