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Application of linker technique to trap transiently interacting protein complexes for structural studies.


ABSTRACT: Protein-protein interactions are key events controlling several biological processes. We have developed and employed a method to trap transiently interacting protein complexes for structural studies using glycine-rich linkers to fuse interacting partners, one of which is unstructured. Initial steps involve isothermal titration calorimetry to identify the minimum binding region of the unstructured protein in its interaction with its stable binding partner. This is followed by computational analysis to identify the approximate site of the interaction and to design an appropriate linker length. Subsequently, fused constructs are generated and characterized using size exclusion chromatography and dynamic light scattering experiments. The structure of the chimeric protein is then solved by crystallization, and validated both in vitro and in vivo by substituting key interacting residues of the full length, unlinked proteins with alanine. This protocol offers the opportunity to study crucial and currently unattainable transient protein interactions involved in various biological processes.

SUBMITTER: Reddy Chichili VP 

PROVIDER: S-EPMC4789767 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Application of linker technique to trap transiently interacting protein complexes for structural studies.

Reddy Chichili Vishnu Priyanka VP   Kumar Veerendra V   Sivaraman J J  

Journal of biological methods 20160101 1


Protein-protein interactions are key events controlling several biological processes. We have developed and employed a method to trap transiently interacting protein complexes for structural studies using glycine-rich linkers to fuse interacting partners, one of which is unstructured. Initial steps involve isothermal titration calorimetry to identify the minimum binding region of the unstructured protein in its interaction with its stable binding partner. This is followed by computational analys  ...[more]

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