A method to trap transient and weak interacting protein complexes for structural studies

Intrinsically Disordered Proteins
Vishnu Priyanka Reddy ChichiliJ Sivaraman

Abstract

Several key biological events adopt a "hit-and-run" strategy in their transient interactions between binding partners. In some instances, the disordered nature of one of the binding partners severely hampers the success of co-crystallization, often leading to the crystallization of just one of the partners. Here, we discuss a method to trap weak and transient protein interactions for crystallization. This approach requires the structural details of at least one of the interacting partners and binding knowledge to dock the known minimum binding region (peptide) of the protein onto the other using an optimal-sized linker. Prior to crystallization, the purified linked construct should be verified for its intact folding and stability. Following structure determination, structure-guided functional studies are performed with independent, full-length unlinked proteins to validate the findings of the linked complex. We designed this approach and then validated its efficacy using a 24 amino acid minimum binding region of the intrinsically disordered, neuron-specific substrates, Neurogranin and Neuromodulin, joined via a Gly-linker to their interacting partner, Calmodulin. Moreover, the reported functional studies with independent full-l...Continue Reading

References

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Dec 12, 2012·Protein Science : a Publication of the Protein Society·Vishnu Priyanka Reddy ChichiliJ Sivaraman

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Citations

Jul 7, 2015·Acta Crystallographica. Section F, Structural Biology Communications·Bostjan KobeSimon J Williams
Aug 15, 2013·Scientific Reports·Vishnu Priyanka Reddy ChichiliJ Sivaraman

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Methods Mentioned

BETA
X-ray
NMR
co-crystallization
PCR
size exclusion chromatography
dynamic light scattering
Surface
Isothermal Titration Calorimetry
Circular Dichroism
pull-down

Software Mentioned

DALI
Cluspro
Auto Dock
3D
COOT
Garden
Deep View
ShelxC
Dock Suite

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