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Monitoring Protein-RNA Interaction Dynamics In Vivo at High Temporal Resolution Using χCRAC

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Nucleotide utilization requirements that render ClpB active as a chaperone.

Urko del Castillo¹, José Angel Fernández-Higuero, Sergio Pérez-Acebrón

¹Unidad de Biofísica (CSIC-UPV/EHU), and Departamento de Bioquímica y BiologíaMolecular (UPV/EHU), Facultad de Ciencia y Tecnología, Universidad del País Vasco, P.O. Box 644, Bilbao, Spain.

|January 21, 2010

Summary

The ClpB chaperone protein requires four active subunits for function. Even with inactive subunits, ClpB maintains chaperone activity if one ring has functional nucleotide binding sites.

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Area of Science:

Molecular biology
Protein biochemistry
Cellular stress response

Background:

ClpB is an AAA+ superfamily protein forming a homohexamer.
It utilizes ATP hydrolysis for its chaperone activity.
Previous studies explored ClpB nucleotide utilization, but requirements for subunit cooperation remain unclear.

Purpose of the Study:

To investigate the nucleotide utilization requirements of ClpB.
To determine the minimum number of functional subunits and ATP binding sites for ClpB activity.

Main Methods:

Utilized an experimental approach for random subunit incorporation into the ClpB hexamer.
Assessed chaperone activity with varying combinations of wild-type and mutant ClpB subunits.

Main Results:

A single non-ATP-binding/hydrolyzing subunit significantly reduced chaperone activity.
The wild-type hexamer tolerated two mutant subunits that could only hydrolyze ATP in one ring.
Functional cooperation appears to require at least four subunits, with one ring possessing active nucleotide binding sites.

Conclusions:

ClpB chaperone activity is dependent on the cooperative function of its subunits.
At least four subunits are necessary for full ClpB function.
The presence of active nucleotide binding sites in at least one ring is crucial for ClpB's cooperative mechanism.