Occupation of nucleotide in the binding pocket is critical to the stability of Rab11A

Protein Expression and Purification
Young-Cheul ShinHyun Ho Park

Abstract

The Ras superfamily of small G proteins is a family of guanosine triphosphatases (GTPases) and each GTPase has conserved amino acid sequences in the enzymatic active site that are responsible for specific interactions with GDP and GTP molecules. Rab GTPases, which belong to the Ras superfamily, are key regulators of intracellular vesicle trafficking via the recruitment of effector molecules. Here, we purified wild type, active mutant and inactive mutant of Rab11A. In this process, we found that the inactive mutant (Rab11A S25N) had low stability compared with wild type and other mutants. Further analysis revealed that the stability of Rab11A S25N is dependent on the occupation of GDP in the nucleotide binding pocket of the protein. We found that the stability of Rab11A S25N is affected by the presence of GDP, not other nucleotides, and is independent of pH or salt in FPLC buffer. Our results provide a better understanding of how GTPase can be stable under in vitro conditions without effector proteins and how proper substrate/cofactor coordination is crucial to the stability of Rab11A. Successful purification and proposed purification methods will provide a valuable guide for investigation of other small GTPase proteins.

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