title: Occupation of nucleotide in the binding pocket is critical to the stability of Rab11A.
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.
authors.affiliation: Department of Physiology and Biomedical Sciences, Institute of Human-Environment Interface Biology, Seoul National University College of Medicine, Seoul 110-799, South Korea.
authors.forename: Young-Cheul
authors.initials: YC
authors.lastname: Shin
authors.affiliation: School of Biotechnology and Graduate School of Biochemistry at Yeungnam University, Gyeongsan 38541, South Korea.
authors.forename: Chang Min
authors.initials: CM
authors.lastname: Kim
authors.affiliation: School of Biotechnology and Graduate School of Biochemistry at Yeungnam University, Gyeongsan 38541, South Korea.
authors.forename: Jae Young
authors.initials: JY
authors.lastname: Choi
authors.affiliation: Department of Physiology and Biomedical Sciences, Institute of Human-Environment Interface Biology, Seoul National University College of Medicine, Seoul 110-799, South Korea. Electronic address: jhjeon2@snu.ac.kr.
authors.forename: Ju-Hong
authors.initials: JH
authors.lastname: Jeon
authors.affiliation: School of Biotechnology and Graduate School of Biochemistry at Yeungnam University, Gyeongsan 38541, South Korea. Electronic address: hyunho@ynu.ac.kr.
authors.forename: Hyun Ho
authors.initials: HH
authors.lastname: Park
mesh_terms: D020134:Catalytic Domain; D006153:Guanosine Diphosphate / Q000378:metabolism*; D006801:Humans; D009154:Mutation; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000302:isolation & purification; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D000099193:rab11 GTP-Binding Proteins
chemical_list: D006153:Guanosine Diphosphate; D011994:Recombinant Proteins; D020691:rab GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins
keywords: Membrane trafficking; Protein stability; Rab11; Small G protein