Plant annexins form calcium-independent oligomers in solution.
The oligomeric state in solution of four plant annexins, namely Anx23(Ca38), Anx24(Ca32), Anx(Gh1), and Anx(Gh2), was characterized by sedimentation equilibrium analysis and gel filtration. All proteins were expressed and purified as amino-terminal His(n) fusions. Sequencing of the Anx(Gh1) construct revealed distinct differences with the published sequence. Sedimentation equilibrium analysis of Anx23(Ca38), Anx24(Ca32), and Anx(Gh1) suggests monomer-trimer equilibria for each protein with association constants in the range of 0.9 x 10(10)-1.7 x 10(11) M(-2). All four proteins were subjected to analytical gel filtration under different buffer conditions. Observations from this experiment series agree quantitatively with the ultracentrifugation results, and strongly suggest calcium independence of the annexin oligomerization behavior; moreover, binding of calcium ions to the proteins seems to require disassembly of the oligomers. Anx(Gh2) showed a different elution profile than the other plant annexins; while having only a very small trimer content, this annexin seems to exist in a monomer-dimer equilibrium in solution.
Hofmann
Andreas
A
Protein Structure Section, Macromolecular Crystallography Laboratory, National Cancer Institute, Frederick, Maryland 21702, USA. hofmanna@ncifcrf.gov
Ruvinov
Sergei
S
Hess
Sonja
S
Schantz
Rodolphe
R
Delmer
Deborah P
DP
Wlodawer
Alexander
A
D000595:Amino Acid Sequence; D017302:Annexins / Q000737:chemistry*; D001483:Base Sequence; D002118:Calcium / Q000737:chemistry*; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics; D003368:Gossypium / Q000737:chemistry*; D007700:Kinetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D012997:Solvents / Q000737:chemistry; D014461:Ultracentrifugation / Q000379:methods
D017302:Annexins; D011993:Recombinant Fusion Proteins; D012997:Solvents; D002118:Calcium