title: Reversible transition between the surface trimer and membrane-inserted monomer of annexin 12.
abstract: Under mildly acidic conditions, annexin 12 (ANX) inserts into lipid membranes to form a transbilayer pore [Langen, R., et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 14060]. In this study, we have addressed the question of the oligomeric state of ANX in this transbilayer conformation by means of Forster-type resonance energy transfer (FRET). Two single-cysteine mutants (K132C and N244C) were labeled with either Alexa-532 (donor) or Alexa-647 (acceptor). The labels were positioned at the sites thought to be on the cis side of the known transmembrane regions [Ladokhin, A. S., et al. (2002) Biochemistry 41, 13617]. If the pore were comprised of an annexin oligomer, efficient energy transfer should be observed. Fluorescence excitation spectra of several mixtures of donor- and acceptor-labeled ANX were recorded under various conditions. Spectroscopic hallmarks of oligomerization-related FRET were established by following a well-documented transition of ANX from the soluble monomer to surface trimer upon addition of calcium at neutral pH. These hallmarks, however, were not detected for the membrane-inserted form of ANX at pH 4.5, suggesting that the transbilayer form is a monomer. This implies that the pore is formed by several transmembrane regions of the same ANX molecule. FRET and other fluorescence experiments demonstrate that the transitions between the surface trimer and membrane-inserted monomer are reversible. This reversibility, in combination with the absence of oligomerization in the water-soluble and inserted state, makes ANX a good experimental model for thermodynamic studies of folding and stability of membrane proteins.
authors.affiliation: Department of Biochemistry and Molecular Biology, Kansas University Medical Center, Kansas City, Kansas 66160-7421, USA.
authors.forename: Alexey S
authors.initials: AS
authors.lastname: Ladokhin
authors.forename: Harry T
authors.initials: HT
authors.lastname: Haigler
mesh_terms: D000818:Animals; D017302:Annexins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002118:Calcium / Q000737:chemistry; D003545:Cysteine / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer; D006829:Hydra; D008563:Membrane Lipids / Q000737:chemistry* / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000737:chemistry / Q000378:metabolism; D010715:Phosphatidylglycerols / Q000737:chemistry / Q000378:metabolism; D010718:Phosphatidylserines / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013499:Surface Properties; D013816:Thermodynamics
chemical_list: D017302:Annexins; D008563:Membrane Lipids; D010713:Phosphatidylcholines; D010715:Phosphatidylglycerols; D010718:Phosphatidylserines; C060037:1-palmitoyl-2-oleoylglycero-3-phosphoglycerol; D003545:Cysteine; D002118:Calcium; C028694:1-palmitoyl-2-oleoylphosphatidylcholine