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Acidic pH‐induced folding of annexin VI is a prerequisite for its insertion into lipid bilayers and formation of ion channels by the protein molecules
Author(s) -
Golczak Marcin,
Kicinska Anna,
Bandorowicz-Pikula Joanna,
Buchet Rene,
Szewczyk Adam,
Pikula Slawomir
Publication year - 2001
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fsb2fj000523fje
Subject(s) - chemistry , membrane , circular dichroism , lipid bilayer , biophysics , bilayer , phospholipid , hydrophobic effect , ion channel , liposome , crystallography , biochemistry , receptor , biology
Annexin VI (AnxVI), a member of a family of Ca 2+ ‐ and lipid‐binding proteins, can interact with membranes in a Ca 2+ ‐independent manner and behave as a membrane integral protein. The molecular mechanisms for such behavior of AnxVI remain, however, elusive. By determining the surface pressure of phospholipid monolayers in the presence of AnxVI in a Ca 2+ ‐free medium, it was observed that AnxVI can penetrate the hydrophobic region of the membrane at mildly acidic pH. A pH‐dependent binding of the protein was also observed with asolectin liposomes and a pull‐down assay (half maximal binding at pH 5.3). The Ca 2+ ‐independent, pH‐induced binding of AnxVI to membranes was a prerequisite for the formation of ion channels by AnxVI, as characterized by the planar lipid bilayer method. Below pH 6.0 and in the nanomolar concentration range, AnxVI formed voltage‐dependent ion channels with no specificity to the transported ions. These channels were inhibited by neither La 3 + nor Zn 2+ . The single channel conductance amounted to 24.3 pS under symmetric ionic conditions. Measurements of the enhancement of fluorescence of a hydrophobic probe, 2‐(p‐toluidino)naphthalene‐6‐sulfonic acid, in the presence of AnxVI as a function of pH revealed that AnxVI exposes hydrophobic surfaces below pH 6.0. This condition would allow penetration of the protein into the hydrophobic core of membrane. With the aid of circular dichroism and Fourier transform infrared spectroscopy, it was determined that the changes in hydrophobicity of AnxVI were accompanied by a decrease of α‐helix content, the appearance of new β‐sheet structures and an increase of a number of β‐turn segments within the AnxVI molecule. Changes of secondary structure of AnxVI were corroborated by an increase in the accessibility of one of the AnxVI tryptophan residues to fluorescence quenchers at pH 4.6, compared with pH 7.4. Our observations provide further insight into some aspects of the molecular interactions between AnxVI and membrane lipid, permitting the suggestion that AnxVI may be involved in some pathological processes where acidification of the cytosol and/or disruption of intracellular ion homeostasis are detected.