title: Visualizing the Ca2+-dependent activation of gelsolin by using synchrotron footprinting.
abstract: Radiolytic protein footprinting with a synchrotron source is used to reveal detailed structural changes that occur in the Ca(2+)-dependent activation of gelsolin. More than 80 discrete peptides segments within the structure, covering 95% of the sequence in the molecule, were examined by footprinting and mass spectrometry for their solvent accessibility as a function of Ca(2+) concentration in solution. Twenty-two of the peptides exhibited detectable oxidation; for seven the oxidation extent was seen to be Ca(2+) sensitive. Ca(2+)titration isotherms monitoring the oxidation within residues 49-72 (within subdomain S1), 121-135 (S1), 162-166 (S2), and 722-748 (S6) indicate a three-state activation process with a intermediate that was populated at a Ca(2+) concentration of 1-5 microM that is competent for capping and severing activity. A second structural transition with a midpoint of approximately 60-100 microM, where the accessibility of the above four peptides is further increased, is also observed. Tandem mass spectrometry showed that buried residues within the helical "latch" of S6 (including Pro-745) that contact an F-actin-binding site on S2 and buried F-actin-binding residues within S2 (including Phe-163) are unmasked in the submicromolar Ca(2+) transition. However, residues within S4 that are part of an extended beta-sheet with S6 (including Tyr-453) are revealed only in the subsequent transition at higher Ca(2+) concentrations; the disruption of this extended contact between S4 and S6 (and likely the analogous contact between S1 and S3) likely results in an extended structure permitting additional functions consistent with the fully activated gelsolin molecule.
authors.affiliation: Department of Physiology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461-1602, USA.
authors.forename: Janna G
authors.initials: JG
authors.lastname: Kiselar
authors.forename: Paul A
authors.initials: PA
authors.lastname: Janmey
authors.forename: Steven C
authors.initials: SC
authors.lastname: Almo
authors.forename: Mark R
authors.initials: MR
authors.lastname: Chance
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D018260:Gelsolin / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017356:Synchrotrons
chemical_list: D018260:Gelsolin; D010446:Peptide Fragments; D011994:Recombinant Proteins; D002118:Calcium