Visualizing the Ca2+-dependent activation of gelsolin by using synchrotron footprinting

Janna G. Kiselar, Paul A. Janmey, Steven C. Almo, Mark R. Chance

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Abstract

Radiolytic protein footprinting with a synchrotron source is used to reveal detailed structural changes that occur in the Ca2+-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 asa function of Ca2+ concentration in solution. Twenty-two of the peptides exhibited detectable oxidation; for seven the oxidation extent was seen to be Ca2+ sensitive. Ca2+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 Ca2+ concentration of 1-5 μM that is competent for capping and severing activity. A second structural transition with a midpoint of ≈60-100 μM, 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-actinbinding site on S2 and buried F-actin-binding residues within S2 (including Phe-163) are unmasked in the submicromolar Ca2+ transition. However, residues within S4 that are part of an extended β-sheet with S6 (including Tyr-453) are revealed only in the subsequent transition at higher Ca2+ 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.

Original languageEnglish (US)
Pages (from-to)3942-3947
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume100
Issue number7
DOIs
StatePublished - Apr 1 2003

ASJC Scopus subject areas

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