Actin polymerization overshoots and ATP hydrolysis as assayed by pyrene fluorescence

F. J. Brooks, A. E. Carlsson

Research output: Contribution to journalArticlepeer-review

Abstract

We investigate via stochastic simulation the overshoots observed in the fluorescence intensity of pyrene-labeled actin during rapid polymerization. We show that previous assumptions about pyrene intensity that ignore the intensity differences between subunits in different ATP hydrolysis states are not consistent with experimental data. This strong sensitivity of intensity to hydrolysis state implies that a measured pyrene intensity curve does not immediately reveal the true polymerization kinetics. We show that there is an optimal range of hydrolysis and phosphate release rate combinations simultaneously consistent with measured polymerization data from previously published severing and Arp2/3 complex-induced branching experiments. Within this range, we find that the pyrene intensity curves are described very accurately by the following average relative intensity coefficients: 0.37 for F-ATP actin; 0.55 for F-ADP + Pi actin; and 0.75 for F-ADP actin. Finally, we present an analytic formula, which properly accounts for the sensitivity of the pyrene assay to hydrolysis state, for estimation of the concentration of free barbed ends from pyrene intensity curves.

Original languageEnglish (US)
Pages (from-to)1050-1062
Number of pages13
JournalBiophysical journal
Volume95
Issue number3
DOIs
StatePublished - Aug 1 2008
Externally publishedYes

ASJC Scopus subject areas

  • Biophysics

Fingerprint Dive into the research topics of 'Actin polymerization overshoots and ATP hydrolysis as assayed by pyrene fluorescence'. Together they form a unique fingerprint.

Cite this