Effects of binding factors on structural elements in F-actin.
Understanding the dynamics of the actin filament is essential to a detailed description of their interactions and role in the cell. Previous studies have linked the dynamic properties of actin filaments (F-actin) to three structural elements contributing to a hydrophobic pocket, namely, the hydrophobic loop, the DNase I binding loop, and the C-terminus. Here, we examine how these structural elements are influenced by factors that stabilize or destabilize F-actin, using site-directed spin-labeled (SDSL) electron paramagnetic resonance (EPR), fluorescence, and cross-linking techniques. Specifically, we employ cofilin, an actin destabilizing protein that binds and severs filaments, and phalloidin, a fungal toxin that binds and stabilizes F-actin. We find that cofilin shifts both the DNase I binding loop and the hydrophobic loop away from the C-terminus in F-actin, as demonstrated by weakened spin-spin interactions, and alters the environment of spin probes on residues of these two loops. In contrast, although phalloidin strongly stabilizes F-actin, it causes little or no local change in the environment of the loop residues. This indicates that the stabilizing effect of phalloidin is achieved mainly through constraining structural fluctuations in F-actin and suggests that factors and interactions that control these fluctuations have an important role in the cytoskeleton dynamics.
Scoville
Damon
D
Department of Chemistry and Biochemistry and Molecular Biology Institute, University of California, Los Angeles, California 90095, USA.
Stamm
John D
JD
Altenbach
Christian
C
Shvetsov
Alexander
A
Kokabi
Kaveh
K
Rubenstein
Peter A
PA
Hubbell
Wayne L
WL
Reisler
Emil
E
D015081:2-Naphthylamine / Q000031:analogs & derivatives / Q000378:metabolism; D051339:Actin Depolymerizing Factors / Q000378:metabolism*; D000199:Actins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000648:ultrastructure; D000595:Amino Acid Sequence; D002384:Catalysis; D003432:Cross-Linking Reagents / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D005456:Fluorescent Dyes / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010590:Phalloidine / Q000378:metabolism*; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000166:cytology / Q000235:genetics; D013696:Temperature
D051339:Actin Depolymerizing Factors; D000199:Actins; D003432:Cross-Linking Reagents; D004220:Disulfides; D005456:Fluorescent Dyes; D010590:Phalloidine; C038518:acrylodan; D015081:2-Naphthylamine