Hydrophobic loop dynamics and actin filament stability.
It has been postulated that the hydrophobic loop of actin (residues 262-274) swings out and inserts into the opposite strand in the filament, stabilizing the filament structure. Here, we analyzed the hydrophobic loop dynamics utilizing four mutants that have cysteine residues introduced at a single location along the yeast actin loop. Lateral, copper-catalyzed disulfide cross-linking of the mutant cysteine residues to the native C374 in the neighboring strand within the filament was fastest for S265C, followed by V266C, L267C, and then L269C. Site-directed spin labeling (SDSL) studies revealed that C265 lies closest to C374 within the filament, followed by C266, C267, and then C269. These results are not predicted by the Holmes extended loop model of F-actin. Furthermore, we find that disulfide cross-linking destroys L267C and L269C filaments; only small filaments are observed via electron microscopy. Conversely, phalloidin protects the L267C and L269C filaments and inhibits their disulfide cross-linking. Combined, our data indicate that, in solution, the loop resides predominantly in a "parked" position within the filament but is able to dynamically populate other conformational states which stabilize or destabilize the filament. Such states may be exploited within a cell by filament-stabilizing and -destabilizing factors.
Scoville
Damon
D
Department of Chemistry and Biochemistry and Molecular Biology Institute, University of California, Los Angeles, California 90095, USA.
Stamm
John D
JD
Toledo-Warshaviak
Dora
D
Altenbach
Christian
C
Phillips
Martin
M
Shvetsov
Alexander
A
Rubenstein
Peter A
PA
Hubbell
Wayne L
WL
Reisler
Emil
E
D008841:Actin Cytoskeleton / Q000737:chemistry / Q000235:genetics / Q000648:ultrastructure*; D019943:Amino Acid Substitution; D004578:Electron Spin Resonance Spectroscopy; D057927:Hydrophobic and Hydrophilic Interactions; D008027:Light; D008854:Microscopy, Electron; D010590:Phalloidine / Q000494:pharmacology; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000737:chemistry; D012542:Scattering, Radiation; D013113:Spin Labels
D013113:Spin Labels; D010590:Phalloidine