title: Conformational dynamics of loop 262-274 in G- and F-actin.
abstract: According to the original Holmes model of F-actin structure, the hydrophobic loop 262-274 stabilizes the actin filament by inserting into a pocket formed at the interface between two protomers on the opposing strand. Using a yeast actin triple mutant, L180C/L269C/C374A [(LC)(2)CA], we showed previously that locking the hydrophobic loop to the G-actin surface by a disulfide bridge prevents filament formation. We report here that the hydrophobic loop is mobile in F- as well as in G-actin, fluctuating between the extended and parked conformations. Copper-catalyzed, brief air oxidation of (LC)(2)CA F-actin on electron microscopy grids resulted in the severing of thin filaments and their conversion to amorphous aggregates. Disulfide, bis(methanethiosulfonate) (MTS), and dibromobimane (DBB) cross-linking reactions proceeded in solution at a faster rate with G- than with F-actin. Cross-linking of C180 to C269 by DBB (4.4 A) in either G- or F-actin resulted in shorter and less stable filaments. The cross-linking with a longer MTS-6 reagent (9.6 A) did not impair actin polymerization or filament structure. Myosin subfragment 1 (S1) and tropomyosin inhibited the disulfide cross-linking of phalloidin-stabilized F-actin. Electron paramagnetic resonance measurements with nitroxide spin-labeled actin revealed strong spin-spin coupling and a similar mean interspin distance ( approximately 10 A) in G- and in F-actin, with a broader distance distribution in G-actin. These results show loop 262-274 fluctuations in G- and F-actin and correlate loop dynamics with actin filament formation and stability.
authors.affiliation: Department of Chemistry and Biochemistry and Molecular Biology Institute, University of California, Los Angeles, Los Angeles, California 90095, USA.
authors.forename: Alexander
authors.initials: A
authors.lastname: Shvetsov
authors.forename: John D
authors.initials: JD
authors.lastname: Stamm
authors.forename: Martin
authors.initials: M
authors.lastname: Phillips
authors.forename: Dora
authors.initials: D
authors.lastname: Warshaviak
authors.forename: Christian
authors.initials: C
authors.lastname: Altenbach
authors.forename: Peter A
authors.initials: PA
authors.lastname: Rubenstein
authors.forename: Kálmán
authors.initials: K
authors.lastname: Hideg
authors.forename: Wayne L
authors.initials: WL
authors.lastname: Hubbell
authors.forename: Emil
authors.initials: E
authors.lastname: Reisler
mesh_terms: D008841:Actin Cytoskeleton / Q000187:drug effects / Q000648:ultrastructure; D000199:Actins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D001643:Bridged Bicyclo Compounds / Q000378:metabolism / Q000494:pharmacology; D003432:Cross-Linking Reagents; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D057927:Hydrophobic and Hydrophilic Interactions; D008698:Mesylates / Q000378:metabolism / Q000494:pharmacology; D015879:Myosin Subfragments / Q000378:metabolism / Q000494:pharmacology; D010590:Phalloidine / Q000378:metabolism; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000166:cytology / Q000235:genetics; D014335:Tropomyosin / Q000378:metabolism / Q000494:pharmacology
chemical_list: D000199:Actins; D001643:Bridged Bicyclo Compounds; D003432:Cross-Linking Reagents; D004220:Disulfides; D008698:Mesylates; D015879:Myosin Subfragments; D014335:Tropomyosin; D010590:Phalloidine; C074502:methanethiosulfonate; C037094:dibromobimane; D003545:Cysteine