Structure and dynamics of the actin filament.
The structures of filamentous Mg-ATP-actin (F actin) in the presence and absence of KCl have been mapped with hydroxyl radicals (*OH) generated by synchrotron X-ray radiolysis. Proteolysis and mass spectrometry (MS) analysis revealed 52 reactive side-chain sites from 27 distinct peptides within actin. The reactivities of these probe sites with *OH in the F-actin states are compared with those of Mg-ATP-G-actin (monomers) analyzed previously [Guan, J.-Q. et al. (2003) Biochemistry 42, 11992-12000]. Filament-dependent protection within subdomains 2, 3, and 4 and at the C terminus is consistent with longitudinal contacts of monomers within the filament helical structure as predicted by the Holmes model. In the absence of KCl, the extent of filament-dependent protection rarely reached 3-fold, consistent with a highly dynamic filament characterized by relatively weak interactions between actin protomers. However, in the presence of KCl, the extents of protection are significantly increased, consistent with a well-ordered, more tightly packed filament structure. Filament-dependent enhancements of reactivity not predicted by the Holmes model are seen for a peptide that overlaps the "hydrophobic plug" (H-plug) region and for a peptide that forms contacts with the polyphosphate moiety of the bound nucleotide. Overall, these data are both consistent with and complementary to a recent deuterium-exchange MS study of filamentous actin [Chik, J. K., and Schriemer, D.C. (2003) J. Mol. Biol. 334, 373-385], which also did not detect any burial of the H plug upon formation of filaments.
Guan
Jing-Qu
JQ
Center for Synchrotron Biosciences, Albert Einstein College of Medicine of Yeshiva University, 1300 Morris Park Avenue, Bronx, New York 10461, USA.
Takamoto
Keiji
K
Almo
Steven C
SC
Reisler
Emil
E
Chance
Mark R
MR
D008841:Actin Cytoskeleton / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D000199:Actins / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D008027:Light; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry; D008958:Models, Molecular; D010084:Oxidation-Reduction; D011108:Polymers / Q000378:metabolism; D011189:Potassium Chloride / Q000737:chemistry; D023302:Protein Footprinting; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011817:Rabbits; D012542:Scattering, Radiation; D013329:Structure-Activity Relationship; D013816:Thermodynamics
D000199:Actins; D011108:Polymers; D011189:Potassium Chloride; D000255:Adenosine Triphosphate; D008274:Magnesium