Effect of polymerization on the subdomain 3/4 loop of yeast actin.
The Holmes F-actin model predicts a polymerization-dependent conformation change of a subdomain 3/4 loop with a hydrophobic tip (residues 266-269), allowing interaction with a hydrophobic surface on the opposing strand of the filament producing filament stabilization. We introduced cysteines in place of Val(266), Leu(267), and Leu(269) in yeast actin to allow attachment of pyrene maleimide. Pyrene at each of these positions produced differing fluorescence spectra in G-actin. Polymerization decreased the fluorescence for the 266 and 267 probes and increased that for the 269 probe. The direction of the fluorescence change was mirrored with a smaller and less hydrophobic probe, acrylodan, when attached to 266 or 269. Following polymerization, increased acrylamide quenching was observed for pyrene at 266 or 267 but not 269. The 267 probe was the least accessible of the three in G- and F-actin. F-actin quenching was biphasic for the 265, 266, and 269 but not 267 probes, suggesting that in F-actin, the pyrene samples multiple environments. Finally, in F-actin the probe at 266 interacts with one at Cys(374) on a monomer in the opposing strand, producing a pyrene excimer band. These results indicate a polymerization-dependent movement of the subdomain 3/4 loop partially consistent with Holmes' model.
Musib
Runa
R
Department of Biochemistry, University of Iowa, Iowa City, Iowa 52242, USA.
Wang
Gufeng
G
Geng
Lei
L
Rubenstein
Peter A
PA
D000178:Acrylamides / Q000737:chemistry; D000199:Actins / Q000737:chemistry* / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D007700:Kinetics; D007930:Leucine / Q000737:chemistry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009218:Myosins / Q000378:metabolism; D009841:Oligonucleotides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D014633:Valine / Q000737:chemistry
D000178:Acrylamides; D000199:Actins; D009841:Oligonucleotides; D009218:Myosins; D007930:Leucine; D014633:Valine; D003545:Cysteine; D002118:Calcium