Effect of polymerization on the subdomain 3/4 loop of yeast actin

Runa Musib; Gufeng Wang; Lei Geng; Peter A Rubenstein

doi:10.1074/jbc.M203096200

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Effect of polymerization on the subdomain 3/4 loop of yeast actin

Journal article

Open access

Peer reviewed

Effect of polymerization on the subdomain 3/4 loop of yeast actin

Runa Musib, Gufeng Wang, Lei Geng and Peter A Rubenstein

The Journal of biological chemistry, Vol.277(25), pp.22699-22709

06/21/2002

DOI: 10.1074/jbc.M203096200

PMID: 11959868

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Abstract

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.

Protein Structure, Tertiary

Oligonucleotides - chemistry

Mutagenesis, Site-Directed

Calcium - metabolism

Actins - metabolism

Models, Molecular

Spectrometry, Fluorescence

Cysteine - chemistry

Plasmids - metabolism

Acrylamides - chemistry

Leucine - chemistry

Saccharomyces cerevisiae - metabolism

Valine - chemistry

Time Factors

Actins - chemistry

Protein Binding

Myosins - metabolism

Kinetics

Details

Title: Subtitle: Effect of polymerization on the subdomain 3/4 loop of yeast actin
Creators: Runa Musib - Department of Biochemistry, University of Iowa, Iowa City, Iowa 52242, USA
Gufeng Wang
Lei Geng
Peter A Rubenstein
Resource Type: Journal article
Publication Details: The Journal of biological chemistry, Vol.277(25), pp.22699-22709
DOI: 10.1074/jbc.M203096200
PMID: 11959868
NLM abbreviation: J Biol Chem
ISSN: 0021-9258
eISSN: 1083-351X
Publisher: United States
Grant note: GM-33689 / NIGMS NIH HHS
Language: English
Date published: 06/21/2002
Academic Unit: Stead Family Department of Pediatrics; Biochemistry and Molecular Biology; Chemistry; Internal Medicine
Record Identifier: 9984025394202771

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