Mapping the G-actin binding surface of cofilin using synchrotron protein footprinting

Jing Qu Guan, Sergeui Vorobiev, Steven C. Almo, Mark R. Chance

Research output: Contribution to journalArticle

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Abstract

Cofilin is an actin regulatory protein that binds to both monomeric and filamentous actin, and has filament severing activity. Although crystal structures for the monomeric forms of both G-actin and cofilin have been described, the structure of the binary cofilin-G-actin complex is not available. Synchrotron protein footprinting is used to identify specific side chain residues on the cofilin surface that are buried in the formation of the cofilin-G-actin binary complex. Exposure to synchrotron X-rays results in stable oxidative modifications of aromatic, aliphatic, and sulfur-containing side chains, with the rate of modification for a particular residue being dependent on its intrinsic reactivity and solvent accessibility. The rates of modification were monitored for a number of peptides generated by digestion of oxidized cofilin, both in isolation and in its binary complex with G-actin. After binding to G-actin takes place, a significant decrease in modification rates, indicating protection of side chain groups, is seen for cofilin peptides corresponding to residues 4-20, 10-17, 83-96, 91-105, and 106-117. A number of other peptides show no change in reactivity, and are presumed to represent regions distal to the binding site. Tandem mass spectrometry demonstrates that residues Leu 13, Pro 94, Met 99, and Leu 108 and 112 directly participate in the binding interface. These results are generally consistent with, and complementary to, the results of previous site-directed mutagenesis studies and extend our understanding of the G-actin binding surface of cofilin.

Original languageEnglish (US)
Pages (from-to)5765-5775
Number of pages11
JournalBiochemistry
Volume41
Issue number18
DOIs
StatePublished - May 7 2002

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Protein Footprinting
Actin Depolymerizing Factors
Synchrotrons
Actins
Proteins
Peptides
Mutagenesis
Tandem Mass Spectrometry
Site-Directed Mutagenesis
Actin Cytoskeleton
Sulfur
Mass spectrometry
Digestion
Crystal structure
Binding Sites
X-Rays

ASJC Scopus subject areas

  • Biochemistry

Cite this

Mapping the G-actin binding surface of cofilin using synchrotron protein footprinting. / Guan, Jing Qu; Vorobiev, Sergeui; Almo, Steven C.; Chance, Mark R.

In: Biochemistry, Vol. 41, No. 18, 07.05.2002, p. 5765-5775.

Research output: Contribution to journalArticle

Guan, Jing Qu ; Vorobiev, Sergeui ; Almo, Steven C. ; Chance, Mark R. / Mapping the G-actin binding surface of cofilin using synchrotron protein footprinting. In: Biochemistry. 2002 ; Vol. 41, No. 18. pp. 5765-5775.
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