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NMR Analysis of Actin Binding Protein Cofilin
Author(s) -
Audette Nicholas Jack,
Yan Si,
kudryashov Dmitri,
Polenova Tatyana
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.989.2
Subject(s) - cofilin , actin , chemistry , cytoskeleton , actin cytoskeleton , nuclear magnetic resonance spectroscopy , biophysics , actin binding protein , biochemistry , microbiology and biotechnology , biology , cell , stereochemistry
The cytoskeleton and its associated proteins are vital to numerous physiological functions including cell migration, mitosis, polarization, and organelle transport and have been connected to numerous physiological disorders. Despite their importance, the function and structure of these proteins are not fully understood because they are not completely soluble and F‐actin lacks long range order and resists crystallization. Proteins of ADF/cofilin family are vital regulators of the actin cytoskeleton in eukaryotes. Binding of cofilin results in dramatic reorganization of F‐actin structure and potentiates filament severing and its accelerated depolymerization from the pointed end. The long term goal of this project is to generate high resolution information on the interaction between cofilin and F‐actin through solid state nuclear magnetic resonance (NMR) spectroscopy. In this experiment, isotopically labeled yeast cofilin was expressed and purified in S. cerevisieae for solution NMR of the free protein. This provides detailed resonance assignments, structural information, and backbone dynamics of the protein. Approximately 60 g of 15 N labeled cofilin has been produced at high purity and initial HSQC experiments have been run to assist resonance assignments of the protein backbone. Additionally 15 N‐ 1 H dipolar lineshapes have been measured in a residue‐specific way in U‐ 15 N, 13 C cofilin sample to examine the backbone mobility of the protein on micro‐ to nanosecond timescales.