The effect of the S14A mutation on the conformation and thermostability of Saccharomyces cerevisiae G-actin and its interaction with adenine nucleotides

Xin Chen; Junmin Peng; Mehrdad Pedram; Charles A Swenson; Peter A Rubenstein

doi:10.1074/jbc.270.19.11415

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The effect of the S14A mutation on the conformation and thermostability of Saccharomyces cerevisiae G-actin and its interaction with adenine nucleotides

Journal article

Open access

Peer reviewed

The effect of the S14A mutation on the conformation and thermostability of Saccharomyces cerevisiae G-actin and its interaction with adenine nucleotides

Xin Chen, Junmin Peng, Mehrdad Pedram, Charles A Swenson and Peter A Rubenstein

The Journal of biological chemistry, Vol.270(19), pp.11415-11423

05/12/1995

DOI: 10.1074/jbc.270.19.11415

PMID: 7744778

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Abstract

The actin Ser14 hydroxyl is one of a number of ligands that binds to the gamma-phosphate of ATP thereby stabilizing the actin.ATP complex. In yeast actin, conversion of Ser14 to Ala (S14A), causes a temperature-sensitive phenotype in vivo and temperature-sensitive polymerization defects in vitro (Chen, X., and Rubenstein, P. A. (1995) J. Biol. Chem. 270, 11406-11414). Here, using a new luciferase-based procedure, we show that the mutation results in a 40-60-fold decrease in actin's affinity for ATP. The mutation causes a decrease in the intrinsic ATPase activity of both Ca- and Mg-G-actin at 30 degrees C and alters the protease susceptibility of sites on subdomain 2. Ca-S14A-actin but not Mg-S14A-actin binds etheno-ATP at 37 degrees C. Intrinsic tryptophan fluorescence measurements show that at 37 degrees C, Mg-S14A-actin but not the calcium form unfolds. CD measurements show the mutation causes a decrease in the apparent denaturation temperature for Ca-actin from 57 to 45 degrees C and for the magnesium form a decrease from 52 to 40 degrees C. Based on a re-examination of actin's crystal structure coordinates, we propose that the Ser14 hydroxyl forms a polar bridge between the ATP gamma-phosphate and the amide nitrogen of Gly74, thus conferring additional stability on the actin small domain.

Thermodynamics

Amino Acid Sequence

Protein Denaturation - drug effects

Rabbits

Alanine

Models, Structural

Actins - biosynthesis

Calcium - pharmacology

Saccharomyces cerevisiae - genetics

Drug Stability

Serine

Actins - metabolism

Muscle, Skeletal - metabolism

Hot Temperature

Magnesium - pharmacology

Saccharomyces cerevisiae - metabolism

Point Mutation

Animals

Adenosine Triphosphate - metabolism

Actins - chemistry

Protein Conformation

Kinetics

Binding Sites

Details

Title: Subtitle: The effect of the S14A mutation on the conformation and thermostability of Saccharomyces cerevisiae G-actin and its interaction with adenine nucleotides
Creators: Xin Chen - University of Iowa
Junmin Peng
Mehrdad Pedram
Charles A Swenson
Peter A Rubenstein
Resource Type: Journal article
Publication Details: The Journal of biological chemistry, Vol.270(19), pp.11415-11423
DOI: 10.1074/jbc.270.19.11415
PMID: 7744778
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: 05/12/1995
Academic Unit: Iowa Technology Institute; Stead Family Department of Pediatrics; Biochemistry and Molecular Biology; Internal Medicine
Record Identifier: 9984025400102771

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