HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Feng, L. Articles by Rubenstein, P. A. Search for Related Content PubMed PubMed Citation Articles by Feng, L. Articles by Rubenstein, P. A. Social Bookmarking                      What's this?

Volume 272, Number 27, Issue of July 4, 1997 pp. 16829-16837
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Fluorescence Probing of Yeast Actin Subdomain 3/4 Hydrophobic Loop 262-274
ACTIN-ACTIN AND ACTIN-MYOSIN INTERACTIONS IN ACTIN FILAMENTS

(Received for publication, February 28, 1997, and in revised form, April 24, 1997)

Li Feng , Eldar Kim ^§ , Wei-Lih Lee , Carl J. Miller ^§ , Bing Kuang , Emil Reisler ^§ and Peter A. Rubenstein

From the  Department of Biochemistry, University of Iowa College of Medicine, Iowa City, Iowa 52242 and the ^§ Department of Chemistry and the Molecular Biology Institute, University of California, Los Angeles, California 90095

Residues 262-274 form a loop between subdomains 3 and 4 of actin. This loop may play an important role in actin filament formation and stabilization. To assess directly the behavior of this loop, we mutated Ser²⁶⁵ of yeast actin to cysteine (S265C) and created another mutant (S265C/C374A) by changing Cys³⁷⁴ of S265C actin to alanine. These changes allowed us to attach a pyrene maleimide stoichiometrically to either Cys³⁷⁴ or Cys²⁶⁵. These mutations had no detectable effects on the protease susceptibility, intrinsic ATPase activity, and thermal stability of labeled or unlabeled G-actin. The presence of the loop cysteine, either labeled or unlabeled, did not affect the actin-activated S1 ATPase activity or the in vitro motility of the actin. Both mutant actins, either labeled or unlabeled, nucleated filament formation considerably faster than wild-type (WT) actin, although the critical concentration was not affected. Whereas the fluorescence of the C-terminal (WT) probe increased during polymerization, that of the loop (S265C/C374A) probe decreased, and the fluorescence of the doubly labeled actin (S265C) was ~50% less than the sum of the fluorescence of the individual fluorophores. Quenching was also observed in copolymers of labeled WT and S265C/C374A actins. An excimer peak was present in the emission spectrum of labeled S265C F-actin and in the labeled S265C/C374A-WT actin copolymers. These results show that in the filaments, the C-terminal pyrene of a substantial fraction of monomers directly interacts with the loop pyrene of neighboring monomers, bringing the two cysteine sulfurs to within 18 Å of one another. Finally, when bound to labeled S265C/C374A F-actin, myosin S1, but not tropomyosin, caused an increase in fluorescence of the loop probe. Both proteins had no effect on excimer fluorescence. These results help establish the orientation of monomers in F-actin and show that the binding of S1 to actin subdomains 1 and 2 affects the environment of the loop between subdomains 3 and 4.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				K. E. Bryan and P. A. Rubenstein Allele-specific Effects of Human Deafness {gamma}-Actin Mutations (DFNA20/26) on the Actin/Cofilin Interaction J. Biol. Chem., July 3, 2009; 284(27): 18260 - 18269. [Abstract] [Full Text] [PDF]

				K.-K. Wen and P. A. Rubenstein Differential Regulation of Actin Polymerization and Structure by Yeast Formin Isoforms J. Biol. Chem., June 19, 2009; 284(25): 16776 - 16783. [Abstract] [Full Text] [PDF]

				M. McKane, K.-K. Wen, I. R. Boldogh, S. Ramcharan, L. A. Pon, and P. A. Rubenstein A Mammalian Actin Substitution in Yeast Actin (H372R) Causes a Suppressible Mitochondria/Vacuole Phenotype J. Biol. Chem., October 28, 2005; 280(43): 36494 - 36501. [Abstract] [Full Text] [PDF]

				K.-K. Wen and P. A. Rubenstein Acceleration of Yeast Actin Polymerization by Yeast Arp2/3 Complex Does Not Require an Arp2/3-activating Protein J. Biol. Chem., June 24, 2005; 280(25): 24168 - 24174. [Abstract] [Full Text] [PDF]

				K. E. Bryan and P. A. Rubenstein An Intermediate Form of ADP-F-actin J. Biol. Chem., January 14, 2005; 280(2): 1696 - 1703. [Abstract] [Full Text] [PDF]

				R. Musib, G. Wang, L. Geng, and P. A. Rubenstein Effect of Polymerization on the Subdomain 3/4 Loop of Yeast Actin J. Biol. Chem., June 14, 2002; 277(25): 22699 - 22709. [Abstract] [Full Text] [PDF]

				A. M. Gordon, E. Homsher, and M. Regnier Regulation of Contraction in Striated Muscle Physiol Rev, April 1, 2000; 80(2): 853 - 924. [Abstract] [Full Text] [PDF]

				C. Landis, N. Back, E. Homsher, and L. S. Tobacman Effects of Tropomyosin Internal Deletions on Thin Filament Function J. Biol. Chem., October 29, 1999; 274(44): 31279 - 31285. [Abstract] [Full Text] [PDF]

				S. Sastry and N. Linderoth Molecular Mechanisms of Peptide Loading by the Tumor Rejection Antigen/Heat Shock Chaperone gp96 (GRP94) J. Biol. Chem., April 23, 1999; 274(17): 12023 - 12035. [Abstract] [Full Text] [PDF]

				J. Buzan, J. Du, T. Karpova, and C. Frieden Histidine-tagged wild-type yeast actin: Its properties and use in an approach for obtaining yeast actin mutants PNAS, March 16, 1999; 96(6): 2823 - 2827. [Abstract] [Full Text] [PDF]

				F. Ressad, D. Didry, G.-X. Xia, Y. Hong, N.-H. Chua, D. Pantaloni, and M.-F. Carlier Kinetic Analysis of the Interaction of Actin-depolymerizing Factor (ADF)/Cofilin with G- and F-Actins. COMPARISON OF PLANT AND HUMAN ADFs AND EFFECT OF PHOSPHORYLATION J. Biol. Chem., August 14, 1998; 273(33): 20894 - 20902. [Abstract] [Full Text] [PDF]

				V. L. Korman, V. Hatch, K. Y. Dixon, R. Craig, W. Lehman, and L. S. Tobacman An Actin Subdomain 2 Mutation That Impairs Thin Filament Regulation by Troponin and Tropomyosin J. Biol. Chem., July 14, 2000; 275(29): 22470 - 22478. [Abstract] [Full Text] [PDF]

				L. S. Tobacman and C. A. Butters A New Model of Cooperative Myosin-Thin Filament Binding J. Biol. Chem., September 1, 2000; 275(36): 27587 - 27593. [Abstract] [Full Text] [PDF]

				J. Strand, M. Nili, E. Homsher, and L. S. Tobacman Modulation of Myosin Function by Isoform-specific Properties of Saccharomyces cerevisiae and Muscle Tropomyosins J. Biol. Chem., September 7, 2001; 276(37): 34832 - 34839. [Abstract] [Full Text] [PDF]