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Ultra short yeast tropomyosins show novel myosin regulation

  • Robin Maytum
    ,
  • V. Hatch
    ,
  • M. Konrad
    ,
  • W. Lehman
    ,
  • Michael A. Geeves
  • University of Kent
Research Output: Contribution to journal Article Peer-review

Open access

Abstract

Tropomyosin (Tm) is an α-helical coiled-coil actin-binding protein present in all eukaryotes from yeast to man. Its functional role has been best described in muscle regulation; however its much wider role in cytoskeletal actin regulation is still to be clarified. Isoforms vary in size from 284 or 248 amino acids in vertebrates, to 199 and 161 amino acids in yeast, spanning from 7 to 4 actin binding sites respectively. In Saccharomyces cerevisiae, the larger yTm1 protein is produced by an internal 38-amino acid duplication, corresponding to a single actin-binding site. We have produced an ultra-short Tm with only 125 amino acids by removing both of the 38 amino acid repeats from yTm1, with the addition of an Ala-Ser extension used to mimic the essential N-terminal acetylation. This short Tm, and an M1T mutant of it, bind to actin with a similar affinity to most Tms previously studied (K50% ∼ 0.5 μm). However, an equilibrium fluorescence binding assay shows a much greater inhibition of myosin binding to actin than any previously studied Tm. Actin cosedimentation assays show this is caused by direct competition for binding to actin. The M1T mutant shows a reduced inhibition, probably due to weaker end-to-end interactions making it easier for myosin to displace Tm. All previously characterized Tms, although able to sterically block the myosin-binding site, are able to bind to actin along with myosin. By showing that Tm can compete directly with myosin for the same binding site these new Tms provide direct evidence for the steric blocking model. *

Publication Information

Output type

Research Output: Contribution to journal Article Peer-review

Original language

English

Pages from-to (Number of pages)

Pages 1902-1910

Journal (Volume, Issue Number)

Journal of Biological Chemistry (Volume 283, Issue 4)

Publication milestones

  • Published - 25/01/2008

Publication status

Published - 25/01/2008

ISSN

0021-9258

External Publication IDs

  • handle.net: 10547/228331
  • Scopus: 38349166409

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