Cloning, expression, and protein interaction of human nebulin fragments composed of varying numbers of sequence modules

J. P. Jin, K. Wang

Research output: Contribution to journalArticle

78 Citations (Scopus)

Abstract

Nebulin, a family of giant myofibrillar proteins of 600-900 kDa, contains a large number of highly conserved sequence repeats of 31-38 amino acids. To investigate the significance of this repeat, human skeletal muscle nebulin cDNA fragments encoding two, six, seven, eight, or fifteen repeat modules were expressed in high yield as nonfusion proteins in Escherichia coli with the pET3d plasmid vector. F-actin cosedimentation and solid phase binding assays demonstrated that all nebulin fragments, except the smallest two-module 67-mer, bound to muscle actin with high affinity under physiological ionic conditions. Solid phase binding assays also revealed that a six-module fragment, NB5, binds to myosin and C-terminal protein but fails to bind to tropomyosin, troponin, and tubulin. Furthermore, the binding of NB5 to actin was inhibited by both tropomyosin and troponin. Immunoelectron microscopic localization of NB5 indicated that this N-terminal region fragment is situated near the distal end of thin filaments in the sarcomere. These results indicate that nebulin is a giant protein with an unprecedently large number of actin-binding sites along its length and is anchored at the C terminus to the Z line in the sarcomere. Nebulin may function as a multifunctional template protein that regulates the length of thin filaments and participates in muscle activities by interacting with actin and myosin filaments in the sarcomere of skeletal muscles.

Original languageEnglish
Pages (from-to)21215-21223
Number of pages9
JournalJournal of Biological Chemistry
Volume266
Issue number31
Publication statusPublished - 1991
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint Dive into the research topics of 'Cloning, expression, and protein interaction of human nebulin fragments composed of varying numbers of sequence modules'. Together they form a unique fingerprint.

  • Cite this