Production of recombinant human tektin 1, 2, and 4 and in vitro assembly of human tektin 1

M. S. Budamagunta, F. Guo, N. Sun, B. Shibata, Paul G FitzGerald, John C Voss, J. F. Hess

Research output: Contribution to journalArticle

Abstract

Proteins predicted to be composed of large stretches of coiled-coil structure have often proven difficult to crystallize for structural determination. We have successfully applied EPR spectroscopic techniques to the study of the structure and assembly of full-length human vimentin assembled into native 11 nm filaments, in physiologic solution, circumventing the limitations of crystallizing shorter peptide sequences. Tektins are a small family of highly alpha helical filamentous proteins found in the doublet microtubules of cilia and related structures. Tektins exhibit several similarities to intermediate filaments (IFs): moderate molecular weight, highly alpha helical, hypothesized to be coiled-coil, and homo- and heteromeric assembly into long smooth filaments. In this report, we show the application of IF research methodologies to the study of tektin structure and assembly. To begin in vitro studies, expression constructs for human tektins 1, 2, and 4 were synthesized. Recombinant tektins were produced in E. coli and purified by chromatography. Preparations of tektin 1 successfully formed filaments. The recombinant human tektin 1 was used to produce antibodies which recognized an antigen in mouse testes, most likely present in sperm flagella. Finally, we report the creation of seven mutants to analyze predictions of coiled-coil structure in the rod 1A domain of tektin 1. Although this region is predicted to be coiled-coil, our EPR analysis does not reflect the parallel, in register, coiled-coil structure as demonstrated in vimentin and kinesin. These results document that tektin can be successfully expressed and assembled in vitro, and that SDSL EPR techniques can be used for structural analysis.

Original languageEnglish (US)
Pages (from-to)3-11
Number of pages9
JournalCytoskeleton
Volume75
Issue number1
DOIs
StatePublished - Jan 1 2018

Fingerprint

Intermediate Filaments
Vimentin
Sperm Tail
In Vitro Techniques
tektins
Kinesin
Cilia
Microtubules
Testis
Chromatography
Proteins
Research Design
Molecular Weight
Escherichia coli
Antigens
Peptides
Antibodies

Keywords

  • coiled-coil structure
  • EPR spectroscopy
  • human tektin
  • in vitro filament assembly

ASJC Scopus subject areas

  • Structural Biology
  • Cell Biology

Cite this

Production of recombinant human tektin 1, 2, and 4 and in vitro assembly of human tektin 1. / Budamagunta, M. S.; Guo, F.; Sun, N.; Shibata, B.; FitzGerald, Paul G; Voss, John C; Hess, J. F.

In: Cytoskeleton, Vol. 75, No. 1, 01.01.2018, p. 3-11.

Research output: Contribution to journalArticle

Budamagunta, M. S. ; Guo, F. ; Sun, N. ; Shibata, B. ; FitzGerald, Paul G ; Voss, John C ; Hess, J. F. / Production of recombinant human tektin 1, 2, and 4 and in vitro assembly of human tektin 1. In: Cytoskeleton. 2018 ; Vol. 75, No. 1. pp. 3-11.
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