Real-time observation of coiled-coil domains and subunit assembly in intermediate filaments

Research output: Contribution to journalArticle

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Abstract

We have utilized electron paramagnetic resonance spectroscopy to study secondary structure, subunit interaction, and molecular orientation of vimentin molecules within intact intermediate filaments and assembly intermediates. Spectroscopy data prove α-helical coiled-coil structures at individual amino acids 316-336 located in rod 2B. Analysis of positions 305, 309, and 312 identify this region as conforming to the helical pattern identified within 316-336 and thus demonstrates that, contrary to some previous predictions, this region is in an α-helical conformation. We show that by varying the position of the spin label, we can identify both intra- and inter-dimer interactions. With a label attached to the outside of the α-helix, we have been able to measure interactions between positions 348 of separate dimers as they align together in intact filaments, identifying the exact point of overlap. By mixing different spin-labeled proteins, we demonstrate that the interaction at position 348 is the result of an anti-parallel arrangement of dimers. This approach provides high resolution structural information (<2 nm resolution), can be used to identify molecular arrangements between subunits in an intact intermediate filament, and should be applicable to other noncrystallizable filamentous systems as well as to the study of protein fibrils.

Original languageEnglish (US)
Pages (from-to)35516-35522
Number of pages7
JournalJournal of Biological Chemistry
Volume277
Issue number38
DOIs
StatePublished - Sep 20 2002

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Intermediate Filaments
Dimers
Spectrum Analysis
Observation
Spin Labels
Electron Spin Resonance Spectroscopy
Vimentin
Proteins
Spectroscopy
Amino Acids
Molecular orientation
Paramagnetic resonance
Conformations
Labels
Molecules

ASJC Scopus subject areas

  • Biochemistry

Cite this

Real-time observation of coiled-coil domains and subunit assembly in intermediate filaments. / Hess, John F.; Voss, John C; FitzGerald, Paul G.

In: Journal of Biological Chemistry, Vol. 277, No. 38, 20.09.2002, p. 35516-35522.

Research output: Contribution to journalArticle

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