title: Analysis of distinct molecular assembly complexes of keratin K8 and K18 by hydrogen-deuterium exchange.
abstract: Keratins are intermediate filament (IF) proteins that form complex filament systems in epithelial cells, thus serving as scaffolding elements and mechanical stress absorbers. The building blocks of keratin IFs are parallel coiled-coil dimers of two distinct sequence-related proteins distinguished as type I and type II keratins. To gain more insight into their structural dynamics, we resorted to hydrogen-deuterium exchange mass spectrometry of keratins K8 and K18, which are characteristic for simple epithelial cells. Using this powerful technique not employed with IFs before, we mapped patterns of protected versus unprotected regions in keratin complexes at various assembly levels. In particular, we localized protein segments exhibiting different hydrogen exchange patterns in tetramers versus filaments. We observed a general pattern of precisely positioned regions of stability intertwining with flexible regions, mostly represented by the non-α-helical segments. Notably, some regions within the coiled-coil domains are significantly more dynamic than others, while the IF-consensus motifs at the end domains of the central α-helical "rod" segment, which mediate the "head-to-tail" dimer-dimer interaction in the filament elongation process, become distinctly more protected upon formation of filaments. Moreover, to gain more insight into the dynamics of the individual keratins, we investigated the properties of homomeric preparations of K8 and K18. The physiological importance of keratins without a partner is encountered in both pathological and experimental situations when one of the two species is present in robust excess or completely absent, such as in gene-targeted mice.
authors.affiliation: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5A, 02-106 Warsaw, Poland.
authors.forename: Aiswarya
authors.initials: A
authors.lastname: Premchandar
authors.affiliation: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5A, 02-106 Warsaw, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Kupniewska
authors.affiliation: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5A, 02-106 Warsaw, Poland.
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Tarnowski
authors.affiliation: Biophysics of Macromolecules, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany.
authors.forename: Norbert
authors.initials: N
authors.lastname: Mücke
authors.affiliation: Molecular Genetics, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany.
authors.forename: Monika
authors.initials: M
authors.lastname: Mauermann
authors.affiliation: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5A, 02-106 Warsaw, Poland.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Kaus-Drobek
authors.affiliation: INSERM U1151, Université Paris Descartes, Paris, France.
authors.forename: Aleksander
authors.initials: A
authors.lastname: Edelman
authors.affiliation: Molecular Genetics, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany.
authors.forename: Harald
authors.initials: H
authors.lastname: Herrmann
authors.affiliation: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5A, 02-106 Warsaw, Poland; Warsaw University, Biology Department, Institute of Genetics and Biotechnology, Miecznikowa 3, Warsaw, Poland. Electronic address: michald@ibb.waw.pl.
authors.forename: Michał
authors.initials: M
authors.lastname: Dadlez
mesh_terms: D000595:Amino Acid Sequence; D003599:Cytoskeleton / Q000378:metabolism; D041961:Deuterium Exchange Measurement; D004847:Epithelial Cells / Q000378:metabolism*; D007382:Intermediate Filaments / Q000378:metabolism*; D007633:Keratins / Q000378:metabolism*; D017434:Protein Structure, Tertiary
chemical_list: D007633:Keratins
keywords: Cytoskeleton; Electron microscopy (EM); Hydrogen–deuterium exchange; Intermediate filament; Keratin