Solid-state NMR studies of the dynamics and structure of mouse keratin intermediate filaments.
The molecular dynamics and structural organization of mouse epidermal keratin intermediate filaments (IF) have been studied via solid-state nuclear magnetic resonance (NMR) experiments performed on IF labeled both in vivo and in vitro with isotopically enriched amino acids. As a probe of the organization of the peripheral glycine-rich end domains of the IF, carbon-13 NMR experiments have been performed on subfilamentous forms (prekeratin) and on IF reassembled in vitro that had been labeled with either [1-13C]glycine or [2-13C]glycine, as more than 90% of the glycines of the keratins are located in the end domains. Although cross-labeling to seryl residues was observed, the proportion of serine located in the end domains is nearly the same as that for glycine. Measurements of carbon relaxation times, nuclear Overhauser enhancements, and signal intensities show that the motions of the peptide backbone in the end domains are effectively isotropic, with average correlation times distributed over the range of 0.2-20 ns. These results indicate that the end domains of IF are remarkably flexible and have little or no structural order. To probe the structural organization of the coiled-coil rod domains of the IF, separate samples of native keratin IF, raised in primary tissue culture, were labeled with L-[1-13C]leucine, L-[2H10]leucine, or L-[2,3,3-2H3]leucine, as greater than 90% of the leucyl residues of the keratin IF types studied are located in the coiled coils which form the central core of IF.(ABSTRACT TRUNCATED AT 250 WORDS)
Mack
J W
JW
Bone Research Branch, National Institute of Dental Research, Bethesda, Maryland 20892.
Torchia
D A
DA
Steinert
P M
PM
D000818:Animals; D003599:Cytoskeleton / Q000648:ultrastructure*; D004817:Epidermis / Q000032:analysis; D005998:Glycine; D007382:Intermediate Filaments / Q000648:ultrastructure*; D007633:Keratins; D007930:Leucine; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008807:Mice, Inbred BALB C; D011498:Protein Precursors
D011498:Protein Precursors; C004818:prekeratin; D007633:Keratins; D007930:Leucine; D005998:Glycine