Structure, stability, and aggregation of paired helical filaments from tau protein and FTDP-17 mutants probed by tryptophan scanning mutagenesis.
By using tryptophan scanning mutagenesis, we observed the kinetics and structure of the polymerization of tau into paired helical filaments (PHFs) independently of exogenous reporter dyes. The fluorescence exhibits pronounced blue shifts due to burial of the residue inside PHFs, depending on Trp position. The effect is greatest near the center of the repeat domain, showing that the packing is tightest near the beta-structure inducing hexapeptide motifs. The tryptophan response allows measurement of PHF stability made by different tau isoforms and mutants. Unexpectedly, the stability of PHFs is quite low (denaturation half-points approximately 1.0 m GdnHCl), implying that incipient aggregation should be reversible and that the observed high stability of Alzheimer PHFs is due to other factors. The stability increases with the number of repeats and with tau mutants promoting beta-structure, arguing for a gain of toxic function in frontotemporal dementias. Fluorescence resonance energy transfer (FRET) was used to analyze the distances of Tyr(310) to tryptophans in different positions. The degree of FRET in the soluble protein was position-dependent, with highest signals within the second and third repeats but low or no signals further away. In PHFs most mutants showed FRET, indicating that tight packing results from assembly of tau into PHFs.
Max-Planck-Unit for Structural Molecular Biology Notkestrasse 85, 22607 Hamburg, Germany.
Li
L
Li
Martin
M
von Bergen
Eva-Maria
EM
Mandelkow
Eckhard
E
Mandelkow
D000544:Alzheimer Disease / Q000378:metabolism* / Q000473:pathology; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000737:chemistry*; D008870:Microtubules / Q000737:chemistry; D016296:Mutagenesis; D016874:Neurofibrillary Tangles / Q000737:chemistry*; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D020449:Repetitive Sequences, Amino Acid; D014364:Tryptophan; D016875:tau Proteins / Q000737:chemistry*
C054369:MAPT protein, human; D008869:Microtubule-Associated Proteins; D016875:tau Proteins; D014364:Tryptophan