Proline-directed Pseudo-phosphorylation at AT8 and PHF1 Epitopes Induces a Compaction of the Paperclip Folding of Tau and Generates a Pathological (MC-1) Conformation
2008; Elsevier BV; Volume: 283; Issue: 46 Linguagem: Inglês
10.1074/jbc.m805300200
ISSN1083-351X
AutoresSadasivam Jeganathan�, Antje Hascher, Subashchandrabose Chinnathambi, Jacek Biernat, Eva‐Maria Mandelkow, Eckhard Mandelkow�,
Tópico(s)Cellular transport and secretion
ResumoTau, a neuronal microtubule-associated protein that aggregates in Alzheimer disease is a natively unfolded protein. In solution, Tau adopts a "paperclip" conformation, whereby the N- and C-terminal domains approach each other and the repeat domain (Jeganathan, S., von Bergen, M., Brutlach, H., Steinhoff, H. J., and Mandelkow, E. (2006) Biochemistry 45, 2283-2293). In AD, Tau is in a hyperphosphorylated state. The consequences for microtubule binding or aggregation are a matter of debate. We therefore tested whether phosphorylation alters the conformation of Tau. To avoid the ambiguities of heterogeneous phosphorylation we cloned "pseudo-phosphorylation" mutants of Tau where combinations of Ser or Thr residues were converted into Glu. These mutations were combined with FRET pairs inserted in different locations to allow distance measurements. The results show that the paperclip conformation becomes tighter or looser, depending on the pseudo-phosphorylation state. In particular, pseudo-phosphorylation at the epitope of the diagnostic antibody AT8* (S199E + S202E + T205E) moves the N-terminal domain away from the C-terminal domain. Pseudo-phosphorylation at the PHF1 epitope (S396E + S404E) moves the C-terminal domain away from the repeat domain. In both cases the paperclip conformation is opened up. By contrast, the combination of AT8* and PHF1 sites leads to compaction of the paperclip, such that the N-terminus approaches the repeat domain. The compaction becomes even stronger by combining pseudo-phosphorylated AT8*, AT100, and PHF1 epitopes. This is accompanied by a strong increase in the reaction with conformation-dependent antibody MC1, suggesting the generation of a pathological conformation characteristic for Tau in AD. Furthermore, the compact paperclip conformation enhances the aggregation to paired helical filaments but has little influence on microtubule interactions. The data provide a framework for the global folding of Tau dependent on proline-directed phosphorylation in the domains flanking the repeats and the consequences for pathological properties of Tau. Tau, a neuronal microtubule-associated protein that aggregates in Alzheimer disease is a natively unfolded protein. In solution, Tau adopts a "paperclip" conformation, whereby the N- and C-terminal domains approach each other and the repeat domain (Jeganathan, S., von Bergen, M., Brutlach, H., Steinhoff, H. J., and Mandelkow, E. (2006) Biochemistry 45, 2283-2293). In AD, Tau is in a hyperphosphorylated state. The consequences for microtubule binding or aggregation are a matter of debate. We therefore tested whether phosphorylation alters the conformation of Tau. To avoid the ambiguities of heterogeneous phosphorylation we cloned "pseudo-phosphorylation" mutants of Tau where combinations of Ser or Thr residues were converted into Glu. These mutations were combined with FRET pairs inserted in different locations to allow distance measurements. The results show that the paperclip conformation becomes tighter or looser, depending on the pseudo-phosphorylation state. In particular, pseudo-phosphorylation at the epitope of the diagnostic antibody AT8* (S199E + S202E + T205E) moves the N-terminal domain away from the C-terminal domain. Pseudo-phosphorylation at the PHF1 epitope (S396E + S404E) moves the C-terminal domain away from the repeat domain. In both cases the paperclip conformation is opened up. By contrast, the combination of AT8* and PHF1 sites leads to compaction of the paperclip, such that the N-terminus approaches the repeat domain. The compaction becomes even stronger by combining pseudo-phosphorylated AT8*, AT100, and PHF1 epitopes. This is accompanied by a strong increase in the reaction with conformation-dependent antibody MC1, suggesting the generation of a pathological conformation characteristic for Tau in AD. Furthermore, the compact paperclip conformation enhances the aggregation to paired helical filaments but has little influence on microtubule interactions. The data provide a framework for the global folding of Tau dependent on proline-directed phosphorylation in the domains flanking the repeats and the consequences for pathological properties of Tau. Microtubules that serve as the tracks for motor proteins are important for the intracellular transport of vesicles, organelles, and protein complexes by motor proteins (2Hirokawa N. Takemura R. Nat. Rev. Neurosci. 2005; 6: 201-214Crossref PubMed Scopus (658) Google Scholar, 3Mandelkow E. von Bergen M. Biernat J. Mandelkow E.M. Brain Pathol. 2007; 17: 83-90Crossref PubMed Scopus (182) Google Scholar). Microtubule dynamics are modulated by microtubule-associated proteins that bind to the surface of microtubules; among these, Tau protein is one of the major microtubule-associated proteins in neurons (4Garcia M.L. Cleveland D.W. Curr. Opin. Cell Biol. 2001; 13: 41-48Crossref PubMed Scopus (210) Google Scholar, 5Binder L.I. Guillozet-Bongaarts A.L. Garcia-Sierra F. Berry R.W. Biochim. Biophys. Acta. 2005; 1739: 216-223Crossref PubMed Scopus (320) Google Scholar). Its expression is strongly up-regulated during neuronal development to promote the generation of cell processes and to establish cell polarity (6Drubin D.G. Kirschner M.W. J. Cell Biol. 1986; 103: 2739-2746Crossref PubMed Scopus (562) Google Scholar). During this phase, Tau becomes sorted into the axon, and it diversifies into 6 different isoforms by alternative splicing (7Lee G. Cowan N. Kirschner M. Science. 1988; 239: 285-288Crossref PubMed Scopus (517) Google Scholar, 8Goedert M. Spillantini M.G. Jakes R. Rutherford D. Crowther R.A. Neuron. 1989; 3: 519-526Abstract Full Text PDF PubMed Scopus (1816) Google Scholar). In Alzheimer disease, Tau becomes hyperphosphorylated, missorted into the somatodendritic compartment, and aggregates into neurofibrillary tangles (9Braak H. Braak E. Acta Neuropathol. (Berl.). 1991; 82: 239-259Crossref PubMed Scopus (11332) Google Scholar).The numerous phosphorylation sites of Tau (10Johnson G.V. Stoothoff W.H. J. Cell Sci. 2004; 117: 5721-5729Crossref PubMed Scopus (421) Google Scholar) can be broadly subdivided into three classes: (i) SP/TP motifs in the flanking regions of the repeat domain are targets of proline-directed kinases such as glycogen synthase kinase3β (11Hanger D.P. Hughes K. Woodgett J.R. Brion J.P. Anderton B.H. Neurosci. Lett. 1992; 147: 58-62Crossref PubMed Scopus (648) Google Scholar, 12Mandelkow E.M. Drewes G. Biernat J. Gustke N. Van Lint J. Vandenheede J.R. Mandelkow E. FEBS Lett. 1992; 314: 315-321Crossref PubMed Scopus (480) Google Scholar), cyclin-dependent kinase 5 (CDK5) (13Baumann K. Mandelkow E.M. Biernat J. Piwnica-Worms H. Mandelkow E. FEBS Lett. 1993; 336: 417-424Crossref PubMed Scopus (417) Google Scholar), or mitogen-activated kinase and its relatives (14Drewes G. Lichtenberg-Kraag B. Doring F. Mandelkow E.M. Biernat J. Goris J. Doree M. Mandelkow E. EMBO J. 1992; 11: 2131-2138Crossref PubMed Scopus (492) Google Scholar). (ii) KXGS motifs in repeats are targets of non-proline directed kinases, such as MARK (15Drewes G. Ebneth A. Preuss U. Mandelkow E.M. Mandelkow E. Cell. 1997; 89: 297-308Abstract Full Text Full Text PDF PubMed Scopus (699) Google Scholar), SAD kinase (16Kishi M. Pan Y.A. Crump J.G. Sanes J.R. Science. 2005; 307: 929-932Crossref PubMed Scopus (260) Google Scholar), or PKA (17Drewes G. Trinczek B. Illenberger S. Biernat J. Schmitt-Ulms G. Meyer H.E. Mandelkow E.M. Mandelkow E. J. Biol. 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Phosphorylation at SP/TP motifs has only a moderate influence on Tau-microtubule interactions but is up-regulated in AD 3The abbreviations used are: AD, Alzheimer disease; DTT, dithiothreitol; FRET, fluorescence resonance energy transfer; GdnHCl, guanidine hydrochloride; IAEDANS, 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid; PHF, paired helical filaments; ThS, thioflavin S; BES, 2-[bis(2-hydroxyethyl)amino]ethanesulfonic acid; PIPES, 1,4-piperazinediethanesulfonic acid. 3The abbreviations used are: AD, Alzheimer disease; DTT, dithiothreitol; FRET, fluorescence resonance energy transfer; GdnHCl, guanidine hydrochloride; IAEDANS, 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid; PHF, paired helical filaments; ThS, thioflavin S; BES, 2-[bis(2-hydroxyethyl)amino]ethanesulfonic acid; PIPES, 1,4-piperazinediethanesulfonic acid. and other tauopathies (3Mandelkow E. von Bergen M. Biernat J. Mandelkow E.M. Brain Pathol. 2007; 17: 83-90Crossref PubMed Scopus (182) Google Scholar, 20Gong C.X. Liu F. Grundke-Iqbal I. Iqbal K. J. Neural. Transm. 2005; 112: 813-838Crossref PubMed Scopus (358) Google Scholar). This characteristic feature can be recognized by various diagnostic antibodies (3Mandelkow E. von Bergen M. Biernat J. Mandelkow E.M. Brain Pathol. 2007; 17: 83-90Crossref PubMed Scopus (182) Google Scholar, 21Augustinack J.C. Schneider A. Mandelkow E.M. Hyman B.T. Acta Neuropathol. (Berl.). 2002; 103: 26-35Crossref PubMed Scopus (713) Google Scholar). Phosphorylation by certain non-proline directed kinases (e.g. by MARK or SADK at the KXGS motifs of the repeat domain, or Ser-214 by PKA) results in a strong reduction of the ability of Tau to bind to microtubules (15Drewes G. Ebneth A. Preuss U. Mandelkow E.M. Mandelkow E. Cell. 1997; 89: 297-308Abstract Full Text Full Text PDF PubMed Scopus (699) Google Scholar, 22Brandt R. Lee G. Teplow D.B. Shalloway D. Abdel-Ghany M. J. Biol. Chem. 1994; 269: 11776-11782Abstract Full Text PDF PubMed Google Scholar, 23Illenberger S. Zheng-Fischhofer Q. Preuss U. Stamer K. Baumann K. Trinczek B. Biernat J. Godemann R. Mandelkow E.M. Mandelkow E. Mol. Biol. Cell. 1998; 9: 1495-1512Crossref PubMed Scopus (272) Google Scholar, 24Zheng-Fischhofer Q. Biernat J. Mandelkow E.M. Illenberger S. Godemann R. Mandelkow E. Eur. J. Biochem. 1998; 252: 542-552Crossref PubMed Scopus (289) Google Scholar) and inhibits the formation of PHFs (25Schneider A. Biernat J. von Bergen M. Mandelkow E. Mandelkow E.M. Biochemistry. 1999; 38: 3549-3558Crossref PubMed Scopus (445) Google Scholar). The region of Tau responsible for microtubule binding comprises the repeat domains (R1-R4) and the proline-rich flanking regions (Fig. 1). The repeat domain is also responsible for forming the core of the PHFs (26von Bergen M. Friedhoff P. Biernat J. Heberle J. Mandelkow E.M. Mandelkow E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5129-5134Crossref PubMed Scopus (752) Google Scholar). The flanking domain upstream of the repeats contributes to MT binding, but can also bind to other proteins, e.g. Pin-1 (27Smet C. Sambo A.V. Wieruszeski J.M. Leroy A. Landrieu I. Buee L. Lippens G. Biochemistry. 2004; 43: 2032-2040Crossref PubMed Scopus (84) Google Scholar) or protein phosphatase 2A (28Sontag E. Nunbhakdi-Craig V. Lee G. Brandt R. Kamibayashi C. Kuret J. White C.L. 3rd, Mumby M.C. Bloom G.S. J. Biol. Chem. 1999; 274: 25490-25498Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar). The N-terminal domain of Tau (∼200 residues) projects away from the microtubule surface (29Chen J. Kanai Y. Cowan N.J. Hirokawa N. Nature. 1992; 360: 674-677Crossref PubMed Scopus (486) Google Scholar) and may serve as an anchor for other cell components such as kinases, membranes, or motor components (30Magnani E. Fan J. Gasparini L. Golding M. Williams M. Schiavo G. Goedert M. Amos L.A. Spillantini M.G. EMBO J. 2007; 26: 4546-4554Crossref PubMed Scopus (165) Google Scholar).In solution, Tau behaves as a "natively unfolded" or "intrinsically disordered" protein (31Schweers O. Schonbrunn-Hanebeck E. Marx A. Mandelkow E. J. Biol. Chem. 1994; 269: 24290-24297Abstract Full Text PDF PubMed Google Scholar). NMR spectroscopy confirmed the paucity of secondary structural elements, but there are motifs in R2 and R3 showing inherent β-structure propensity that coincide with the hexapeptide motifs that nucleate PHF aggregation (26von Bergen M. Friedhoff P. Biernat J. Heberle J. Mandelkow E.M. Mandelkow E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5129-5134Crossref PubMed Scopus (752) Google Scholar, 32Mukrasch M.D. Biernat J. von Bergen M. Griesinger C. Mandelkow E. Zweckstetter M. J. Biol. Chem. 2005; 280: 24978-24986Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). Several observations suggest that Tau cannot simply be a "random coil" in the strict sense. Hints for special conformational states come from the reactivities of antibodies such as Alz50, MC1, Tau-66, MN423, and SM134 that have discontinuous epitopes on Tau. Antibodies Alz50 and MC1 recognize conformations of Tau in brain tissue that occur at an early stage of AD. Their epitopes comprise residues near the N terminus and in the third repeat and this conformation is called "pathological conformation of Tau" as it precedes aggregation (33Carmel G. Mager E.M. Binder L.I. Kuret J. J. Biol. Chem. 1996; 271: 32789-32795Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar, 34Jicha G.A. Bowser R. Kazam I.G. Davies P. J. Neurosci. Res. 1997; 48: 128-132Crossref PubMed Scopus (393) Google Scholar). Similarly, Tau-66 reactivity depends on the elements upstream of the repeat domain and residues in repeat R3 (35Ghoshal N. Garcia-Sierra F. Fu Y. Beckett L.A. Mufson E.J. Kuret J. Berry R.W. Binder L.I. J. Neurochem. 2001; 77: 1372-1385Crossref PubMed Scopus (81) Google Scholar), SMI34 reacts to a folded state of Tau wherein the repeat domain and one of the KSP motifs upstream or downstream from the repeats are required (36Lichtenberg-Kraag B. Mandelkow E.M. Biernat J. Steiner B. Schroter C. Gustke N. Meyer H.E. Mandelkow E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5384-5388Crossref PubMed Scopus (178) Google Scholar) and antibody MN423 requires a truncation site downstream of the repeats (at Glu-391) and the residues within the repeat domain (37Skrabana R. Kontsek P. Mederlyova A. Iqbal K. Novak M. FEBS Lett. 2004; 568: 178-182Crossref PubMed Scopus (40) Google Scholar). We recently characterized this globally folded state of Tau in solution by generating Tau variants containing FRET pairs at different positions and measuring their distance by the fluorescence energy transfer from the donor (tryptophan) to the acceptor (cysteine carrying a dansyl group). This study revealed a double hairpin or "paperclip" conformation where the C terminus was folded near the repeat domain, and the N terminus was folded back near the C terminus (1Jeganathan S. von Bergen M. Brutlach H. Steinhoff H.J. Mandelkow E. Biochemistry. 2006; 45: 2283-2293Crossref PubMed Scopus (299) Google Scholar).Given the results, the next question was whether phosphorylation at critical sites would have an influence on this global conformation of Tau and could induce the pathological state seen by the MC1 antibody. Ideally, it would be desirable to phosphorylate Tau by predetermined sites with 100% efficiency. This cannot be achieved due to the open structure of Tau, which makes many sites accessible to various kinases. Therefore, phosphorylation reactions generally result in a heterogeneous mixture of Tau molecules phosphorylated at different sites and to different extents. To circumvent this ambiguity we generated Glu mutants where phosphorylatable serine or threonine residues were replaced by glutamate. Although glutamate is not a perfect substitute for phosphorylation, it is a reasonable approximation (38Huang W. Erikson R.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8960-8963Crossref PubMed Scopus (131) Google Scholar), and the extent of "pseudo-phosphorylation" is by definition specific and complete. The effect of pseudo-phosphorylation on the aggregation and microtubule binding of Tau has also been studied (39Leger J. Kempf M. Lee G. Brandt R. J. Biol. Chem. 1997; 272: 8441-8446Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 40Haase C. Stieler J.T. Arendt T. Holzer M. J. Neurochem. 2004; 88: 1509-1520Crossref PubMed Scopus (122) Google Scholar). But it has not been studied whether there are changes in the global folding of Tau that could be linked to its properties. We have therefore combined phospho-mutants with FRET donor/acceptor mutants to measure intramolecular distances. Here we report that phosphorylation at critical sites upstream or downstream of the repeats indeed modifies the global conformation of Tau so that the paperclip structure is either opened up or tightened. In the latter case the N-terminal domain approaches the repeats, which is reminiscent of the conformation recognized by conformation-dependent antibody MC1. This has consequences for the aggregation behavior of Tau.EXPERIMENTAL PROCEDURESChemicals and Proteins—Chemicals of biochemical grade such as GdnHCl were purchased from Sigma. The fluorescent label 1,5-IAEDANS was obtained from Invitrogen. Point mutations were made in the htau40 coding sequence carried by plasmid pNG2 by site-directed mutagenesis using the QuikChange kit (Stratagene). The plasmids were sequenced on both strands to confirm the mutations. A number of mutants of the full-length Tau isoform htau40 (4R/2N) that carry mutations for FRET pairs, i.e. tryptophan (donor) and cysteine linked to IAEDANS (acceptor) at different positions, was used in the previous study (1Jeganathan S. von Bergen M. Brutlach H. Steinhoff H.J. Mandelkow E. Biochemistry. 2006; 45: 2283-2293Crossref PubMed Scopus (299) Google Scholar). For the current study, the mutant for mapping the proximities between the N terminus and repeats (N-R) were chosen with tryptophan at residue 310 and cysteine at 17; the mutant for mapping the distance between repeats and the C terminus (R-C) were chosen with tryptophan at residue 432 and cysteine at either 291 or 322; the mutant for mapping the proximities between the N and C terminus (N-C) were chosen with tryptophan at residue 432 and cysteine at 17. These FRET pair mutations were combined with phosphomimic mutations (glutamic acid) (Fig. 1B), for example, phosphorylated epitopes of antibodies: AT8* (Ser-199, Ser-202, Thr-205) (41Biernat J. Mandelkow E.M. Schröter C. Lichtenberg-Kraag B. Steiner B. Berling B. Meyer H.E. Mercken M. Vandermeeren A. Goedert M. Mandelkow E. EMBO J. 1992; 11: 1593-1597Crossref PubMed Scopus (426) Google Scholar, 42Goedert M. Jakes R. Vanmechelen E. Neurosci. Lett. 1995; 189: 167-169Crossref PubMed Scopus (469) Google Scholar), AT100 (Thr-212, Ser-214) (24Zheng-Fischhofer Q. Biernat J. Mandelkow E.M. Illenberger S. Godemann R. Mandelkow E. Eur. J. Biochem. 1998; 252: 542-552Crossref PubMed Scopus (289) Google Scholar, 43Hoffmann R. Lee V.M. Leight S. Varga I. Otvos Jr., L. Biochemistry. 1997; 36: 8114-8124Crossref PubMed Scopus (150) Google Scholar), and PHF1 (Ser-396, Ser-404) (44Greenberg S.G. Davies P. Schein J.D. Binder L.I. J. Biol. Chem. 1992; 267: 564-569Abstract Full Text PDF PubMed Google Scholar, 45Otvos Jr., L. Feiner L. Lang E. Szendrei G.I. Goedert M. Lee V.M. J. Neurosci. Res. 1994; 39: 669-673Crossref PubMed Scopus (405) Google Scholar). Thus to analyze the effect of mimicking phosphoepitopes (AT8*, AT100, and PHF1), constructs were made carrying Glu mutations and FRET pairs at different positions (e.g. N-R, R-C, and N-C). The expression of phosphomimic mutants were done using the BL21(DE3) Escherichia coli strain. The purification of Tau was usually done by making use of a heating step as described earlier (46Barghorn S. Biernat J. Mandelkow E. Methods Mol. Biol. 2005; 299: 35-51PubMed Google Scholar). But FRET mutants of Tau were purified with modifications involving a stepwise ammonium sulfate precipitation but no heating step. Briefly, the cell lysate was initially brought to 25% (NH4)2SO4 and then centrifuged for 45 min at 127,000 × g to clear the supernatant. The supernatant was then adjusted to 55% saturated (NH4)2SO4 to precipitate Tau protein and centrifuged for 45 min at 127,000 × g to collect the pellet that was then dissolved and dialyzed against buffer. Further purification was carried out using the ion exchange column SP Sepharose, followed by a gel filtration column G200 (Amersham Biosciences). The purity of the proteins was analyzed by SDS-PAGE. As an example of terminology, Tau/N-CAT8*+AT100+PHF1 will be used to denote the htau40 mutant carrying a combination of Glu mutations for epitopes of AT8*, AT100, and PHF1 with tryptophan at 432 and cysteine at 17 (N-C).Labeling of Proteins—Protein in 4 m GdnHCl, phosphate-buffered saline buffer (∼100 μm) was incubated with 10 m excess DTT for 10 min at 37 °C. DTT was then removed by size exclusion chromatography (Fast Desalting column, Amersham Biosciences) and the eluted protein was immediately supplemented with ∼20 m excess IAEDANS (dissolved in N,N-di-methylformamide). The labeling reaction was allowed to proceed at room temperature for 2 h, or alternatively "solid state-based labeling" was used (47Kim Y. Ho S.O. Gassman N.R. Korlann Y. Landorf E.V. Collart F.R. Weiss S. Bioconjug. Chem. 2008; 19: 786-791Crossref PubMed Scopus (179) Google Scholar), which is achieved by reducing the protein with DTT, then precipitating with 70% (NH4)2SO4, followed by dissolving the protein pellet with buffer containing IAEDANS. The solution was then dialyzed against phosphate-buffered saline and residual IAEDANS was removed by size exclusion chromatography. The concentration of protein was determined by absorption at 280 nm using the molar extinction coefficient ϵTau = 11,460 to 12,950 m-1 cm-1, depending on the Tau mutants. The amount of bound IAEDANS was determined by the absorption at 336 nm (ϵIAEDANS = 6,100 m-1 cm-1) (48Hudson E.N. Weber G. Biochemistry. 1973; 12: 4154-4161Crossref PubMed Scopus (389) Google Scholar). The protein concentration was corrected for the contribution of the IAEDANS at 280 nm and the stoichiometry was calculated. Typically the labeling stoichiometry was 0.7-0.9. The distances calculated with or without correction for the fractional labeling ratio are within a difference of 10-15% (see below).Fluorescence Spectroscopy—All steady state fluorescence measurements were performed with a Spex Fluoromax spectrophotometer (Polytec, Waldbronn, Germany), using 3 × 3-mm quartz microcuvettes from Hellma (Mühlheim, Germany) with 20 μl sample volumes. Protein was irradiated at 290 nm to excite tryptophan but not tyrosine. In all cases, the experimental parameters were as follows: scan range = 300-550 nm, excitation slit width = 4 nm, emission slit width = 6 nm, integration time = 0.25 s, and photomultiplier voltage = 950 V. Each time 3 spectra were scanned and averaged. A protein concentration of 4 μm was used and checked by SDS-PAGE as a control. In denaturation experiments with GdnHCl, the efficiency was calculated from emission intensities of labeled protein and unlabeled protein at the same GdnHCl concentration. The influence of various GdnHCl concentrations on the fluorimetric properties of tryptophan and IAEDANS was controlled with free dyes alone and in combination. The effects due to GdnHCl as a solvent were minor (<10%) in comparison to the FRET effects and the spectra were corrected for it. The FRET efficiency was measured by the energy transfer, EFRET = (1 − DA/D)(1/fA)(Eq. 1) where DA is the fluorescence intensity of the donor in the presence of the acceptor and D is the fluorescence intensity of donor in the absence of acceptor. The apparent efficiencies were normalized by fA, the fractional labeling with acceptor, as shown in Equation 1. The distance R between donor and acceptor was calculated by the Förster equation, EFRET = [1 + (R/Ro)6]−1(Eq. 2) where the Förster radius Ro is 22 Å for the Trp-IAEDANS pair (49Matsumoto S. Hammes G.G. Biochemistry. 1975; 14: 214-224Crossref PubMed Scopus (58) Google Scholar). In the case of the tryptophan-IAEDANS pair, a small error in the labeling ratio would give distance values that are within acceptable error range even without a correction factor, due to the dependence of the efficiency on the 6th power of the distance. For example, if the measured efficiency is 0.5 with 100% labeling, then r = 22 Å. On the other hand, if the measured FRET efficiency is 0.5 with only 80% labeling (fA = 0.8) and the correction for fractional labeling is applied, then r = 20.2 Å. Note that in unfolded proteins the distance between a given FRET pair shows a wider distribution and the apparent FRET reflects this heterogeneity (50Schuler B. Lipman E.A. Eaton W.A. Nature. 2002; 419: 743-747Crossref PubMed Scopus (777) Google Scholar).CD Spectroscopy—All measurements were carried out with a Jasco J-810 CD spectrometer (Jasco, Groß-Umstadt, Germany) in a cuvette with a path length of 0.1 cm. The scanning speed was 100 nm/min, bandwidth 0.1 nm, and a response time of 4 s. In each experiment, measurements were done at 20 °C and 4 spectra were summed and averaged. The CD spectra were normalized for the concentration at 214 nm using bovine serum albumin as a standard.PHF Assembly—Aggregation was induced by incubating soluble Tau typically in the range of 50 μm in volumes of 20 μl at 37 °C in 20 mm BES, pH 7.4, plus 25 mm NaCl buffer with the anionic cofactor heparin 6000 (molar ratio of Tau to heparin = 4:1). The formation of aggregates was monitored by ThS fluorescence and confirmed by electron microscopy. For ThS fluorescence, 5 μl of 50 μm assembly reaction was diluted to 50 μl with NH4Ac, pH 7.0, containing 20 μm ThS. Fluorescence measurements were done at 25 °C in a Tecan spectrofluorimeter (Crailsheim, Germany) with an excitation wavelength of 440 nm and an emission wavelength of 521 nm (slit width 7.5 nm each) in a black microtiter plate with 384 round wells (ThermoLabsystems, Dreieich, Germany). The background fluorescence was subtracted when needed. For electron microscopy, protein solutions were diluted to 1-10 μm and placed on 600 mesh carbon-coated copper grids for 45 s, washed twice with H2O, and negatively stained with 2% uranyl acetate for 45 s. The samples were examined with a Philips CM12 electron microscope at 100 kV.Microtubule Polymerization Assay—Microtubule assembly was monitored by UV light scattering at an angle of 90° at a wavelength of 350 nm in a black microtiter plate with 384 round wells (ThermoLabsystems) in a Tecan spectrofluorimeter in the presence and absence of Tau. 5 μm Tau was mixed with 30 μm tubulin dimer at 4 °C in microtubule assembly buffer (100 mm Na-PIPES, pH 6.9, 1 mm EGTA, 1 mm MgSO4, 1 mm GTP, 1 mm DTT) in a final volume of 40 μl. The reaction was started by raising the temperature to 37 °C.Western Blotting—The protein samples were added to SDS sample buffer and boiled at 95 °C for 5 min. Equal amounts of protein were loaded onto 10% SDS-polyacrylamide gels for subsequent electrophoresis. The proteins were transferred to a nitrocellulose membrane at 100 V for 40 min. After the transfer, the blot membrane was blocked with 5% milk and incubated overnight at 4 °C with the primary antibody (MC1 at 1:1000 dilution). After washing the unbound primary antibody, the secondary antibody (goat anti-mouse IgM conjugated to horseradish peroxidase at 1:1000 dilution) was incubated with the blot. The blot was then stripped of MC1 antibody and incubated with the pan-Tau antibody K9JA at 1:8000 dilutions (A0024, DAKO, Glostrup Denmark). Protein bands were visualized using chemiluminescence (ECL, Amersham Biosciences).RESULTSProteins and Phosphomimic Mutations—Tau is a natively unfolded protein that does not contain a significant amount of secondary structure (31Schweers O. Schonbrunn-Hanebeck E. Marx A. Mandelkow E. J. Biol. Chem. 1994; 269: 24290-24297Abstract Full Text PDF PubMed Google Scholar). However, it is possible that there are global conformations defined by interactions between the different domains of Tau, as suggested by certain antibodies (e.g. Alz50, MC1) that are diagnostic of a pathological conformation and react with a discontinuous epitope comprising residues near the N terminus and the third repeat (33Carmel G. Mager E.M. Binder L.I. Kuret J. J. Biol. Chem. 1996; 271: 32789-32795Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar, 34Jicha G.A. Bowser R. Kazam I.G. Davies P. J. Neurosci. Res. 1997; 48: 128-132Crossref PubMed Scopus (393) Google Scholar). Indeed, as shown previously, Tau in solution adopts preferred long range interactions between the repeat domain and the C terminus and between the N and C terminus (paperclip conformation), as judged by FRET (1Jeganathan S. von Bergen M. Brutlach H. Steinhoff H.J. Mandelkow E. Biochemistry. 2006; 45: 2283-2293Crossref PubMed Scopus (299) Google Scholar). For mapping FRET distances we inserted Trp as a donor and Cys-dansyl as an acceptor near the N terminus (e.g. residue Y18W or T17C-dansyl), near the C terminus (e.g. V432W), or within the repeats (Cys291-dansyl in R2, Cys322-dansyl in R3 or Y310W in R3). In solution, the C-terminal domain of Tau is unexpectedly close (19-23 Å) to the repeat domain and therefore causes a pronounced FRET signal, whereas the N-terminal domain (residue 17) is not within the FRET range of the repeat domain, but close to the C-terminal tail where it causes FRET (21-24 Å, Fig. 1A).Because the functions of Tau are regulated by phosphorylation we were interested whether this would influence the global conformation. In particular, we wanted to know whether phosphor
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