The microtubule-associated protein tau forms a triple-stranded left-hand helical polymer.

High resolution transmission electron microscopy (TEM) has shown that bovine tau are 2.1 +/- 0.2-nm diameter filaments which are triple-stranded left-hand helical structures composed of three 1.0 +/- 0.2-nm strands. The reported amino acid sequence of human and bovine tau have been computer processed to predict secondary structure. Within the constraints imposed by the images, the secondary structure models and other structural information have been used to calculate tau's maximum and minimum length. The length calculations and secondary structure form the basis for image interpretation. This work indicates that each approximately 1.0-nm strand is a tau polypeptide chain and that the approximately 2.1-nm filament is composed of three separate tau chains (tau3). Bovine tau length measurements indicate that tau trimer filaments are generally longer than a fully extended tau monomer. These measurements indicate that each trimer, tau3, is joined with other trimers to form long tau polymers, (tau3)n. An inverse temperature transition has been found in the circular dichroism spectrum of tau indicating that its structure is less ordered below 20 degrees C and more ordered at 37 degrees C. The implications of this phenomenon with respect to tau's temperature-dependent ability to reconstitute microtubules is discussed and a mechanism for the possible abnormal aggregation of tau into neurofibrillary tangles in Alzheimer's disease is proposed.

* This work was supported in part by New York State Office of Mental Retardation and Developmental Disabilities, National Institutes of Health Grants AG05892, AG08076, AG04220, and NS18105, and a grant from the Alzheimer's Disease Research Program of the American Health Assistance Foundation, Rockville, MD. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed.
have been reported. All of these reports indicate that tau is a family of proteins derived from a single gene and that the heterogeneity in the amino acid chain length is due to alternative RNA splicing (Himmler, 1989). Bovine tau has a sequence of 448 amino acids (46,332 daltons) with variable deletions that can reduce its length by as much as 146 amino acids (Himmler et al., 1989;Himmler, 1989). Human tau has a sequence of 441 amino acids (45,850 daltons) with deletions of 29, 31, 58, and as many as 89 amino acids (Goedert et al., 1989). Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) of tau produces four bands ranging from 55,000 to 62,000 daltons (Cleveland et al., 1977a(Cleveland et al., , 1977b. When tau is partially phosphorylated there can be six to eight bands by SDS-PAGE (Lindwall and Cole, 1984b), with the phosphorylated bands shifted to what appear to be higher molecular weights. Fully denatured tau has a higher apparent molecular weight than a fully denatured equivalent standard globular protein marker by SDS-PAGE. Tau is much less hydrophobic than globular proteins (see "Results and Discussion"), binds less negatively charged SDS, and runs more slowly in an electric field applied to a polyacrylamide gel. The structure of tau was first studied by ultracentrifugation (Cleveland et al., 1977b). This work suggested that it was a rod-shaped molecule with an axial ratio of 20:l. More recently (Hagestedt et al., 1989), paracrystals of phosphorylated and nonphosphorylated tau have been reported. Phosphorylated tau was 90-95 nm in length and 3-6 nm in diameter whereas nonphosphorylated tau was 69-75 nm in length. An even shorter length of 30 nm was reported for undamaged tau indicating that it is an extremely flexible molecule. Tau was also studied in relation to microtubules, and its length was found to be 56.1 f 14.1 nm (Hirokawa et al., 1988). No reference was made in this work to tau's phosphorylation state.
The study of freeze-dried vertically platinum-carbon (Pt-C) replicated isolated bovine tau was undertaken to characterize tau's structure with high resolution transmission electron microscopy (TEM) (Ruben, 1989). Since tau has been found in both Alzheimer neurofibrillary tangles and in paired helical filaments (Grundke-Iqbal et al., 1986a, 1986bIhara et al., 1986;Nukina and Ihara, 1986;Yen et al. 1987;Goedert et al., 1988Goedert et al., , 1989Wischik et al., 1988;Lee et al., 1991), the study of tau's normal structure had to preceed TEM studies of neurofibrillary tangles and paired helical filaments so that their structural relationship to tau could be properly accessed in subsequent work.
In the present study isolated bovine tau preparations are shown to contain 2.1 f 0.2-nm filaments. It is shown that tau is triple-stranded and left-hand helical. Using the amino acid 22019 sequence of bovine tau and/or human tau, computer programs have been used to predict protein secondary structure under "Results and Discussion." Limits imposed by the tau images and circular dichroism estimates of a helix and p structure were used to temper these secondary structure predictions. The secondary structure, even with its limited accuracy, made it possible to identify the three separate strands as different tau sequences.

MATERIALS AND METHODS
Bovine Tau Preparation for TEM-The bovine tau was isolated by heat treatment of three cycled microtubules at pH 2.7, followed by extraction in 2.5% perchloric acid according to Grundke-Iqbal et al. (1986a). These preparations generally revealed in SDS-PAGE three to four bands in the 48,000-62,000-dalton range shown in Fig. 1 (lane I ) and no bands corresponding to microtubule-associated protein 2 (MAP-2). The line at the top of lane 1 is the protein entrance to the gel. Western blots of bovine tau (Fig. 1, lane 2) were developed with monoclonal antibody Tau-1 (Binder et al., 1985) using avidin biotin reagents of Vector (Burlingame, CA) according to Grundke-Iqbal et al. (1986a). Three samples were measured and averaged to yield 78 -t 9 ( n = 3) nmol of phosphate/mg of protein (Iqbal and Grundke-Iqbal, 1990).
The tau used for freeze-drying and vertical Pt-C replication in a mixed state of phosphorylation was at a concentration of 100 pg of protein/ml in 0.15 M NaCl, pH 7, and was deposited on the surface of a 13-mm filter disc with a 0.1-pm porosity (mixture of cellulose acetate and cellulose nitrate) from Millipore Corporation (type: VC, catalog no. WP01300). This sample was washed with distilled water at 18-20 "C, blotted with ashless filter paper to remove excess water, and frozen in liquid propane. It was then freeze-dried in the modified Baker's 300 for 2.5 h a t -80 "C, vertically (80O angle) replicated with 1.04 nm of Pt-C, and backed with rotary deposited (100" angle) 13.8 nm of evaporated carbon (Ruben, 1989). The samples were digested on 80% sulfuric acid, washed, and mounted as previously described (Ruben, 1989).
A second sample of tau in 5 mM Tris-HC1 buffer, pH 7, was prepared by placing three to four large drops on a mica disc at the center of a spinning table top centrifuge. The drops spread rapidly and radially from the center until they disappeared from the disc's surface. The sample disc was then frozen in liquid propane. This sample was freeze-dried and replicated with 0.93 nm of Pt-C and backed with 12.2 nm of rotary deposited carbon. The very long lengths of tau reported in the results were taken from this sample and were 1 2 Stds j ...;.,  measured with a map measurer a t a print magnification of X69,OOO. A JEM lOOCX transmission electron microscope with a lanthanum hexaboride filament, a 400-pm condenser aperture, and a 40-pm objective aperture was used. The fact that this arrangement limited resolution a t 80 kV to 0.66 nm was of no consequence since replica resolution was not better than 0.6-0.7 nm (Ruben, 1989). The micrographs were reversed and printed as described before (Ruben, 1989). Circular Dichroism-The bovine tau prepared for circular dichroism was 160 pg of protein/ml in 25 mM sodium phosphate, pH 6.5, and was also in a mixed state of phosphorylation averaging 78 -I-9 ( n = 3) nmol of phosphate/mg of protein or 3.1 0.4 ( n = 3) phosphates/ 384 amino acid tau (average number of amino acids in tau due to alternative RNA splicing, see table IV) as measured by Iqbal and Grundke-Iqbal (1990). The circular dichroism spectra were recorded as a function of temperature a t 5" intervals from 10 to 80 "C. The sample was equilibrated at each temperature for 15-20 min before two separate CD spectra were recorded and averaged. The circular dichroism equipment and methods have been described before (Ciardelli et al., 1988).
Secondary Structure Analysis-The analysis of the published bovine tau sequence (Himmler et al., 1989;Himmler, 1989) and the published human tau sequence (Goedert et al., 1989) was accomplished using the computer software PC Gene (6.1). @-Turns were analyzed with the Chou and Fasman (1979) @-turn program. The Garnier program for the prediction of protein secondary structure (Garnier et al., 1978) was used to predict CY helix, @-sheet, and coil structure. The evaluation of hydropathic index as a function of was done with the Kyte and Doolittle (1982) program SOAP. This sequence was averaged over a five to nine amino acid interval and program also calculated a grand average of hydrophobicity score (GRAVY) which is either positive or negative and is a measure of a protein's overall hydrophobic (+) or hydrophilic (-) character. This work was used to interpret the images of tau.
Longest and Shortest Tau Monomer Length Calculations Using the Idealized Tau Model in Fig. 6-The triple helix model has a diameter of 2.1 nm (Fig. 6a) where each strand is roughly 1.05 nm in diameter and where the center of each strand is 0.525 nm from the central axis of the 2.1-nm filament. Each tau strand forms a super helix around the filament axis with a 5.4-nm pitch. Using the extended chain spacing of 0.3-0.35 nm (0.325 nm) for each amino acid and 0.95 nm for each @-turn, the longest tau axial lengths in the triple helix filaments were calcuated for bovine tau (448 amino acids, 42 &turns) and human tau (441 amino acids, 42 @-turns). The shortest tau lengths were calculated by assuming that the amino acid sequence has the axial spacings of an CY helix or 0.15 nm between amino acids. This calculation is shown in Table IV along with the shortest length calculation for the spiral conformation first described in elastin by Urry et al. (1969). This helical coil ( p spiral) has a -1.66-nm diameter (could be 0.5 nm less, see Table I11 footnotes) with a pitch of 0.945 nm (Chang and Urry, 1989). The first calculation assumes an CY helical configuration with a tau monomer diameter of 1.05 nm and a pitch in the polymer of 5.4 nm. In the CY helix of each monomer the amino acid chain would follow a path -1.16-nm long/0.54-nm pitch. Each @-turn would occupy a distance of 0.95 nm along this helical path. The length calculations for these two models is shown in Table  IV. Since tau contains -20% CY helix and -38% @-turn structure, and -42% @ spiral residues (or coil) (excludes &turns) (Table II), a composite model (containing the afore mentioned secondary structure) shortest length was also calculated and reported in Table IV. In this calculation, the CY helical regions do not contain @-turns and the @ spiral regions contain all the &turns.

TEM of Isolated Bovine Tau Protein
Examination of the replicas of tau spread on 0.1-pm filter discs reveal the presence of long narrow filaments that are 2.1 f 0.2 nm in diameter (2.7 nm with 0.6 nm of Pt-C coating; Ruben, 1989) (Fig. 2a). The length of the filaments are 120, 300, and 800 nm as far as they can be measured on the surface of the 0.1-pm Millipore filter. The filter surface without any tau present does not contain any of these 2.1-nm filaments.
In Fig. 2b, sections of tau filaments extending across the holes in the filter were examined a t high magnification. Although the filament in panel A was measured as 2.2 nm (2.8 Panel a, bovine tau suspended on a Millipore filter. Isolated bovine tau on a 0.1-m Millipore filter was washed with distilled water (18-20 "C) before freezing. This sample was freeze-dried and vertically replicated with 1.04 nm of Pt/C and backed with 13.8 nm of evaporated carbon. The arrows point to long thin 2.1 f 0.2-nm filaments (2.7 nm with 0.6 nm of Pt-C coating; Ruben, 1989) which are identified as tau. The larger filaments are 10 f 1 and 13 f 1 nm in width. Filters without tau did not contain long thin -2.1-nm or larger filaments extending across the filter holes. X 92,500. b, the structure of bovine tau. Bovine tau suspended over 0.1-pm Millipore filter pores was replicated with 1.04 nm of Pt-C. Panels A-D contain tau filaments extending from left to right. Panels A'-D' contain tracings of tau adjacent to the panels with the same letter. In panels A and A' a 2.2f.3-nm filament (2.8 nm with 0.6 nm of Pt-C coating) shows subfilaments that cross the filament axis in a left-handed direction. The strands have a diameter averaging 1.0 f 0.2 nm (1.6 nm with 0.6 nm of Pt-C coating). Just to the left of the arrows in panels B and B' the filament measures 2.1 f 0.2 nm and shows a lefthanded helical structure. The filament separates into three strands where the arrows are labeled 1-3. These left-hand wrapped strands average 1.0 f 0.2 nm in diameter. The panel C and C' tau filament averages 2.1 f 0.5 nm and contains strands twisted around the filament axis in a left-handed direction. The edges of these strands were too poorly defined to measure. In panels D and D', a left-hand wrapped filament separates into three strands marked 1-3. These strands also average 1.0 f 0.3 nm in diameter. This tau strand is at the bottom of a and is an enlargement of the junction with the 13nm filament. This image was taken from a different micrograph in the tilt series of a. X 500.000. The replicas of tau spread on mica discs revealed a wide range of lengths of tau filaments (Fig. 3) The longest filament lengths were 2030 nm ( n = 2) with other lengths of 1740 ( n = 2), 1600,1380,1330,1240,914,682 nm ( n = 2), and smaller.

The Structure of Tau by Circular Dichroism
The CD spectrum in Fig. 4a shows a large trough in the spectrum at 197 nm which is identified as a coil polypeptide conformation (Greenfield and Fasman, 1969;Johnson, 1987). Because bovine tau is mainly coil conformation (60-66%) the empirical formulas for calculating LY helix and p-sheet are not considered accurate in quantitating these secondary structures. These formulas are only reliable when these structures are in abundance (Taylor and Kaiser, 1987). Nonetheless, these formulas were used on the 10, 40, or 80 "C CD spectra with nearly identical estimates of CY helix (10-12%) and p structure (24-28%) which were also similar to the room temperature values reported by Cleveland et al. (1977b). The CD spectum of bovine tau in Fig. 4a was taken as a function of temperature. Fig. 4b shows that the mean residue ellipticity (6') a t 197-nm increases by 52% from 10 to 80 "C indicating that with increasing temperature random coil is disappearing and is being replaced with a more ordered secondary structure.

Interpretation of Tau Images Using the Secondary Structure of Bovine and Human cDNA-derived Amino Acid Sequences
The long filamentous properties of tau suggest that it should have an unusual distribution of hydrophobic and hydrophilic amino acids in comparison to globular proteins. In Fig. 5a, the hydropathic index is plotted against the amino acid sequence in human tau (Goedert et al., 1989). A similar plot for bovine tau (not shown) looked almost identical to human tau. The tubulin-binding domains of tau, the four 31-amino acid repeats from about amino acid 244 to 368 are less hydrophilic than the sequence from 1 to 244 and not as hydrophobic as  Table IV) (Iqbal and Grundke- Iqbal, 1990). Panel b, inverse temperature transition in bovine tau. The ellipticity at 197 nm as a function of temperature from 10 to 80 "C at 5 "C intervals for the bovine tau in punel a. The progressive increase at 197 nm with increasing temperature indicates that random coil is being replaced with a more ordered structure. This figure indicates that bovine tau undergoes an inverse temperature transition. Most proteins heated from 10 to 80 "C show an increasing negative ellipticity at 197 nm or increasing random coil secondary structure.
the carboxy-terminal (400-441). The grand average of hydropathy, GRAVY, of human tau and bovine tau (Table 11) is -8.68 and -8.52, repectively, whereas water-soluble globular proteins are between +0.3 and -1.0 and average -0.4 on this index (Kyte and Doolittle, 1982). Tau's hydropathic index and the grand average of hydropathy are consistent with an extended conformation exposed along its surface to water and containing no buried globular hydrophobic regions.
Secondary structure predicting programs are useful for showing trends in protein structure, even though they are no better than 63% (Garnier et al., 1978;Busetta and Hospital, 1982;Kabsch and Sander, 1983) to 70% accurate (Chou and Fasman, 1979). Estimates from our circular dichroism measurements and Cleveland et al. (1977b) suggest that tau is 10-12% a helical and 20-27% @ conformation whereas the Garnier program (Garnier et al., 1978) suggests that tau contains 31% a helix and 37-39% @ conformation (Tables I and 11). It is unlikely that triple stranded tau (-2.1-nm diameter) contains @-sheet since each sheet strand could contain only two amino acid chains roughly 1.0-1.3 nm X 0.3-0.6 nm in crosssection to approximate the 1.0 f 0.2-nm strands. Second, it is difficult to understand how these strands could remain as separate strands with unfulfilled hydrogen bonds on both sides of a strand. Third, &sheet is a fully extended conformation (0.3-0.35-nm spacing between amino acids) which would not produce an elastic tau as described by Hagestedt et al. (1989). Finally it has been shown that @-sheet can have a right-handed helical twist with a pitch as short as 9.2-9.6 nm (Fraser and Macrae, 1974;Fraser et al., 1971;Stewart, 1977), but it is hard to understand how these two amino acid strands could cross the 2.1 f 0.2-nm filament axis at 5.4-nm intervals and maintain their @-sheet structure and their separate strand identity. The @ structure in tau is more likely to take the form of @-turns also called @-bends or reverse turns.

Tau Lengths Calculated Using Its Secondary Structure and an Idealized Periodic Conformation
The analysis of the amino acid sequence is consistent with tau's filamentous conformation. The images in Figs. 2 and 3 reinforce this prediction, and in conjunction with the previous analyses, the evidence suggests that the three strands are separate tau monomers in an extended filamentous conformation. Assuming that each 2.1 f 0.2-nm filament is composed of three adjacent tau monomers (tau,), the longest and the shortest tau monomer strand lengths can be calculated (see "Materials and Methods"). The longest tau axial lengths in the triple helix filaments are 106-117 nm (112 nm for 0.325-nm amino acid spacing) or 104-116 nm (110 nm for 0.325-nm amino acid spacing) for bovine tau (448 amino acids, 42 @-turns) and human tau (441 amino acids, 42 @-turns), respectively. Assuming that the full-length tau and tau with deletions are equally represented then the longest average length of human tau is 96.2 nm and the longest average length of bovine tau is 95.8 nm. In Table 111, the longest measured porcine tau monomer paracrystalline length was reported as 90-95 nm. Clearly the tau monomer length within a triple helix model can accomodate the longest measured tau monomer.
The shortest tau lengths were calculated by assuming that the amino acid sequence has the axial spacings of an a helix. This first calculation is shown in Table IV along with the length calculation for the @ spiral conformation first described in elastin by Urry et al. (1969). This helical coil (@ spiral) has a -1.66-nm diameter (its diameter could be 0.5 nm less, see Table I11 footnotes) with a pitch of 0.945 nm. In the a helix the amino acid chain would follow a path -1.16-nm long/ 0.54-nm pitch. The length calculations for these two models is shown in Table IV. Neither of these models by themselves yields lengths close to 30 nm. Since tau contains -20% a helix and -38% @-turn structure, and -42% @ spiral residues (excludes @-turns) (Table 11), a composite model (containing the aforementioned secondary structure) shortest length was calculated in Table IV. The composite model gives estimates of tau's monomer length slightly shorter than 30 nm. The shortest paracrystalline length reported for porcine tau in Table I11 is 30 nm which can be accomodated by the tau monomer length in the triple-helical tau polymer model. A nonphosphorylated porcine tau length of 69-75 nm (Hegestedt et al., 1989) or the other porcine tau length of 56.1 f 14.1 nm (Hirokawa et al., 1988) have also been reported ( M a -t u g proWility pmfile of TAU441G.froa anino acid 1 to amino acid 44l., Ihc y ax15 values npresrnt the pmbablllty p(tum) * 1 W

FIG. 5. Panel a, hydropathic index of human tau. The hydropathic index on the left-hand scale for an averaged interval of five amino
acids is plotted against the amino acid sequence in human tau (Goedert et al., 1989). Positive values are hydrophobic and negative values are hydrophilic. The grand average of hydropathy (GRAVY) for human tau is -8.68 and for bovine tau is -8.52 (Table 11). Panel b, 0-turn probability in the human tau sequence. The probability of a tetrapeptide forming @-turns in human tau as a function of its amino acid sequence. The dashed horizontal line corresponds to a cut off probability of 0.75 X 10" below which a &turn is not predicted. The grouped peaks are often overlapping amino acid sequences from which the most likely &turn has been selected in Table I. Although not shown, the plot for bovine tau looked almost identical to this human tau plot. 111). These lengths roughly correspond to the average of the longest and shortest tau monomer length calculations which are 61.8 nm for bovine tau and 62.4 nm for human tau. The triple-stranded left-hand helical model for tau can accomodate the variety of paracrystalline tau monomer lengths cited in the literature (see Table 111). The shortest length calculation uses the estimated secondary structure of human tau in Table   11. The tau polymer model is not only compatible with tau protein secondary structure estimates in Table 11, but it is also consistant with the reported elastic behavior of porcine tau (Hegestedt et al., 1989). The /3 spiral protein (in contrast to a helix) secondary structure (Table IV) according to Urry (1988) has elastic properties.

Tau F o r m a Polymeric Structure: a New Class of Triple-helical Fibrous Proteins
The images in Fig. 2 indicate that tau forms -2.1-nm filaments that are triple-stranded and left-hand helical. The strand diameter averages -1.0 nm, which is about the diameter of a right-handed a helix (-0.46 nm) enlarged an additional 0.5 nm due to amino acid side chains. An extended coil could also be about this size. A frequently observed 1.8 f 0.2 nm distance between strands that cross the -2.1-nm filament axis at 30-55" angle is generated by the -1.0-nm strands (see Fig. 6, a and d ) . The images, sequence analyses, and length calculations suggest that the quaternary structure of the -2.1nm filament is composed of three left-hand helical extended tau monomers (taus) between 29.2-95.8 nm in length. If it is assumed that tau29 normal length is intermediate or -63 nm (Hirokawa et al., 1988;Hagestedt et al., 19891, then it can be concluded that tau3 forms polymers of many different sizes. Using -63 nm as an estimate of taus's length, then the polymer, (tau,), can have positive integer n values from 2 to 33 (see range of tau lengths in Table 111). Its not likely that n is just limited to these values.
Since (tau,), appears to be a continuous -2.1-nm filament (Fig. 2), the ends of one taua smoothly intermesh with each consecutive tau, unit. The four to six tau sequence lengths (Goedert et al., 1989;Himmler et al., 1989) make this possible if each tau strand is assembled in register on the region with the three or four 31 amino acids repeat sequences, with the NHz-terminals at one end and the COOH-terminals at the other. This should align the 2 cysteines in each tau sequence to form a disulfide bond with an adjacent tau strand. Joining the tau trimers, NH2-terminal to COOH-terminal would orient the taus within the (tau3), with the same chain polarity and equidistant spacing between repeat regions. In addition the COOH-terminal domains are predominantly positively charged (17+ basic versus 12-acidic amino acids) and the NH2-terminal domains are negatively charged (27-acidic versus 4+ basic amino acids) (Himmler et al., 1989) so that opposite charge attraction could drive the polymerization process (see Table V). However compelling these arguments are, the alternative method of coupling NHz-terminal to NH2terminal and COOH-terminal to COOH-terminal, although less probable, cannot be completely ruled out. Nevertheless, each tau strand has a complex secondary structure, has little tertiary structure, and assembles with a triple-stranded lefthand helical quaternary structure. This structure helps explain why tau needs to be isolated in a disulfide bond-reducing medium and is stable at temperatures of -100 "C. Tau is also soluble and stable when heated in a pH 2.7 buffer (Grundke-Iqbal et al., 1986a) because it contains roughly equal numbers of basic and acidic amino acids leading to a soluble positively charged molecule at high temperature whereas MAP-2 precipitates with only 213 the number of basic amino acids versus acidic residues. Globular proteins with extensive hydrophobic domains also precipitate under these conditions. It should also be pointed out that this kind of protein structure is new and is quite different from the 1.5-nm triple-stranded righthanded helix of collagen (see Fig. 6b) and a 2-2.5-nm triplestranded a helix-coiled coil suggested for a-keratin and T3 tail fibers (see Fig. 6c) (Crick, 1953;Fraser et al., 1962;Takahashi and Ooi, 1988). Neither of these structures is consistent with the secondary structure predicted from tau's primary sequence, with the tau images or with circular dichroism results. Strong evidence for a triple-stranded lefthand helical fibrous protein like tau has not been reported before. Although the model of tau is presented as a regular structure to facilitate the calculation of its maximum and minimum length, all of the tau samples frozen from an initial temperature of 18-20 "C were only partially regular (Fig. 6d).

Implications of a n Elastin-like Inverse Temperature
Transition in Tau Elastin's Similarities to Tau Suggest That Tau Shortens from 20 to 37 "C and Becomes Elastic-Elastin is filamentous, Predicted &turn and a helical regions of human tau Four consecutive amino acids are underlined showing the location in the human tau sequence of the predicted @-turns from the Chou and Fasman (1979) computer program. The 8-turn probability map is shown in Fig. 5b. The predicted sequences of a helix are in boldface letters in the sequence. Although these sequences (24 residues) frequently overlap @-turns (1-4 residues), we have chosen to ignore the incompatibility of these two structures and report the results of the Garnier et al. (1978) program. In compiling Table 11, we have retained all the &turn predictions at the expense of a! helix except for the one at residue 3-6 which was omitted. If a helix sequences are 4 residues or fewer it has been assumed arbitrarily that they are not likely to retain this configuration. 8 sheet secondary structure is assumed to be incompatible with the 1.0-nm strand diameter in the tau images (see "Discussion"). Secondary structure which is neither 8-turn or a helix we assume to be coil or 8 spiral. This same approach was used in judging secondary structure for all the proteins listed in Table 11. yesL.J +6.85 Chou andFasman, 1979. Garnier et al., 1979. e The coil trough at 197 nm diminishes as the temperature is raised from 10 to 80 "C, the inverse of normal protein denaturation which increases the trough at 197 nm in the CD spectrum. Kyte and Doolittle, 1982;(-) hydrophilic, (+) hydrophobic. e Goedert et al., 1989. ' Himmler et al., 1989. Lewis et al., 1988. Raju and Anwar, 1987. Starcher et al. (1973, estimated from CD spectrum. J Urry et al., 1969Urry et al., , 1985 has a secondary and quaternary structure similar to tau's (Tables I1 and 111), and its length like porcine tau's (Table  111) is variable. Because elastin, tropoelastin, and bovine a elastin are very hydrophobic (GRAVY = +6.85, Table 11) and first assemble into small filaments, at temperatures of 24-40 "C the small filaments condense into larger 0.5-9-pm wide fibers. Nonetheless, bovine elastin, tropoelastin, and a synthetic peptide containing -200 copies of elastin's pentapeptide repeat are water soluble at temperatures less than 24 "C. At temperatures between 24-40 "C, these proteins condense into aggregates of parallel fibrils which are a coascervate of -37% protein and -63% water (Volpin, 1977;Urry, 1988). During the temperature increase from 24 to 40 "C, circular dichroism has shown that the elastin structure or pentapeptide polymer change from a less ordered internal state to a more ordered internal state (Urry et al., 1969(Urry et al., , 1985Starcher et al., 1973). This entropic process has been called an inverse temperature transition where ordered clathrate water leaves the protein at higher temperature, the protein becomes more ordered and the removed water becomes disordered increasing the water and protein's overall entropy (Urry, 1990). In this process elastin and a polypentapeptide elastin-like polymer are shortened by 22 and by 55%, respectively (Table 111) and develop elastomeric force (Urry et al., 1986;Urry, 1988). This process is reversible. By lowering the temperature to 20 "C, elastin and the elastin-like pentapeptide polymer extend (with a (3 spiral rise of 2.16 nm/turn of 2.9-3.0 pentapeptide repeats; Chang and Urry, 1989) and looses its elastomeric properties. By rewarming to 40 "C, elastin and the elastin-like pentapeptide polymer resume their shortened length (with a (3 spiral rise of 0.945 nm/turn of 2.9-3.0 pentapeptide repeats; Chang and Urry, 1989) with the return of elastomeric properties. The inverse temperature transition is a finely tuned entropic transition which begins at 24 "C and is complete at 37 "C or body temperature. By chemically synthesizing an additional hydrophobic group in the pentapeptide repeat, the inverse temperature transition occurs at a temperature 15 "C lower (Urry, 1988). If a hydrophilic group is chemically synthesized into the pentapeptide repeat, the inverse temperature transition occurs at a temperature 45 "C higher (Urry et al., 1988). The circular dichroism spectra showing with increasing temperature a decrease in the random coil, in the present study, directly establishes that the tau protein also undergoes an inverse temperature transition which should also be affected  Hirokawa et al., 1988. Voter andErickson, 1982. e Gottlieb and Murphy, 1985. ' Hirokawa, 1986. Ranachandran and Santhanam, 1957. Cleary and Cliff, 1978 ' Gotte et al., 1968. j Starcher et al., 1973 ' Serafini-Fracassini et al., 1977. ' Urry et al., 1986. Using numbers in Chang and Urry, 1989, the 8 spiral strand diameters were calculated assuming amino acid side chains added 0.5 nm to the diameter like they do with an a helix. The diameter of the 8 spiral would be reduced if the side chains were parallel to or pointed inwards toward the filament axis. The diameters for these configurations would be 0.98 and 1.16 nm and would be closer to the size of 1.0 nm reported for the triple-helix strand diameter. The length of the composite model is calculated assuming that the shortest tau would contain 38% 8-turns, -42% j3 spiral residues (coil) not including the &turns, and -20% a helix. According to the definition of B spiral, this model would be -80% B spiral, and -20% a helix. by similar changes in amino acid composition.

SGDTSPRHLSNVSEIDMVDEATLADEVSASLAKQGL
peptide polymer, undergoes an inverse temperature transition Microtubule Assembly and Stability-Since the circular di- (Fig, 4, a and b ) which starts in a less ordered extended state chroism of tau demonstrates a progressive increase in mean at 10 "C and is transformed to a shortened more ordered state residue ellipticity (e) at 197 nm with increasing temperature at 37 "C (Table 111) and at higher temperatures with an upper from 10 to 80 "C, tau, like elastin and the elastin-like pentalimit of -80 "C. This process has important implications for 1 b 3 FIG. 6. Panel a, idealized periodic triple-stranded left-hand helical tau model. This tau model idealizes the structural features of tau that have been observed in Figs. 2 and 6d. The diameter of 2.1 nm is composed of three -1.0-nm strands which cross the filament axis a t -1.8-nm intervals with a pitch of -5.4 nm. Panel b, triple-stranded collagen model. In b the model for collagen is shown with a 1.5-nm diameter. Three 0.7-nm strands cross the axis a t -2.9-nm intervals along the axis with a pitch of 8.6 nm. The collagen strands also contain a triplet repeat motive, (Gly-X-Y), where X and Y are frequently proline and hydroxy proline. These residues form a lefthanded helix with a pitch of 0.87-0.90 nm with each amino acid extending 0.295-0.30 nm axially (Ramachandran, 1963;Schulz and Schirmer, 1979). This is very near the fully extended amino acid distance of 0.35 nm (-17% longer than 0.3 nm) found in 8-sheet and probably explains why rat tail collagen can only extend about 1.5-17% before it ruptures (Kastelic and Baer, 1980). Panel c, triplestranded a-keratin model. In panel c, the model for a-keratin is shown with a 1.9-nm diameter. Three 0.95-nm strands cross the axis at  Goedert et al., 1989. ' Himmler et al., 1989. Lewis et al., 1988 how tau functions in reconstituting microtubules. Tubulin, at 37 "C, in the presence of buffers and tau, assembles into microtubules. If the temperature is lowered below 20 "C, the microtubules disassemble.
This process also occurs with MAP-2 and other microtubule-associated proteins. MAP-2s secondary structure suggests that it should, like tau and elastin, undergo an inverse temperature transition from 20 to 37 "C (see Tables 11, 111, and V). We plan to investigate this CD spectrum-temperature relationship in the near future.
Additional phosphorylation shifts the inverse temperature transition to higher mean temperature (Urry, 1990). It is not surprising, then, that heavily phosphorylated tau (Lindwall and Cole, 1984a) and other heavily phosphorylated microtubule-associatedproteins (Jameson et al., 1980) do not promote microtubule assembly a t 37 "C. This suggests that the shorter, more ordered tau or MAPS a t 37 "C binds more strongly than the longer less ordered state a t lower temperature and that heavy phosphorylation can make tau or MAPS more hydrophilic and prevent their transition to a shorter more ordered state at 37 "C.
An elastic tau is needed to stabilize very long axonal microtubules that are subject to constant bending, as at knee and elbow joints. It is unlikely that individual tau monomers with only a single binding domain could restore microtubule structure elastically after a bending insult. Each tau has a single region of three or four repeating sequences, totaling 93 or 124 amino acids, that bind to tubulin in microtubules (Aizawa et al., 1988). A tau polymer oriented axially along the microtubule would connect tau monomers which each extend over a t least seven or eight (-63 nm) 8-nm tubulin dimers with binding domains approximately 13.1 or 17.5-nm long. Such an elastic tau polymer repeated around the microtubule periphery would stabilize the microtubule and maintain microtubule structure through a bending process. We are investicoil of three a helices has a diameter of 1.9-2.5 nm and is composed of three helices each 0.95-1.0 nm in diameter which left-hand twist around each other (Crick, 1953) with a pitch of 18.6-20 nm (Crick, 1953;Fraser et al., 1962). Panel d, triple-stranded left-hand helical tau is not perfectly regular. Isolated freeze-dried vertically replicated (1.04 nm of Pt-C) bovine tau prepared as in Fig. 2a. The 2.1-nm diameter has been enlarged by 0.6 nm of Pt-C (Ruben, 1989) to 2.7 nm and the -1.0-nm strands, enlarged to 1.6 nm by 0.6 nm of Pt-C, cross the axis a t -1.8-nm intervals in a partially regular region. An optical diffraction pattern of this filament was attempted and was unsuccessful. The traced tau filament is recorded below the halftone image. The edges of the strands are black and the strand widths are not perfectly regular. The arrows labeled 1-3 point to separate strands in the triple-stranded left-handed tau helix. The coil structure or 8 spiral structure in the strands of tau monomer can in theory be -6.3-nm intervals along the axis with a pitch of 19 nm. The coiled stretched as thin as the backbone amino acid chain. X 1,800,000.

The MAP Tau Forms a Three-stranded Left-hand Helical Polymer 22027
gating the prediction that tau polymer is longitudinally oriented on microtubules and that it has a functional role in the axons.

Alzheirner Neurofibrillary Tangle Formation
The inverse temperature transition has important theoretical implications for neurofibrillary tangle formation from tau in Alzheimer's disease. Elastin and the elastin-like pentapeptide polymer form coacervates at 24-37 "C. It is possible by making the amino acid composition of tau more hydrophobic that tau can aggregate like elastin at 24-37 "C instead of promoting microtubule assembly. Shifting the coascervation phenomena to lower temperatures by 10-15 "C may require the replacement or elimination of only 1 aspartate or glutamate/100 amino acid residues (Urry, 1990). It seems probable then that some tau produced in the nerve cell body of Alzheimer's disease victims contains a slighty more hydrophobic sequence which self-aggregates and condenses into neurofibrillary tangles at 37 "C and neutral pH. This mechanism could be triggered if tau was phosphorylated in the wrong position leaving a long sequence of about 100-150 amino acids more hydrophobic. Tau from Alzheimer neurofibrillary tangles is abnormally phosphorylated (Grundke-Iqbal et al., 1986b;Iqbal et al., 1986Iqbal et al., , 1989 and is unable to reconstitute microtubules at 37 "C (Iqbal et al., 1986;Nieto et al., 1990). We know that tau that is functionally active in microtubule assembly contains a number of phosphate groups (Lindwall and Cole, 1984a;Iqbal and Grundke-Iqbal, 1990). The number and location of the phosphorylation sites on both normal and abnormally phosphorylated tau are yet to be determined.