Protofilaments

Berliner, E., Young, E., Anderson, K., et al., 1995. Failure of a. single-headed kine.sin to track parallel to microtubule protofilaments. Nature 373 718-721. 

Centrosomes, also called the microtubule organizing centre, are protein complexes that contain two cen-trioles (ringlike structures) and y- tubulin. They serve as nucleation points for microtubular polymerization and constrain the lattice structure of a microtubule to 13 protofilaments. They are responsible for organizing the mitotic spindle during mitosis. 

A microtubule is a hollow tube of 25 nm diameter formed by 13 protofilaments. Each protofilament consists of polymerized a- and (3-tubulin heterodimers. Microtubules are polarized and have a plus and a minus end. 

When tubulin heterodimers are assembled into microtubules, they form linear protofilaments with the P-tubulin subunit of one tubulin molecule linking covalently with the a-subunit of the next. Direct examination by electron microscopy of tannic acid-treated specimens has shown that micrombules in neurons and the A-microtubules of cilia and flagella have 13 protofilaments arranged side to side to form a cylinder around what appears to be an empty lumen. 

Protofilaments (composed of alpha and beta tubulin subunits)... 

Li L, Darden TA, Bartolotti L, Kominos D, Pederson LG. An atomic model for the pleated beta-sheet structure of Abeta amyloid protofilaments. Biophys J 1999 76 2871-2878. 

Figure 18 Structural model for A0 (1-40) protofilaments, derived by energy minimization with constraints based on solid-state NMR data, (a) Ribbon representation of residues 8-40, viewed down the long axis of the protofilament, (b) Atomic representation of residues 1-40. From Ref. 141.

Goda S, Takano K, Yamagata Y, et al. Amyloid protofilament formation of hen egg lysozyme in highly concentrated ethanol solution. Protein Sci. 2000 9 369-375. 

With regard to microtubular ultrastructure, micro filaments (5-7 run in diameter) are composed of filamentous actin. The tubule-like structures are formed by a, P-tubulin heterodimers. The wall is composed of 13 parallel protofilaments. Various microtubule-associated proteins and motor proteins (kinesin and dynein) are bound to the wall. The microtubule is a polar structure, i.e., plus and minus ends. 

Mandelkow EM, Mandelkow E. (1980) Subunit structure and conformations of tubulin protofilaments, in Electron Microscopy at Molecular Dimensions, State of the Art and Strategies for the Future (Baumeister W, Vogell W, eds.), Springer-Verlag, Berlin, Germany, 1980, pp. 117-125. 

Fig. 4. New structural models for amyloid and prion filaments with the parallel and in-register arrangement of //-strands in the //-sheets. //-Strands are denoted by arrows. The filaments are formed by hydrogen-bonded stacks of repetitive units. Axial projections of single repetitive units corresponding to each model are shown on the top. Lateral views of the overall structures are on the bottom. (A) The core of a //-helical model of the //-amyloid protofilament (Petkova et al., 2002). Two such protofilaments coil around one another to form a //-amyloid fibril. (B) The core of a //-helical model of the HET-s prion fibril (Ritter et al., 2005). The repetitive unit consists of two //-helical coils. (C) The core of a superpleated //-structura l model suggested for yeast prion Ure2p protofilaments and other amyloids (Kajava et al., 2004).

Chiti, F., Webster, P., Taddei, N., Clark, A., Stefani, M., Ramponi, G., and Dobson, C. M. (1999). Designing conditions for in vitro formation of amyloid protofilament and fibrils. Proc. Natl. Acad. Sd. USA 96, 3590-3594. 

Inouye, H., Bond, J. E., Deverin, S. P., Lim, A., Costello, C. E., and Kirschner, D. A. (2002). Molecular organization of amyloid protofilament-like assembly of betabellin 15D Helical array of beta-sandwiches. Biophys.J. 83, 1716-1727. 

In many amyloid systems, filament polymorphism has been observed by EM (e.g., Goldsbury et al., 1997, 2000). Structural variations may be expressed in terms of long-range axial repeats (Goldsbury et al, 2005 Jimenez et al., 2001), diameter (Louis et al., 2005), and/or number of protofilaments (Jimenez et al., 2002). Solid-state NMR has also been used to detect slight structural differences in Alzheimer s /1-peptide filaments... 

Malisauskas, M., Zamotin, V.,Jass, J., Noppe, W., Dobson, C. M., and Morozova-Roche, L. A. (2003). Amyloid protofilaments from the calcium-binding protein equine lysozyme Formation of ring and linear structures depends on pH and metal ion concentration. / Mol. Biol. 330, 879-890. 

A prime example of a Refolding model is that of the insulin protofilament (Jimenez et al., 2002). Insulin is a polypeptide hormone composed of two peptide chains of mainly o -helical secondary structure (Fig. 3A Adams et al., 1969). Its chains (21- and 30-amino acids long) are held together by 3 disulfide bonds, 2 interchain and 1 intrachain (Sanger, 1959). These bonds remain intact in the insulin amyloid fibrils of patients with injection amyloidosis (Dische et al., 1988). Fourier transform infrared (FTIR) and circular dichroic (CD) spectroscopy indicate that a conversion to jS-structure accompanies insulin fibril formation (Bouchard et al., 2000). The fibrils also give a cross-jS diffraction pattern (Burke and Rougvie, 1972). 

Jimenez et al. (2002) proposed a molecular model for the insulin protofilament based on these data and on electron cryomicroscopy (cryo-EM) reconstructions of insulin fibrils. The fibrils show a number of twisted morphologies that seem to be alternative packings of similar protofilaments. The protofilaments have cross sections of 30x40 A. The authors suggest a complete conversion to / -structure and model the amyloid monomer as having four jS-strands (Fig. 3B). Each insulin chain contributes two of these jS-strands, and the chains align in a parallel stack, constrained by the interchain disulfide bonds. One pair of stacked /i-stran ds is curved... 

Fig. 3. Refolding model of insulin protofilaments, from Jimenez et al. (2002). (A) Ribbon diagram of the crystal structure of porcine insulin (PDB ID code 3INS), generated with Pymol (DeLano, 2002). The two chains are shown as dark and light gray with N- and C-termini indicated. The dotted lines represent the three disulfide bonds 1 is the intrachain and 2 and 3 are the interchain bonds. (B) Cartoon representation of the structure of monomeric insulin in the fibril, as proposed by Jimenez et al. (2002). The thick, arrowed lines represent /1-strands, and thinner lines show the remaining sequence. The disulfide bonds are as represented in panel A, and N- and C-termini are indicated. (Components of this panel are not to scale.) (C) Cartoon representation of an insulin protofilament, showing a monomer inside. The monomers are proposed to stack with a slight twist to form two continuous /(-sheets. (Components of this panel, including the protofilament twist, are not to scale.) In the fibril cross sections presented byjimenez et al. (2002), two, four, or six protofilaments are proposed to associate to form the amyloid-like fibrils.

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