Biochemistry - Chemical Reactions in Living Cells, Volume 1 - D. Metzler 1980

How molecules join together
Macromolecular packing
Helical structures

If the angle formed by two subunits when binding a to j differs slightly from the angle corresponding to a closed ring, a helical Structure is formed, as shown in Fig. 4-6, B. A single turn of the helix may contain either an integer or a non-integer number of subunits (as in the helical structure shown in the figure). Each successive subunit attaches to the preceding one through the same heterologous contacts of type aj, though other interactions may also occur in this case. If the subunit surfaces are complementary and their geometry favors The formation of additional contacts, groups located in different PARTS OF THE molecule (e.g., b and k) can join each other, forming another heterologous bond. The formation of a third heterologous bond, cl, between two other Regions of the subunit surfaces is also possible. If the contacts aj, bk, and cl are sufficiently strong (i.e., if the areas of the complementary subunit surfaces are large enough and the surfaces themselves are highly complementary), extremely strong microtubule structures can form (e.g., eukaryotic flagella, Fig. 1-5). When the interactions are not as strong, labile microtubule structures—which constantly assemble and disassemble—are frequently observed within Cells.

The geometry of subunits within helices can be visualized by constructing a PROJECTION OF THE unwrapped surface of the structure, as illustrated in Fig. 4-6, C. In this diagram, the subunits forming the helix depicted in Fig. 4-6, B lie on a plane obtained by mentally cutting the cylinder along its axis and flattening the cylindrical surface. In the example considered, the number of subunits per helical turn is approximately 4.3, though this number can also be an integer. The bk interactions between subunits along the fiber axis can sometimes be stronger than the aj-type interactions. In such cases, the microtubule "ruptures" at the ends due to the breaking of the aj bonds. If this phenomenon occurs in eukaryotic flagella, it can be directly observed using an Electron microscope.

Fig. 4-7 illustrates four beautiful types of helical structures formed from individual molecular fragments. These are the E. coli pilus, the Actin filament (F-actin) from Muscle fiber, the bacterial flagellum (E. coli), and the tobacco mosaic virus virion. Each of these structures is believed to consist of A large number of protomers of a single type. The structure of the virus particle has been studied in the most detail. In particular, The sequence of the 158 amino acid residues forming each viral protein subunit is known (mol. weight = 17,500); the number of subunits per particle is approximately 2,200, forming a rod ~300 nm in length. An RNA chain containing approximately 6,600 NUCLEOTIDES is located within the helical groove (thus, there are 3 nucleotides per protein subunit) [34–36a]. As in the simpler helical STRUCTURE OF THE bacterial pilus [37], the protein subunits of the virus particle appear to be linked to one another solely by heterologous bonds.

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FIG. 4-7. Some helical structures built from protein subunits. A. E. coli pilus: helical pitch 2.3 nm, aperture diameter 2.0–2.5 nm; Molecular Weight of a single subunit 17,000; subunits per helical turn. B. Muscle F-actin: helical pitch 70 nm; two chains; molecular weight of a single subunit 60,000; 13 subunits per helical turn. C. Bacterial flagellum: helical pitch 25 nm; aperture diameter 6 nm; five chains; molecular weight of a single subunit 40,000; 11 subunits per helical turn. D. Tobacco mosaic virus: length 300 nm (approximately 2,200 subunits); helical pitch 2.5 nm; molecular weight of a single subunit 17,500; 16.3 subunits per helical turn.

The structure of the protein coat of the filamentous bacteriophage Pf1 was studied in detail relatively recently [38, 39]. The molecular weight of the protein subunits of this bacteriophage (Supplement 4-B) is ~5,000. They have the shape of a-helices 7 nm in length, forming a left-handed helical structure within the bacteriophage with a pitch of 1.5 nm and 4.4 subunits per turn (Fig. 4-8). The protein "rods" are positioned at an angle to the helix axis and point toward the interior of the helix. Each rod contacts two others located 5 (—5) and 9 (—9) units further down the helical path (Fig. 4-8, A, B). This arrangement results in lock-and-key-type hydrophobic interactions between the subunits (Ch. 2, Sec. B.3,d). It is highly probable that bacterial pili and flagella possess precisely this structure rather than the one depicted in Fig. 4-7.

FIG. 4-8. A. Proposed arrangement of 1.0X7.0 nm a-helical protein rods in the protein coat of the filamentous bacteriophage Pf1 [38]. The dashed line at the top of the figure represents the left-handed a-helix on which the N-termini of the rods are presumed to lie. B. Schematic representation of the arrangement of a-carbon atoms in adjacent a-helical rods; the atoms are projected onto a plane parallel to the axis of the virus particle. The circled numbers denote amino acid residue numbers. Using three subunits as an example, the relative positioning of one subunit (0) and the fifth (—5) and ninth (—9) subunits located further down the helical path is shown [39].

Both helical Viruses and bacterial pili can be viewed as single helices of subunits, sometimes referred to as open-ended helices. The actin filament (Fig. 4-7) consists of two chains built from subunits; these chains are twisted around each other [40], meaning the structure has two open ends. The flagella of E. coli and Salmonella Bacteria (Fig. 4-7, C) can be envisioned as five chains twisted together around the same axis (one of the chains in the figure is shaded for clarity). However, these same flagella can also be viewed as structures formed by 11 parallel strands twisted into a helix with a significantly larger pitch [41]. Bacterial flagella possess many remarkable properties (see Supplement 4-B). For instance, in electron micrographs they typically appear as "superhelices" with a pitch of ~2.5 µm. What FEATURES OF MOLECULAR packing might this fact indicate? It would be excellent if the reader were to ponder and resolve this question independently, without rushing to the answer.

Supplement 4-A

Microtubules and the Action of Colchicine

An essential component of the Cytoplasm is microtubules—hollow rods with an outer diameter of 24±2 nm and an inner diameter of 13–15 nm. Their most striking form is found in the flagella and cilia of Eukaryotic cells (Fig. 1-5). The stable microtubules of cilia appear to be an integral part of the apparatus responsible for flagellar movement^a,b. (The illustration on the right is taken from work^b.) Labile (i.e., assembling and subsequently disassembling) microtubules are found most frequently in the cytoplasm of motile cells (e.g., in amoeboid pseudopodia). The mitotic spindle (Ch. 15, Sec. G.9) is a set of microtubules that ensure chromosome movement in a dividing Cell. Microtubules are also found in the division planes of plant cells.

A large number of microtubules are contained within the long axons of Nerve Cells. Here, they presumably facilitate the rapid transport of Proteins and other substances from The Cell body to the axon. Microtubules of unknown function have also been discovered in many sensory cells. It has recently been demonstrated that microtubules are present in the cytoplasm of A wide variety of cells. Using the indirect fluorescent antibody method, Weber et al.^c obtained the micrograph of mouse embryo fibroblasts shown below (kindly provided by the author).

Cells were fixed with formaldehyde, dehydrated, and treated with Antibodies obtained by immunizing rabbits with microtubule protein. The cells were then treated with fluorescent goat antibodies specific for rabbit y-globulin (Supplement 5-E) and photographed in their own fluorescence light. It is hypothesized that movements in systems containing microtubules are mediated by other proteins. For example, the "arms" on ciliary microtubules (Fig. 1-5) catalyze ATP Hydrolysis, resembling the muscle protein Myosin in this regard. Ciliary movement is likely driven by the sliding of microtubules, analogous to the sliding of fibrils in Skeletal Muscle (Sec. E).

All microtubules are constructed from tubulins—dimers consisting of structurally similar subunits (a, ß) with a molecular weight of 60,000; microtubules also contain a minor amount of a high-molecular-weight protein^d. The microtubules themselves likely represent groups of parallel strands of end-to-end connected tubulin molecules. Each tubulin dimer molecule binds two GTP molecules, binding one of them more tightly. In this respect, tubulin resembles actin, whose subunits are of approximately the same size. However, the Amino acid sequences of these proteins differ significantly.

It is generally accepted that labile Cytoplasmic microtubules exist in dynamic equilibrium with monomeric or dimeric units. It is known, for instance, that microtubules can assemble or disassemble depending on metabolic conditions. Their assembly requires GTP, the hydrolysis of which appears to be an essential part of the assembly process^e. Recently published data on the phosphorylation of microtubule proteins indicate that this process is highly complex.

Of particular interest is the interaction of microtubules with the alkaloid colchicine, which is produced by various members of the Liliaceae family.

This compound, containing a tropolone ring in its structure, specifically and tightly binds to tubulin. A striking consequence of this binding in the living cell is the destruction of labile microtubules, including those involved in forming the mitotic spindle. When dividing cells are treated with colchicine, Cell Division is arrested at metaphase (Ch. 15, Sec. G.9), and the resulting daughter cells exhibit a high degree of ploidy. All this has led to the widespread use of colchicine as an agent for producing tetraploid varieties of flowering plants. Similar effects on microtubules are exerted by the antineoplastic agents vincristine and vinblastine—Alkaloids synthesized by the lesser periwinkle (Vinca minor)^g.

The microtubules of eukaryotic cilia (and flagella) have a different structure. Throughout most of the cilium, they exist as fused pairs: the A-tubule bears an "arm," and the B-tubule attaches to it via shared subunits located at the center of the structure. As with labile microtubules, Two Types of tubulin molecules have been isolated, but how they fit into the structure of paired microtubules remains an open question.

^a Snyder J. A., McIntosh J. R., Annu. Rev. Biochem., 45, 699–720 (1976).

^b Bryan J., Fed. Proc., Fed. Am. Soc. Exp. Biol., 33, 152–157 (1974).

^c Weber K., Pollack R., Bibring T., PNAS, 72, 459–463 (1975).

^d Murphy D. B., Borisy G. G., PNAS, 72, 2696–2700 (1975).

^d Jacobs M., Smith H., Taylor E. W., JMB, 89, 455—468 (1974).

^e Marguhs T. N., JACS, 96, 899—902 (1974).

^g Wilson L., Bamberg J. R., Mizel S. B., Grisham L. M., Creswell К. M., Fed. Proc, Fed. Am. Soc. Exp. Biol., 33, 158—166 (1974).