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1 .LECTURE 2 (Continued): THE SUPRAMOLECULAR ORGANISATION AND FUNCTIONS OF FIBROUS PROTEINS We have already seen that the most common secondary structures can, if extended, lead to linear polymers. The -sheet is the basic theme of silk, the -helix occurs in wool, and the polyproline helix in tendon. But how these basic units assembled in the fibres? Is there a hierarchical organisation of the kind found in globular proteins? Astbury began to address these questions in the 1930s and showed that fibres such as wool and silk gave characteristic and X-ray patterns and these led Pauling to use the terms -helix and -sheet in the 1950s when he suggested their molecular structures. 5.1 Silk -fibroin TIBS 7, 105-108 (1982) The haploid genome of the silkworm Bombyx mori contains only one copy of the gene for fibroin, the major protein of silk, but nevertheless produces 109 copies of this protein during larval development. Fibroin from different silkworm species has a chain of Mr 350-415,000. It consists of about 50 antiparallel -sheet regions, with a repeated amino acid sequence pattern, interspersed with irregular stretches of up to 100-200 residues. Figure 5.1.1. Structure of silk. The sequence of the crystalline -sheet regions is typically (in single-letter amino acid code): -[ GAGAGSGAAG(SGAGAG)8Y]50- or [GAGAGS]nconsisting only of glycine (G), serine (S) and alanine (A), with an occasional tyrosine (Y). The -sheets place the alternating glycines all on one face of the sheets. The sheets are thought to pack on top of one another so that glycines pack against glycines, the side-chains fitting in between each other. Similarly, the faces with alanine and serine also pack against each other. Figure 5.1.2. The packing of sidechains of alanines and glycines between alternately between sheets in silk. This nicely accounts for the properties of silk. It is elastic, due to the disordered regions, but only in a limited way, and then shows great tensile strength (force applied along 2 the polypeptide backbone). It is also flexible, because only H-bonds and van der Waals forces hold the strands together. -helical coiled coils TIBS (1986) 11, 245-248 The most extensive -helices in proteins occur not singly but in combination: 2 or 3 helices lie parallel and in register and fold together to form a superhelix or coiled coil. These occur in the myosin of muscle and other eukaryotic cells; in the intermediate filaments of the cytoskeleton (a multi-gene family including -keratins of hair and wool); and in fibrinogen, a key component of the blood-clotting cascade. 5.2.1 -keratins and related proteins (e.g. Darnell pp 845-847, Stryer III) The -keratins of wool, hair and skin are members of a group of structural proteins dubbed intermediate filaments (IF). They are the simplest class of structural proteins that probably all contain a 2-stranded coiled coil, and amino acid/DNA sequencing indicates that 3 or 4 such helical regions are usually interspersed with other, non-helical, linker regions. The amino acid composition is much less monotonous than that of silk fibroin. There is an abundance of residues favouring an -helix, such as leucine, alanine and glutamate, and no proline at all. The sequence contains a hierarchy of repeating units. The basic repeat unit is 7 residues long (abcdefg) in which a and d are hydrophobic (usually leucine) and b, c and f are often charged. In a right-handed -helix of 3.6 residues per turn (to be a perfect repeat it would need to have 3.5 amino acids per turn), this produces an amphipathic helix. Thus, two (or three) parallel -helices can coil round each other with the hydrophobic regions packing together between them. Figure 5.2.1. Coiled coils of -keratin. The -helices are relatively easily extended. They can be stretched by breaking the hydrogen bonds from 3.6 residues per turn towards the extended strand with 2.2 residues per turn. As hairdressers discovered many years ago, this can be assisted by steaming, which helps break the hydrogen bonds. Keratins (Mr 70,000) have a particularly high -helical content, and keratin fibres can be extended to about twice their original length. The helices are cross-linked with disulphide bridges, and these provide both a resistance to stretch and a restoring force when the stress is removed. 3 The lamins A, B and C are the major proteins of the nuclear lamina, which provides a supportive structural network on the nucleocytoplasmic side of the nuclear envelope. Increased phosphorylation of lamins occurs before the disintegration of the nuclear envelope during mitosis. 5.2.2 Myosin The thick filaments of striated muscle are a further example where the coiled coil is used as a rigid rod, but here it is also exploited as a ruler for regular assembly of a complex structure. The thick filaments, as revealed by electron microscopy, are primarily composed of bundles of molecules of myosin (Figure 5.2.2). Myosin consists of two identical "heavy chains" each of Mr 230,000 (2100 amino acids), and is organised as a modular structure, consisting of a double-headed globular region joined to a very long rod, which is hinged in two places (Figure 5.2.2). Figure 5.2.2. Structure of myosin The C-terminal 1300 amino acid residues of each polypeptide chain form a two-stranded coiled coil with only a single interruption, at a central hinge region. These helical regions have a well-defined heptad repeat, similar to that described above. Thus, two parallel myosin chains pack closely together with the hydrophobic residues buried, producing a mechanically rigid rod. A further regularity occurs every 28 residues (7 x 4) where alternating bands of positive and negative charge are produced on the surface of the 4 supercoil. There is a further repeat every 196 residues (7 x 4 x 7). These latter two repeats account for the self-assembly of about 250 myosin molecules into a thick filament, in which the central 'bare region' is about as long as a myosin tail, and in which individual globular myosin heads protrude in a regular helical array at intervals of 14 nm along the filament axis. There are no covalent bonds formed between the myosin molecules in the bundle. In contrast to the thin filaments contain actin (a globular protein) and the regulatory molecules troponin and tropomyosin (Figure 5.2.3). The tropomyosin of thin filaments (Tn) is composed of relatively short helical rods (Mr 70,000), which lie in the grooves of actin filaments. Each tropomyosin molecule controls the interaction of seven actin molecules with myosin, and in turn is controlled by the troponin complex. The latter contains Tn-I (binds to actin); Tn-T (binds to tropomyosin); and Tn-C (calciummodulated). See Stryer III, pp 921-937 for discussion Figure 5.2.3. Interactions of tropomyosin with fibrous actin. The actin-dependent ATP-hydrolyase (ATPase) activity is localised in the globular heads of myosin (labelled S1) each of which also binds two different "light chains". The enzyme-catalysed reaction involves a profound conformational change in the structure of S1. It was one of the first enzymes for which it was realised that the slowest step is not the breaking or making of chemical bonds. The important feature of the cycle is that actin has a high affinity for myosin and myosinADP-Pi, but a low affinity for myosin-ATP complex. Actin stimulates the powerstroke step, the conformation change leading to ADP release, from 0.02 s-1 to 20 s-1. Actin alternately binds to and is released from myosin as ATP is hydrolysed. 5 Figure 5.2.4. The role of myosin in the cycle of muscle contraction. Thus the coiled-coil structures of myosin "heavy chains" and tropomyosin, provide relatively rigid structural rods, with sequence patterns that assure the assembly of the globular active components at regular intervals along the chains. Note particularly the importance of segmental flexibility. The presence of the two non-helical hinge regions facilitates the packing of the myosin and allows the S1 ATPase to alter its contacts with actin during the power stroke of muscle contraction. 5.3 Proteins of connective tissue 6 Collagen, the most abundant protein in mammals, is rigid and resistant to stretch. Elastin, on the other hand, is the main component of elastic fibres, which are reversibly extensible to several times their length. 5.3.1 The structure of collagen. We have seen in section 2.4 that type 1 collagen consists of two chains of 1(I) and one of 2(I), forming a left-handed helices of equal length, aligned parallel, and coiled into right-handed superhelix.The conformation adopted by the individual chains is dictated by the proline and hydroxyproline, the sidechains of which replace the peptide NH and disfavour -helices and -strands. Examination of the tropocollagen structure explains why glycine is required: each of the three chains in turn contributes a side-chain to the congested centre of the superhelix, and only glycine can fit. There is H-bonding between the chains, perpendicular to the axis of the superhelix. The collagen superfamily of proteins now contains at least 19 proteins formally defined as collagens and an additional ten proteins that are collagen-like. The most abundant collagens form extracellular fibrils or network structures. Among these the most important types are: 7 A. fibril forming with structures as described above (types I, II, III, V and XI) B. network-forming with longer triple-helical structures and found in basement membranes (types IV, VIII and X) C. associated with and on surface of fibrils with interrupted or shorter regions of triple helix (IX, XII,XIV,XVI & XIX.) D. beaded filaments (type VI) E. collagen-anchoring fibrils for basement membranes (VII) The collagen genes contain about 50 exons, mainly of about 54 or 108 base-pairs or some near-multiple. The structure has almost certainly evolved by duplication of a 54 bp exon (six turns of helix). However, the single gene for collagen Type X has no introns at all! Non-collagen collagens include C1q, surfactant protein, mannan binding protein and the acetyl cholinesterase tail. 5.3.2. Biosynthesis of collagen Type I collagen is synthesised as pro-collagen chains (Mr 140,000). In the lumen of the rough endoplasmic reticulum, a short signal peptide is cleaved, and some of the proline and lysine residues are hydroxylated. Ascorbic acid is a cofactor for proline hydroxylase (hence the effects of scurvy). The C-domain is also glycosylated to mark the protein as "for export". In the Golgi, a proportion of the hydroxylysine residues are modified by addition of a galactosyl unit, and in some cases also a further glucosyl unit. Pro-collagen is formed after specific inter-chain disulphide links are made between the C-terminal peptides of individual pro- chains. The aligned helical regions fold spontaneously and pro-collagen bundles are exported from the fibroblasts by exocytosis. In the extracellular space, specific pro-collagen peptidases sever the N- and C-terminal extension peptides to produce tropocollagen. 8 Figure 5.3.1. Biosynthesis of collagen 5.3.3. Assembly of collagen Electron microscopy indicates that cross-striations in type I collagen fibres are at 6768 nm intervals. As in fibrin, this indicates a staggered array as the tropocollagen molecule is much longer than this. Analysis of the tropocollagen sequence indicates that charged and uncharged residues are periodically clustered along the axis of the triple helix at intervals of 67 nm (about every 230 amino acids). If two tropocollagen molecules are aligned in parallel, a displacement of one by 67 nm maximises the number of inter-chain electrostatic and H-bonds. There are gaps between the ends of the tropocollagen molecules about 40 nm which may be important in bone synthesis and/or cross-linking. The degree of cross-linking affects the mechanical strength and flexibility of the resulting collagen fibre. Covalent cross-links are formed within a tropocollagen molecule and between adjacent molecules (usually at their ends). All cross-links are initiated from lysine residues which are oxidised by lysyl oxidase (copper-requiring) to reactive aldehydes. 5.3.4 Mutations in collagen and disease Many mutations in collagen genes have been identified in many diseases, mostly of the bone and connective tissue. For example, the majority of the 200 mutations found in type I chains give rise to osteogenesis imperfecta (OI), which is characterised by brittle bones, but also abnormal teeth, thin skin, weak tendons and hearing loss. The disease may not be lethal, but leads to repeated fractures and deformities of limbs. In most of these diseases a codon for the obligate glycine in the -Gly-X-Y- sequence is substituted by a more bulky residue, but in other mutations there are insertions, deletions or splicing errors. The substitutions cause errors in the zipper-like assembly of the triple helix, so that the nonfolded material is degraded. This also affects unmutated collagen, which is not correctly incorporated, in a process known as procollagen suicide, a dominant negative effect. Other mutations lead to kinks in the triple helix that delay fibril formation. For a review see Prockop and Kivirikko (1995) Annu. Rev. Biochem. 64: 403- 434