Download Notes - Part 2.

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.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
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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.
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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
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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.
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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
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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:
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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.
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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